University of Southern California Dissertations and Theses

Ameloblastin-protein interactions pattern enamel matrix

(USC Thesis Other)

Ameloblastin-protein interactions pattern enamel matrix

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content
AMELOBLASTIN-PROTEIN INTERACTIONS PATTERN
ENAMEL MATRIX

by

Shuhui Geng

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIOFACIAL BIOLOGY)

December 2015

Copyright 2015 Shuhui Geng

ii

Acknowledgements

I would like to thank my mentor Dr. Malcolm L. Snead for his guidance and
expertise throughout my graduate training process. His encouragement, advice and
support always motivated me at difficult times during my research. He leads me into the
academic world and gives me confidence to move on in scientific research.
I would also like to thank my committee members, Dr. Michael Paine, Dr. Janet
Moradian-Oldak, Dr. Baruch Frenkel, and Dr. Harold Slavkin for their support and
guidance. Special thanks to Dr. Michael Paine for sharing his experience with yeast two-
hybrid assay and assisting me when I preformed this experiment.
I am deeply grateful to my aunt, Dr. Yaping Lei for her expertise. She helped me
master techniques of molecular cloning, histology, cell culture study, lentivirus
transduction, as well as animal studies. Her scientific knowledge, technical support and
collaboration have been invaluable to my studies. And her love supported me to finish
my research program.
I would like to acknowledge my colleagues Dr. Xin Wen and Dr. Zhan Huang for
their technical support and trouble-shooting discussion. Thanks to Dr. Bridget Samuels
for her time in helping me revise and finish this dissertation. Also thanks for all the help
from CCMB members during this entire time.
Finally, many thanks would go to my family in China. Their love, understanding
and constant support have given me freedom and courage to pursue my PhD and get this
far.

iii

Table of Contents

Acknowledgements ii

List of Figures iv

List of Tables vi

Abstract vii

Chapter 1: Introduction 1

1.1 Enamel Formation 1

1.2 Ameloblastin 4

1.3 Structure and Properties of Ameloblastin 6

1.4 Function of Ameloblastin in Enamel Formation 7

Chapter 2: Protein Interaction between Ameloblastin and Proteasome
Subunit Alpha Type 3 can Facilitate Redistribution of
Ameloblastin Domains within Forming Enamel 10

2.1 Introduction 10

2.2 Material and Methods 15

2.3 Results 24

2.4 Discussion 46

Chapter 3: Altering the Ameloblastin-Psma3 Interaction by Knockdown
of Psma3 using Silencing RNAs 52

3.1 Introduction 52

3.2 Material and Methods 54

3.3 Results 61

3.4 Discussion 72

Chapter 4: Immunodetection of Psma3 in Exosomes Recovered from
Ameloblast-like LS8 Cell Culture Medium 75

4.1 Introduction 75

4.2 Material and Methods 77

4.3 Results 78

4.4 Discussion 80

Chapter 5: Summary 83

References 89

iv

List of Figures

Figure 1. Amino acid sequence of human ameloblastin and mouse ameloblastin
protein. 5
Figure 2. Idealized schematic of cellular fabrication showing the organization of
mouse enamel extracellular matrix with HAP crystallite bundles within
enamel rod-to-interrod microstructure. 9
Figure 3. Principle of yeast-two hybrid assay. 14
Figure 4. Histology of mouse incisors and immunolocalization of ameloblastin
in incisors. 30
Figure 5. Histology of mouse incisors and immunolocalization of proteasome
subunit alpha type 3 (Psma3) in incisors. 32
Figure 6. Confocal micrographs reveal co-localization of ameloblastin and
proteasome subunit alpha type 3 (Psma3) in Tomes' processes. 34
Figure 7. Higher magnification view of the co-localization of ameloblastin and
proteasome subunit alpha type 3 (Psma3) in Tomes' processes. 36
Figure 8. Co-immunoprecipitation analysis of ameloblastin and proteasome
subunit alpha type 3 (Psma3). 38
Figure 9. Expression of ameloblastin fusion proteins in yeast cells and
identifiction of the ameloblastin binding site for proteasome
subunit alpha type 3 (Psma3). 40
Figure 10. Digestion of ameloblastin with 20S proteasome in vitro. 42
Figure 11. Map of shRNA cloning vector pGFP-C-shLenti. 60
Figure 12. Efficient transduction of lentivirus in LS8 cells. 64
Figure 13. Lentiviral transduction does not lead to massive cell death. 66
Figure 14. Knockdown of proteasome subunit alpha type 3 (Psma3) in LS8 cells. 68

v

Figure 15. Organ culture of mouse incisors transduced with the control
lentivirus and most effective shRNA lentivirus (LVC). 70
Figure 16. Western blot analysis of Psma3 in exosomes from LS8 cells. 79
Figure 17. Schematic of the possible mechanism of Psma3 export from
ameloblastin into the enamel extracellular matrix. 82

vi

List of Tables

Table 1. Controls used in the yeast two-hybrid assay and phenotypes of
reporter genes for each control. 21
Table 2. Assays for testing protein interactions in the yeast two-hybrid system. 23
Table 3. Lentiviral transfer plasmids. 59

vii

Abstract

Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites
aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the
hardest tissue in the vertebrate body; however, it starts development as a self-organizing
assembly of matrix proteins that control crystallite habit. Ameloblastin, the second most
abundant enamel matrix protein, is initially distributed uniformly across the cell boundary
but redistributes to the lateral margins of the extracellular matrix following secretion,
thus producing cell-defined boundaries within the matrix and the mineral phase. In this
study, I investigate ameloblastin-protein interactions that are hypothesized to facilitate
redistribution of ameloblastin responsible for enamel matrix patterning.
In chapter 2, I present results of a yeast two-hybrid assay which identified that
proteasome subunit alpha type 3 (Psma3) interacts with ameloblastin. Confocal
microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast
secretory ends. Co-immunoprecipitation assays of mouse ameloblast cell lysates with
either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein
engineering demonstrated that only the ameloblastin carboxyl terminus interacts with
Psma3. Furthermore, I found that 20S proteasome digestion of ameloblastin in vitro
generates an N’-terminal cleavage fragment consistent with the in vivo pattern of
ameloblastin distribution. These findings suggest a novel pathway participating in control
of protein distribution within the extracellular space that serves to regulate the protein-
mineral interactions essential to biomineralization.

viii

Using an in vitro cell and organ culture system, I studied the knockdown of Psma3
mediated by shRNA interference with the long-term goal of exploring the effects of
ameloblastin-Psma3 interaction on the formation of patterned enamel structure. In
Chapter 3, I report on the use of lentiviral transduction to deliver shRNAs into target
cells. In the cell culture model, flow cytometric analysis showed that above 80%
lentiviral transduction efficiency was achieved in ameloblast-like LS8 cells. An apoptosis
assay did not detect significant cell death after lentiviral transduction. Immunodetection
of LS8 cell lysate demonstrated that the most effective shRNA molecule produced
approximately 50% knockdown of Psma3 proteins. In a lentiviral transduced mouse
incisor organ culture model, immunohistochemistry revealed that shRNA-mediated gene
silencing resulted in the mosaic elimination of Psma3 in incisor ameloblasts. The in vitro
models provide the foundation for future in vivo studies of the function of ameloblastin-
Psma3 interaction.
Additionally, I performed Western blot analysis to detect the presence of Psma3 in
exosomes recovered from LS8 cell culture medium. The existence of Psma3 in LS8 cell
exosomes suggests the possibility of Psma3 export by ameloblasts into extracellular
space. This finding may introduce a link between exosome biology and enamel
formation.
In summary, this study provides a molecular basis for interpreting the mechanism
by which ameloblastin redistribution participates in enamel microstructure formation.

1

Chapter 1
Introduction

Tooth enamel is the most highly mineralized tissue in the vertebrate body. It is
formed by biomineralization of a protein extracellular matrix precursor that is ultimately
replaced by mineral. Substituted hydroxyapatite (HAP) nanocrystallites with scarce
residual organic material between them form enamel. The crystallites are arranged along
their long axes and tightly packed together to form bundles. A bundle of crystallites is
formed by a single ameloblast cell and constitutes the basic enamel structural unit, called
a rod. The crystallites that surround the enamel rods, which are oriented normal to others,
are called interrod enamel. Enamel rods and interrods are woven together in a continuum
to form highly organized hierarchical structure that permits enamel to resist a lifetime of
wear and fracture during mastication (Baldassarri et al., 2008; Boyde, 1987, 1997; White
et al., 2001).

1.1 Enamel Formation
Enamel formation, or amelogenesis, is a complex biomineralization process that
occurs within an organic extracellular matrix (ECM) (Robinson et al., 1981). During
development, ameloblast cells, differentiated from inner enamel epithelium, synthesize
the matrix proteins and secrete them into the extracellular space through the Tomes’
process, the distal membrane projection of the ameloblast (Tomes, 1849). Once released

2

into the extracellular space, these matrix proteins undergo self-assembly to produce an
organized matrix that is competent to guide the initiation, growth and arrangement of
HAP crystallites in the highly patterned rod-interrod microstructure observed in mature
enamel (Bartlett et al., 2006; Chun et al., 2010a; Fincham et al., 1995; Gibson et al.,
2001; Paine and Snead, 1997). To date, two main groups of enamel matrix proteins have
been identified. These are the amelogenin proteins and the non-amelogenin proteins, the
latter including ameloblastin, enamelin, amelotin and perhaps other, yet-to-be-named
proteins (Eastoe, 1979; Fincham et al., 1999; Iwasaki et al., 2005; Moffatt et al., 2006;
Smith et al., 1995; Termine et al., 1979). The domain protein of enamel matrix is
amelogenin. Amelogenin undergoes self-assembly to form nanospheres, which further
assemble to higher order structure microribbons. The chain alignment of amelogenin
nanospheres functions as a scaffold for extracellular matrix organization, controlling the
growth and orientation of HAP crystallites (Du et al., 2005; Moradian-Oldak and
Goldberg, 2005).
In addition to protein self-assembly, ameloblast cell-to-matrix interactions also
influence the matrix since the Tomes’ process is always in contact with the forming
enamel matrix (Smith and Nanci, 1995). Integrins are the well-know receptors located on
the Tomes’ process that contribute to the cell-to-matrix interaction. In particular, the
lectin-like domain in amelogenin protein can recognize and bind to N-acetyl glucosamine
residues of the receptors located on the Tomes’ processes (Paine et al., 2002;
Ravindranath et al., 1999; Snead, 2015). Moreover, Ameloblastin has been reported to
server as a critical cell adhesion molecule for connecting the ameloblasts and the enamel
matrix (Beyeler et al., 2010; Fukumoto et al., 2004). One ameloblast cell defines one

3

enamel rod. The Tomes’ process of the ameloblast determines the boundaries of each
corresponding enamel rod, while the interrod matrix is deposited among adjacent
ameloblasts, thereby unifying the bioceramic tissue into a continuum which imparts
enamel’s unique material properties (Boyde, 1987; Fong et al., 2003; Paine et al., 2000;
Warshawsky and Smith, 1971; White et al., 2001; Zhu et al., 2006).
During enamel formation, the ameloblast circadian rhythm entrains a daily round of
protein synthesis followed by a similar period for mineral deposition occurring in the
anti-phase of the circadian cycle (Boyde, 1979; Lacruz et al., 2012), thereby allowing
incremental enamel matrix formation to be coupled to incremental HAP mineral
conversion. Proteolytic processing of the enamel matrix proteins starts upon secretion,
and removal of the protein matrix is essential to the expansion of the HAP mineral phase
(Bartlett et al., 2004; Lacruz et al., 2011; Lacruz et al., 2013; Paine et al., 2008; Simmer
and Fincham, 1995; Smith, 1998).

4

1.2 Ameloblastin
Ameloblastin (Snead, 1996), also known as amelin (Fong et al., 1996) or sheathlin
(Hu et al., 1997), is the most abundant of the non-amelogenin enamel matrix proteins.
Ameloblastin protein is highly expressed by the secretory-stage ameloblasts and
diminishes in abundance during the maturation stage (Fukumoto and Yamada, 2005;
Krebsbach et al., 1996; Uchida et al., 1991). The amino acid sequence for ameloblastin is
well conserved among many species (Cerny et al., 1996; Hu et al., 1997; Krebsbach et
al., 1996). So far, no other proteins with similar sequence to ameloblastin have been
characterized.
The human ameloblastin gene is located in the calcium-binding phosphoprotein
gene cluster on chromosome 4q21 (NCBI). Two isoforms are expressed due to alternative
splicing. The major isoform has 447 amino acids (Fig. 1a). In mouse, the ameloblastin
gene locus maps to chromosome 5 near other genes associated with mineralization
(Krebsbach et al., 1996). Alternative splicing of the gene creates two transcripts variants
that encode two isoforms. The longer mouse ameloblastin isoform has 422 amino acids
(Fig. 1b). Human and mouse ameloblastin share high amino acid sequence similarities.
Several post-translational modification have been identified, including phosphorylation
(Krebsbach et al., 1996), hydroxylated prolines, and O-linked glycosylation (Kobayashi
et al., 2007). A signal peptide is present at the N-terminus of ameloblastin, which is
cleaved and removed as the protein is secreted into the extracellular space.

5

Figure 1

Figure 1. Amino acid sequences of human ameloblastin (a) and mouse ameloblastin
protein (b). The signal peptide sequence for each protein is underlined. The arrowhead
between arginine 222 (R222) and leucine 223 (L223) in the human ameloblastin
sequence represents the presumptive cleavage site for generating the human ameloblastin
N-terminal and the C-terminal domain.

6

1.3 Structure and Properties of Ameloblastin
Ameloblastin belongs to the intrinsically disordered protein family. The primary
structure of ameloblastin consists of two distinct domains: a basic N-terminal domain
and an acidic C-terminal domain. The N-terminal domain is relatively stable and has
tendency to adopt a helical conformation. Most proline-rich motifs which are important
for signaling activity are located at the N-terminal region (Wald et al., 2011). An N-
terminal segment encompassing residues 36-72 plays a key role in ameloblastin self-
assembly (Wald et al., 2013). While the short-lived C-terminal domain is primarily
responsible for the structural disorder of the entire ameloblastin molecule. The C-terminal
part contains abundant sites for calcium binding (Vymetal et al., 2008; Yamakoshi et al.,
2001), and also serves as a local phosphate source for HAP crystallites growth (Lee et al.,
2003). These two parts can be liberated from the entire ameloblastin molecule by
proteolysis and server different functions in different subcompartments in forming
enamel matrix. Although the structure of ameloblastin is not fully understood,
bioimformatic studies revealed that a large part of ameloblastin is unstructured. This
structural disorder plays an important role in ameloblastin-protein interactions, which
achieved through protein self-assembly and protein-protein interactions.
To date, intact ameloblastin has not been isolated in vivo, a fact believed to be due
to the immediate protease processing by matrix metalloproteinase 20 (a.k.a. enamelysin
or MMP20) in the extracellular space (Chun et al., 2010b; Iwata et al., 2007). In
developing porcine tooth, ameloblastin is cleaved by MMP-20 initially at one of three
amino acid residues: after glutamine
130
or after arginine
170
or after alanine
222
, to form the

7

N-terminal domain and the C-terminal domain. These initial cleavage products are then
cleaved for a second or a third time forming still smaller fragments.

1.4 Function of Ameloblastin in Enamel Formation
Ameloblastin has been shown to play a critical role in normal enamel formation. In
mouse models that expressed truncated ameloblastin, the formed enamel is significantly
thinned compared to wild-type enamel. Furthermore, the ameloblasts were found to
detach from the forming enamel matrix and form epithelium tumors (Fukumoto et al.,
2004; Smith et al., 2009; Wazen et al., 2009). The enamel hypoplasia and the neoplastic
potential of the ameloblasts could be rescued by transgenic expression of normal full-
length ameloblastin (Chun et al., 2010a). In addition, investigators have reported that
overexpressing ameloblastin resulted in enamel structural imperfections, with disturbance
of the rod-interrod organization (Paine et al., 2003). These findings indicate that
ameloblastin is essential for rod morphology and correct enamel microstructure
organization.
Although it is clear that ameloblastin is required for normal enamel formation, the
underlying molecular mechanism remains unknown. It has been documented that the
ameloblastin cleavage products redistribute into different areas within the enamel rod,
producing a pattern. Using immunohistochemistry, previous studies have found that full-
length ameloblastin and its C’-terminal cleavage products first accumulate within the
newly formed rods, producing a “reverse honeycomb” pattern (Fig. 2a) (Hu and
Yamakoshi, 2003; Murakami et al., 1997; Nanci et al., 1998). Toward the deeper layer of

8

the immature enamel matrix, the N’-terminal cleavage products localize around the
peripheral boundaries of the ameloblasts to form a “honeycomb” pattern (Fig.2b) (Hu and
Yamakoshi, 2003; Nanci et al., 1998; Uchida et al., 1991). These observations suggest
that the redistribution of ameloblastin domains is responsible for establishing the enamel
rod-interrod boundaries.

9

Figure 2

Figure 2. Idealized cartoon of cellular fabrication showing the organization of
mouse enamel extracellular matrix with HAP crystallite bundles within enamel rod-
to-interrod microstructure. Panel (a) shows developing porcine enamel with newly
secreted ameloblastin localized to all of the area of the Tomes’ processes (Murakami et
al., 1997), but soon becomes localized exclusively to the perimeter of each enamel rod as
a sheath showed in panel (b) (Hu and Yamakoshi, 2003; Uchida et al., 1991). Panel (c)
is a tangential (en face) and sagittal section cartoon view of forming mouse enamel. The
boundary (sheath) of each enamel rod corresponds to the lateral limit of each ameloblast
cell’s Tomes’ process. The enamel rod is the basic unit of enamel and is created by a
single ameloblast cell. Within a rod, the crystallites are packed parallel to one another
along their long axis. The interrod crystallites form within the matrix deposited among
adjacent ameloblasts, thereby giving rise to the continuity of the highly patterned rod-to-
interrod enamel microstructure (Boyde, 1979).

10

Chapter 2
Protein Interaction between Ameloblastin and Proteasome Subunit
Alpha Type 3 Can Facilitate Redistribution of Ameloblastin Domains
within Forming Enamel

2.1 Introduction
Enamel is the hardest tissue in the vertebrate body composed of thousands of
hydroxyapatite (HAP) nanocrystallites with scarce residual organic material between
them. The crystallites are aligned parallel along their long axis and tightly packed
together, with each bundle formed under the control of a single ameloblast cell. As a
consequence of cellular fabrication, the boundaries between cells give rise to a
hierarchically integrated microstructure of crystallite bundles, which, in rodents, forms a
decussating pattern of woven bioceramic structure (Boyde, 1987; Warshawsky and
Smith, 1971). This highly organized hierarchical structure provides enamel with its
unique material properties of wear resistance, fracture toughness and in rodents, self-
sharpening edges (Baldassarri et al., 2008; Boyde, 1997; Chun et al., 2010a; Fong et al.,
2003; Imbeni et al., 2005; Simmer et al., 2010; Smith et al., 2005; White et al., 2001;
White et al., 2005).
Enamel starts development as a self-organizing assembly of matrix proteins that
control crystallite habit. Ameloblastin (Snead, 1996), also known as amelin (Fong et al.,
1996) or sheathlin (Hu et al., 1997), is the most abundant of the non-amelogenin enamel
matrix proteins. During enamel formation, ameloblastin protein is highly expressed by

11

the secretory-stage ameloblasts and secreted into the extracellular space, where
ameloblastin is processed by matrix metalloproteinase 20 (a.k.a. enamelysin or MMP20)
(Chun et al., 2010b; Iwata et al., 2007) to form cleavage fragments. Remarkably, the
ameloblastin cleavage products redistribute into different areas within the enamel rod,
producing a pattern. Full-length ameloblastin and its C’-terminal cleavage products first
accumulate within the newly formed rods, producing a “reverse honeycomb” pattern
(Fig. 2) (Hu and Yamakoshi, 2003; Murakami et al., 1997; Nanci et al., 1998). In
contrast, the N’-terminal cleavage products localize around the peripheral boundaries of
the ameloblasts to form a “honeycomb” pattern (Fig.2) (Hu and Yamakoshi, 2003; Nanci
et al., 1998; Uchida et al., 1991). Moreover, in mouse models that express a truncated
ameloblastin (Fukumoto et al., 2004; Smith et al., 2009; Wazen et al., 2009) or
overexpress ameloblastin (Paine et al., 2003), the resulting enamel shows structural
imperfections, with disturbances to the canonical pattern of rod-interrod boundaries. In
the truncated ameloblastin animal, rescue of the enamel rod microstructure abnormalities
has been achieved with expression of a full-length ameloblastin transgene (Chun et al.,
2010a). These observations suggest that the distribution of ameloblastin domains within
the forming enamel matrix play important roles in establishing the enamel microstructure
comprising the rod-interrod pattern of organization and hence in producing the favorable
material properties found in mature enamel.
Based on these observations, we hypothesized that the N’-terminal ameloblastin
domain undergoes redistribution to the ameloblast cell periphery, thus serving to
segregate the forming enamel matrix into individual units (rods) of enamel
microstructure. Enamel does not remodel; therefore, correctly forming the matrix through

12

protein self-assembly in the extracellular space is essential to properly forming the
mineral phase, which must function for the life of the animal. I hypothesize that
ameloblastin redistribution is controlled either by interactions with heretofore-unknown
proteins within the matrix or with proteins localized to Tomes’ processes, the secretory
ends of the ameloblast cells that contact the matrix. The purpose of this investigation was
to identify these previously unknown enamel matrix proteins that interact with
ameloblastin during amelogenesis and to elucidate their expression and localization in
developing mouse enamel.
In order to identify ameloblastin-interacting proteins, I performed a yeast two-
hybrid assay to screen an ameloblast cDNA library using human ameloblastin as the bait.
The yeast two-hybrid assay was developed by Fields and Song in 1989 (Fields and Song,
1989), and is based on the fact that the GAL4 transcription factor can be split into two
separable domains: a DNA binding domain (BD) and a DNA activating domain (AD)
allowing each GAL4 domain to fuse with a query protein to ascertain its interaction(s)
with others. Should the two query proteins interact with one another, the two separated
transcription factor fragments are brought back into proximity to one another and the
GAL4 factor activates transcription, providing a marker and selection strategy to identify
the yeast colony harboring the putative interacting protein partner(s) (Fig. 3). In my
assay, the GAL4 BD is fused to human ameloblastin, and the GAL4 AD is fused to an
unknown protein encoded by a cDNA from an ameloblast cDNA library. If the unknown
protein interacts with ameloblastin, the two separated transcription factor domains
reconstitute the GAL4 factor to activate the reporter/selection genes. The yeast two-
hybrid assay has previously been successfully deployed to identify the interacting protein

13

partners for amelogenin and enamelin
(Paine et al., 1998; Paine and Snead, 1997; Wang
et al., 2005).
I report here that the proteasome subunit alpha type 3 (Psma3) interacts with
ameloblastin in the yeast two-hybrid assay. Using confocal microscopy, I confirmed the
localization of Psma3 to the ameloblast secretory end piece known as Tomes’ processes,
a physical site where ameloblastin is also present. The interaction of ameloblastin with
Psma3 was corroborated by co-immunoprecipitation assay of total mouse ameloblast
lysates using either an ameloblastin- or Psma3-specific antibody to identify the reciprocal
partner protein. Protein engineering was used to define the carboxyl terminus of
ameloblastin as the region interacting with Psma3. Finally, I performed in vitro
proteasome digestion assays to investigate the potential functional significance of the
ameloblastin-Psma3 interaction.

14

Figure 3

Figure 3. Principle of yeast two-hybrid assay (Invitrogen)

15

2.2 Material and Methods
Construction of plasmids
To construct “bait plasmids” for the expression of human ameloblastin full-length
protein (FL, amino-acid residues 27-447, GenBank #AAF73048.1), N’-terminal protein
domain (Np, amino-acid residues 27-222), and C’-terminal protein domain (Cp, amino-
acid residues 223-447) (Chun et al., 2010b; Iwata et al., 2007; Vymetal et al., 2008; Wald
et al., 2011), the corresponding cDNAs were prepared without the ameloblastin signal
peptide which can contribute to false positive interactions. The DNA sequence for each
of the three proteins was amplified from the human cDNA template (clone sc304427,
OriGene Technologies Inc., Rockville, MD), and the PCR products cloned into the
pDEST32 vector (ProQuest Two-Hybrid System, Invitrogen, Grand Island, NY)
containing the GAL4 BD gene sequence using Gateway subcloning technology, as
previously described (Landy, 1989). The inserted cDNA fragments were preserved in
their correct reading frame with the GAL4 BD nucleotide sequence. The constructed
plasmids were transferred into yeast cells where they expressed the following individual
fusion proteins: GAL4 BD-FL; GAL4 BD-Np; and GAL4 BD-Cp.
In this study, the term “prey plasmid” refers to a collection of plasmids containing
cDNAs expressed by the mouse enamel organ epithelial cells. To prepare an enamel
organ epithelial cDNA library, mandibular first molars were dissected from mouse pups
(Swiss Webster) at post-natal day 3 (PN3). The odontogenic epithelia were micro-
dissected from the underlying mesenchyme and used to isolate total RNA using an
RNeasy Plus Mini Kit (QIAGEN). The RNA sample was used to construct a normalized
cDNA library using SMART synthesis techniques that favor full-length cDNAs. The

16

dscDNAs were ligated in the pDEST22 vectors (ProQuest Two-Hybrid system,
Invitrogen) containing the GAL4 AD gene sequence. The sequence representation of this
cDNA library is about 1.75×10
6
and the average insert size is around 2.3 kilobases.

Yeast two-hybrid assay
A ProQuest Two-Hybrid system (Invitrogen, Grand Island, NY) was used to
perform the yeast two-hybrid assay, using the MaV203 yeast strain as the host. The
MaV203 strain contains three reporter genes (lacZ, HIS3, and URA3), which provide four
phenotypes by which to discover and selectively recover the cDNA encoding an
unknown interacting protein. Four control plasmids were used to flank positive and
negative control protein-to-protein interactions and establish their affinity:
pEXP32/Krev1, pEXP22/RalGDS-wt, pEXP22/RalGDS-m1, and pEXp22/RalGDS-m2
(Table 1).
I performed the yeast two-hybrid assay by screening the ameloblast cDNA library
using the full-length human ameloblastin as the bait, according to the manufacturer’s
recommended protocol (Invitrogen). Briefly, the bait plasmid pDEST32-FL and library
plasmids were co-transferred into yeast strain MaV203 to provide double-transformed
yeast colonies. The double-transformed yeast colonies that grew on auxotrophic plates
lacking leucine, tryptophan and histidine and supplemented with 80 mM 3-Amino-1,2,4-
Triazole (3AT, Sigma-Aldrich, St. Louis, MO) were selected and re-plated on their
respective selective plates to confirm the expression of the reporter/selection genes
(Table 2). Positive yeast colonies containing ameloblastin-interacting protein candidates
were identified according to the phenotypes indicating the activation of the three reporter

17

genes. In addition, auxotrophic plates containing 0.2 wt % 5-fluoroorotic acid (5-FOA,
Sigma-Aldrich, St. Louis, MO) were used to eliminate false positives. From these
candidates, the corresponding prey plasmids were isolated and used to transform fresh
MaV203 yeast cells in order to confirm the protein interactions in a second round of
screening.
For each prey plasmid, the nucleotide sequence was obtained for the cDNA
encoding an ameloblastin-interacting protein identified in the yeast two-hybrid assay. The
nucleotide sequence for each putative cDNA was analyzed for similarity to previously
identified genes using the BLAST algorithm searched against the mouse genome plus
transcript database as the comparison reference.

Immunolocalization of proteins
Hemi-mandibles dissected from post-natal day 3 (PN3) mouse pups (Swiss
Webster) were fixed with 4% paraformaldehyde in PBS overnight at 4°C. Tissues were
decalcified with osmotically balanced 10% EDTA (pH 7.4) for 3 days and embedded in
paraffin for sectioning. Sagittal and tangential sections of 5 µm in thickness were
prepared for immunofluorescence. Immunostaining procedures followed a published
protocol (Ausubel, 2005). In brief, sections were blocked with 5% BSA/PBS containing
0.1% Tween 20 for 1 hour before incubation with primary antibody. Polyclonal primary
antibodies were used to detect ameloblastin (M-300, sc-50534, Santa Cruz
Biotechnology, Dallas, TX) and Psma3 (A-17, sc-54707, Santa Cruz Biotechnology,
Dallas, TX). The locations of the primary antibody immunoglobulins were detected by
Alexa Fluor 594 (Invitrogen) or FITC-conjugated (Santa Cruz Biotechnology) secondary

18

antibodies. Nuclei were stained with DAPI in mounting medium (Vector Laboratories,
Burlingame, CA). Tissue sections incubated with secondary antibody alone were used as
negative controls. Fluorescent microscopy (Leica DMI3000 B, Leica Microsystems,
Buffalo Grove, IL) and confocal microscopy (Leica TCS SP5 II, Leica Microsystems)
were used to record immunofluorescent signal. To visualize the Tomes’ processes more
readily, sections tangential to the ameloblast long axis from PN3 mouse mandibular
incisors were used to assess co-localization of ameloblastin and putative ameloblastin-
interacting proteins. 3-D reconstructions of a Z-stack of adjacent images were created
using Leica software (version 2.7.3.9723, Leica Microsystems).

Co-immunoprecipitation and Western blot analysis
Enamel organ epithelia were dissected from PN3 mouse mandibular incisors, then
solubilized into lysis buffer containing 20 mM Tris (PH 7.4), 150 mM NaCl, and 1%
Triton X-100. Protein concentration was determined using a Pierce BCA Protein Assay
Kit (Thermo Scientific, Rockford, IL). The procedure for co-immunoprecipitation (Zhou
et al., 2000) and analysis by Western blot (Zhou and Snead, 2000) has been described and
was used without modification. Cell lysates were subject to immunoprecipitation with an
anti-ameloblastin antibody (N-18, sc-33100, Santa Cruz Biotechnology, Dallas, TX) or
an anti-Psma3 antibody (A-17); the immunoprecipitant was resolved to size by PAGE
and analyzed by Western blotting with anti-Psma3 antibody (EPR5455, ab109532,
Abcam, Cambridge, MA) or anti-ameloblastin antibody (M-300) accordingly. The
negative control consisted of identical lysate incubated with precipitation beads (Santa
Cruz Biotechnology, Dallas, TX) without the primary antibody.

19

Interactions with yeast-synthesized fusion proteins
The three human ameloblastin bait plasmids (pDEST32-FL, pDEST32-Np,
pDEST32-Cp) were separately transformed into yeast MaV203 cells and cultured on
selection media (Leu- plate). Western blot analysis was used to confirm the expression of
ameloblastin fusion proteins in yeast using a GAL4 BD-specific antibody (15-6E10A7,
ab135397, Abcam, Cambridge, MA). The prey plasmid pDEST22-Psma3 was co-
transformed into MaV203 cells and cultured on selection medium (Trp- plate). Total
yeast proteins were recovered as a lysate as previously described (Li and Fields, 1993)
and subjected to co-immunoprecipitation. Anti-ameloblastin primary antibody (M-300)
was used to recover the interacting proteins onto precipitation beads (Santa Cruz
Biotechnology) and Western blot analysis was used to determine the binding domain of
the Psma3 with ameloblastin by anti-Psma3 antibody (A-17). Lysate samples incubated
with precipitation beads (Santa Cruz Biotechnology) but without primary antibody were
used as the negative controls.

In vitro proteolysis
Recombinant human ameloblastin (rhAMBN) (Abnova, Taiwan) was incubated
with purified 20S proteasome (Enzo Life Sciences, Farmingdale, NY) at a proteasome-to-
substrate molar ratio of 1:1 in 50 mM Tri-HCl buffer (pH 7.0) at 37°C. After 1 or 3h, the
reactions were stopped by boiling in SDS loading buffer, and analyzed by Western
blotting with an ameloblastin N’-terminal antibody (N-18, Santa Cruz Biotechnology)
and a C’-terminal antibody (ab116347, Abcam). Proteasome digestion of proline-rich

20

homeodomain protein (PRH) was performed using the same procedure to confirm the
enzymatic activity of the purified 20S proteasome (Bess et al., 2003). The PRH proteins
and anti-PRH mouse polyclonal antibody were kind gifts provided by Dr. P-S Jayaraman
(University of Bristol, Bristol, U.K.). Densitometry of PAGE-resolved protein bands was
obtained by optical scanning and the relative intensity of the bands was quantified using
ImageJ densitometry (http://rsb.info.nih.gov/ij/index.html).

To test the inhibition of proteolytic activity, 50 µM epoxomicin (Enzo Life
Sciences), a proteasome specific inhibitor, was added to the mixture of rhAMBN and 20S
proteasome. The reactions were carried out at 37°C and terminated after 3h. Proteolysis
was analyzed by Western blotting with the two ameloblastin-specific antibodies
described above. Incubation of rhAMBN with 20S proteasome in the absence of
epoxomicin at 37°C for 3h was used as the control.

21

Table 1
Controls Purpose Phenotypes
GAL4 BD
plasmid
GAL4 AD plasmid β-Gal His- Ura- 5-FOA
pEXP32/Krev1 pEXP22/RalGDS-wt Strong
positive

Blue + + −
pEXP32/Krev1 pEXP22/RalGDS-m1 Weak positive Faint blue

+ + −
pEXP32/Krev1 pEXP22/RalGDS-m2

Negative White − − +
pDEST32 pDEST22 Negative self-
activation

White − − +
pDEST32-FL pDEST22 Test of self-
activation
White − − +

Table 1: Controls used in the yeast two-hybrid assay and phenotypes of reporter
genes for each control. Four control plasmids, pEXP32/Krev1, pEXP22/RalGDS-wt,
pEXP22/RalGDS-m1, and pEXP22/RalGDS-m2, were used to bracket the affinity of an
unknown interacting protein. The positive control is based on the interaction of Krev1
and RalGDS protein. Mutation of the RalGDS protein diminishes (RalGDS-m1) or
breaks (RalGDS-m2) the interaction with the Krev1 protein. The backbone of
pEXP32/Krev1 is a pDEST32 vector containing the GAL4 Binding Domain coding gene,
while the backbone of pEXP22/RalGDS-wt, pEXP22/RalGDS-m1, and
pEXP22/RalGDS-m2 is a pDEST22 vector containing the GAL4 Activation Domain
coding gene. The empty vectors pDEST32 and pDEST22 were used as the negative
controls for vector self-activation. The plasmid pair of pDEST32-FL and pDEST22 was
used to test self-activation of the bait pDEST32-FL. Positive interaction in yeast MaV203
cells activates the transcription of three gene reporters (lacZ, HIS3, and URA3). The lacZ
gene expresses β-galactosidase, which generates blue coloration in the X-gal assay.

22

Induction of the HIS3 gene allows the yeast transformants to grow on histidine dropout
(null) plates. Induction of the URA3 gene endows the yeast transformants the ability to
grow on uracil dropout (null) plates, with growth blocked on 5-FOA-containing plates.
Abbreviations: β-Gal, β-galactosidase; His-, histidine auxotrophy; Ura-, uracil
auxotrophy; 5-FOA, 5-fluoroorotic acid; “+”, growth; “-”, no growth.

23

Table 2
Test Assay Plates Phenotypes
Interaction No interaction
lacZ induction
(β-Gal activity)

X-gal assay YPAD containing filter
paper
Blue White
HIS3 induction Histidine
auxotrophy
Leu-/Trp-/His- containing
80mM 3AT

Formed
colony
No colony
URA3 induction

Uracil
auxotrophy
Leu-/Trp-/Ura- Formed
colony
No colony
URA3 induction 5-FOA
sensitivity
Leu-/Trp- containing
0.2 wt% 5-FOA
No colony Formed colony

Table 2: Assays for testing protein interactions in the yeast two-hybrid system. Four
specific assays with the corresponding metabolic selection plates were used to test the
induction of three reporter genes in yeast cells. Reporter gene activation provided four
unique phenotypes with which to assess the putative protein interactions: (i) blue colony
coloration for the X-gal assay; (ii) growth on the Leu- /Trp- /His- with 80 mM 3AT
plates; (iii) growth on the Leu- /Trp- /Ura- plates; and (iv) failure to grow on Leu- /Trp-
with 0.2 wt% 5-FOA plates.
Abbreviations: β-Gal, β-galactosidase; Leu-/Trp-/His-, auxotrophic plate lacking leucine,
tryptophan, and histidine; Leu-/Trp-/Ura-, auxotrophic plate lacking leucine, tryptophan,
and uracil; Leu-/Trp-, auxotrophic plate lacking leucine and trypthophan; 3AT, 3-Amino-
1,2,4-Triazole; 5-FOA, 5-fluoroorotic acid.

24

2.3 Results
Proteasome subunit alpha type 3 (Psma3) interacts with ameloblastin
Using a yeast two-hybrid assay, I screened 6.4×10
6
colonies from a mouse
ameloblast cDNA library using full-length human ameloblastin protein as bait, which
yielded 43 colonies that grew on auxotrophic plates, indicating a protein-to-protein
interaction. These 43 candidates were recovered and inoculated onto selective
auxotrophic plates to corroborate the interaction and to eliminate false positive reactions.
Secondary screening eliminated 36 candidates as false positives but identified seven
colonies that met all of the criteria for protein-to-protein interaction between the
ameloblastin and its interacting partner protein: (i) blue colony coloration for the X-gal
assay; (ii) growth on the Leu- /Trp- /Ura- plates; (iii) growth on the Leu- /Trp- /His- with
80 mM 3AT plates; and (iv) failure to grow on Leu- /Trp- with 0.2 wt% 5-FOA plates.
Nucleotide sequencing revealed that each of the seven candidates were preserved in the
same reading frame as the GAL4 AD, supporting the notion that the encoded protein
could interact with ameloblastin. The BLAST algorithm used for comparison of the
cDNA nucleotide sequences of the seven candidates revealed that all of the candidates
encoded the proteasome subunit alpha type 3 (Psma3): four clones encoding full length
Psma3 (255 amino acids) and the remaining three encoding various lengths of the amino
terminal segment of Psma3, with the most truncated protein being 216 amino acids.

Distribution of ameloblastin in the developing tooth
I used a rabbit polyclonal antibody (M-300, Santa Cruz Biotechnology) to examine
the distribution of ameloblastin in the developing PN3 mouse mandibular incisors by

25

immunofluorescence. Sagittal sections (Fig. 4a-f) revealed ameloblastin expression to be
limited to the ameloblast cell layer (Fig. 4d). The intensity of immunoreactivity increased
in the area corresponding to the secretory-stage ameloblast cells (Fig. 4e) relative to the
pre-secretory stage (Fig. 4f). Intense immunostaining was found in the distal membrane
projections of secretory ameloblast cells, an area of membrane specialization in
ameloblasts known as Tomes’ processes (Fig. 4e). Moreover, on tangential sections (Fig.
4g-k) that favor imaging en face visualization of the Tomes’ processes (Fig. 2),
immunostaining revealed ameloblastin distribution in a decussating pattern,
corresponding to the positional pattern of Tomes’ processes and most easily visualized in
3D reconstruction of a confocal Z-stack of images (Fig. 4j, k). This decussating staining
pattern corresponds precisely to the arrangement of enamel rods observed in mature
mouse enamel, suggesting the ameloblastin was localized to the secretory surface of a rod
with little ameloblastin appearing in the interrod matrix shared among adjacent
ameloblasts. Negative controls showed an absence of fluorochrome indicating no specific
immunolocalization of the target antigen.

Expression of Psma3 protein in the developing tooth
To analyze the expression and localization of Psma3 in the developing tooth, I
again performed imaging experiments on sagittal (Fig. 5a-e) and tangential sections (Fig.
5f-j) of mouse mandibular incisors at PN3. Using a goat polyclonal antibody (A-17,
Santa Cruz Biotechnology), Psma3 protein was detected in the cytoplasm of ameloblasts
and in Tomes’ processes (Fig. 5d). Tomes’ processes showed robust immunostaining
(Fig. 5e). To better observe the localization and distribution of Psma3, I used confocal

26

microscopy to perform 3D reconstruction of a Z-stack of tissue sections cut tangential to
the ameloblast long axis (en face) (Fig. 5i, j). The results showed a distribution pattern for
Psma3 localized to the Tomes’ processes, which was identical to the location observed
for ameloblastin distribution in the same region of forming enamel. The immunostaining
for Psma3 revealed a decussating pattern localized to the matrix that corresponds to
Tomes’ processes, a pattern consistent with the position of enamel rods in mature mouse
enamel. The interrod matrix, representing the shared secretory products of adjacent
ameloblasts, revealed little Psma3 staining. Negative controls showed no specific
immunolocalization of the target antigen.

Co-localization of ameloblastin and Psma3 in the developing tooth
Immunofluorescent staining was performed to examine the co-localization of
ameloblastin and Psma3 using polyclonal antibodies M-300 and A-17, respectively, on
tangential sections (for orientation see Fig. 4g-i and Fig. 5f-h) of mouse mandibular
incisors at PN3. At the resolution of the confocal microscope, immunostaining results
indicated that ameloblastin and Psma3 co-localized in the cytoplasm of secretory
ameloblasts and the Tomes’ processes, with intense immunostaining observed in Tomes’
processes (Fig. 6). The co-localized immunostaining pattern identified for Tomes’
processes revealed a decussating organization of alternating rows of ameloblasts, a
pattern that corresponds to the rod arrangement in mature mouse enamel, where one
ameloblast is responsible for creation of one enamel rod. A higher magnification view of
the co-localization of ameloblastin and Psma3 in the Tomes’ processes is shown in figure
7. Negative controls showed no specific immunolocalization of the target antigen.

27

Interaction between ameloblastin and Psma3 in vitro
To independently corroborate the interaction between ameloblastin and Psma3, a
co-immunoprecipitation assay was performed using a lysate prepared from PN3 mouse
incisor enamel organ epithelium. In this assay, neither Psma3 nor ameloblastin protein
was over-expressed; rather, native physiologic expression levels were assayed. Psma3
protein was co-immunoprecipitated with ameloblastin by a polyclonal anti-ameloblastin
antibody (N-18) and detected in Western blot analysis using a monoclonal anti-Psma3
antibody (EPR5455) (Fig. 8a). The same experimental procedure, reciprocally performed,
detected the ameloblastin protein in an immunocomplex containing Psma3. In the
reciprocal experiment, ameloblastin was co-immunoprecipitated with Psma3 by a Psma3-
specific antibody (A-17) and detected by an anti-ameloblastin antibody (M-300) in
Western blot analysis (Fig. 8b). Taken together, these data confirm Psma3’s interaction
with ameloblastin at the protein level in ameloblast cell lysate expressing physiologically
relevant levels of Psma3 and ameloblastin.

The binding site of Psma3 to ameloblastin maps to the carboxyl-terminal domain
MaV203 yeast cells were separately transformed with the expression plasmids
pDEST32, pDEST32-FL, pDEST32-Np, and pDEST32-Cp. Total yeast proteins were
recovered and subjected to Western blot for detection of ameloblastin GAL4-BD fusion
proteins (Fig. 9a). The GAL4 BD protein alone was detected as an 18kDa protein (Fig.
9a-lane 2), the GAL4 BD-FL fusion protein (561AA) was recognized as a band of ~65
kDa (Fig. 9a-lane 3), the GAL4 BD-Np fusion protein (351AA) as a band of ~22 kDa

28

(Fig. 9a-lane 4), and the GAL4 BD-Cp fusion protein (372AA) as a band of near 24 kDa
as a monomer and as a ~53 kDa as a dimer (Fig. 9a-lane 5). These data confirmed that the
yeast transformants expressed the ameloblastin FL, Np, and Cp fusion proteins.
To investigate the interaction site of Psma3 and ameloblastin, lysate prepared from
yeast cells expressing Psma3 fusion protein was separately mixed with lysate prepared
from yeast cells expressing ameloblastin-FL, -Np, or -Cp fusion protein (Fig. 9a). The
resulting protein mixtures were used for co-immunoprecipitation analysis (Fig. 9b). The
results showed that Psma3 was co-immunoprecipitated with both ameloblastin-FL and -
Cp using an anti-ameloblastin antibody (M-300). The GAL4 AD-Psma3 fusion protein
was detected as a protein band of ~50 kDa in the immunoprecipitated complex by an
anti-Psma3 antibody (A-17) in Western blot analysis. These data indicated that the
binding site of Psma3 was restricted to the carboxyl-terminal domain of the ameloblastin
protein.

Proteolysis of ameloblastin by 20S proteasome in vitro
To investigate the biological consequence of the ameloblastin-Psma3 interaction, I
examined whether ameloblastin is a substrate for proteasome activity. An in vitro
digestion assay was set up between rhAMBN and purified 20S proteasome. In the
presence of the 20S proteasome, the densitometrically measured intensity of full-length
ameloblastin protein detected by both anti-N’-terminal and anti-C’ terminal ameloblastin
antibody decreased after 1h incubation, and still further after 3h incubation time (Fig.
10a). Moreover, digestion with the 20S proteasome resulted in generating a smaller
molecular weight ameloblastin fragment that was detected exclusively by the N’-terminal

29

specific antibody (Fig. 6a-lane 8). I verified activity of the 20S proteasome using PRH
proteins, a substrate known to be degraded by 20S proteasome (Fig 10b). The ability of
the 20S proteasome to digest ameloblastin was blocked by the proteasome specific
inhibitor epoxomicin (Fig. 10c). In the presence of epoxomicin, the mass of rhAMBN
protein was not significantly changed after incubation with 20S proteasome at 37°C for
up to 3h.

30

Figure 4

31

Figure 4. Histology of mouse incisors and immunolocalization of ameloblastin in
incisors. Sagittal (a-f) and tangential (g-k) sections were prepared from post-natal day 3
(PN3) mouse mandibles. Panel (a) is a schematic diagram of a tissue section of the jaw
cut on the sagittal plane. Panels (b) and (c) are hematoxylin-eosin-stained sagittal
sections showing a mandibular incisor with panel (c) being a higher magnification of the
boxed area in panel (b), and arrows identifying Tomes’ processes. Panels (d-f) show
representative immunofluorescent staining of ameloblastin (red fluorochrome) in sagittal
sections with DAPI staining used for nuclear localization. Immunoreactivity showed
increasing intensity in the area corresponding to the secretory-stage ameloblast cells (e)
relative to the pre-secretory-stage cells (f). Panels (e) and (f) are higher-magnification
views of the corresponding boxed areas in (d). Arrows in panel (e) indicate intense
immunostaining in the Tomes’ processes.
Panel (g) is a schematic diagram of a tissue section of the jaw cut on the tangential
section. Panels (h) and (i) are hematoxylin-eosin-stained tangential sections showing a
mandibular incisor with panel (i) being a higher-magnification view of the boxed area in
panel (h). Panels (j) and (k) are 3D reconstructions of confocal Z-stack images from
tangential sections, in which the immunodetection signal reveals ameloblastin antigen
distributed across each Tomes’ process (arrows), forming a decussating pattern of
staining, which corresponds to the rod arrangement in mature mouse enamel. Panel (k) is
a higher-magnification view of the area boxed in (i).
Abbreviations: Am, ameloblast; EM, enamel matrix; D, dentine; pD, pre-dentine; P, pulp;
Od, odontoblast; TP, Tomes’ process.

32

Figure 5

33

Figure 5. Histology of mouse incisors and immunolocalization of proteasome
subunit alpha type 3 (Psma3) in incisors. Sagittal (a-e) and tangential (f-j) sections
were prepared from PN3 mouse mandibles. Panel (a) is a schematic diagram of a sagittal
section through the jaw. Panels (b) and (c) are hematoxylin-eosin-stained sagittal
sections showing a mandibular incisor with panel (c) being a higher-magnification view
of the approximate area in panel (b) shown by the black arrow. Arrows in panel (c)
identify the Tomes’ processes. Panels (d, e) show representative immunofluorescent
staining for Psma3 (green fluorochrome) in a sagittal section with DAPI staining used for
nuclear localization. Psma3 was detected in the cytoplasm of ameloblasts and Tomes’
processes. The higher-magnification image panel (e) shows that intense
immunoreactivity was observed in the distal membrane projections of the secretory
ameloblasts, i.e. Tomes’ processes (arrows).
Panel (f) is a schematic diagram of the plane from a tangential section of the jaw. Panels
(g) and (h) are a hematoxylin-eosin-stained tangential section showing a mandibular
incisor with panel (h) being a higher-magnification view of the boxed area in panel (g).
Panels (i) and (j) are 3D reconstructions of confocal Z-stack images of immunostaining
of Psma3 from tangential section. Panel (j) is a higher-magnification view of the
approximate area boxed in (i). In these reconstruction images, Psma3 was detected and
localized to each of the Tomes’ processes (arrows), producing a decussating pattern
consistent with the arrangement of rods in mature mouse enamel.
Abbreviations: Am, ameloblast; EM, enamel matrix; D, dentine; pD, pre-dentine; Od,
odontoblast; TP, Tomes’ process.

34

Figure 6

35

Figure 6. Confocal micrographs reveal co-localization of ameloblastin and
proteasome subunit alpha type 3 (Psma3) in Tomes’ processes. Immunofluorescent
staining performed on tangential sections of PN3 mouse mandibular incisors shows that
ameloblastin (red fluorophore, panel a) and Psma3 (green fluorophore, panel b) co-
localized to the Tomes’ processes of ameloblasts. The co-localization of ameloblastin and
Psma3 is revealed in a merged image as a yellow fluorescence signal (panel d). Panels
(a), (b), and (d) are 3D reconstructions of confocal Z-stack images of the 10µm thick
immunostained section. Panel (c) is the bright-field view of the stained section showing
the Tomes’ processes and their arrangement. In panels (a-d), arrows point to identical
Tomes’ processes in each panel. The co-localized immunostaining of ameloblastin and
Psma3 in Tomes’ processes (panel d) revealed a decussating pattern of protein
localization corresponding to alternating rows of ameloblasts, a pattern that is consistent
with the rod arrangement in mature mouse enamel.

36

Figure 7

37

Figure 7. Higher magnification view of the co-localization of ameloblastin and
proteasome subunit alpha type 3 (Psma3) in Tomes’ processes. Reconstructions of
confocal Z-stack images in 3-dimensions created from tangential section of
immunodetected protein (a-d), identifying ameloblastin (red fluorophore, panels a) and
Psma3 (green fluorophore, panels b) co-localized to the Tomes’ processes of ameloblasts
in a merged image (yellow fluorescent signal, panels d). Panel (c) is a bright-field view
of the area showing the Tomes’ processes and their arrangement. The co-localized
immunostaining of ameloblastin and Psma3 in Tomes’ processes revealed a decussating
pattern of protein localization, corresponding to the arrangement of rods in mature mouse
enamel. Top panels are lower magnification images with the boxed area shown in higher
magnification in panels a-d. In panels (a-d), arrows point to identical Tomes’ processes
in each panel.

38

Figure 8

39

Figure 8. Co-immunoprecipitation analysis of ameloblastin and proteasome subunit
alpha type 3 (Psma3). Ameloblast cell lysates prepared from PN3 mouse incisor enamel
organ epithelium were subjected to immunoprecipitation (a, b). Total cell lysate was used
as the positive control. Cell lysate incubated with Protein A/G PLUS-Agarose beads
served as the negative control. Panel (a) shows that Psma3 was co-immunoprecipitated
with ameloblastin by an ameloblastin-specific antibody (N-18, Santa Cruz
Biotechnology) and probed by a Psma3 antibody (EPR5455, Abcam) in Western blot.
Panel (b) shows that ameloblastin was reciprocally co-immunoprecipitated with Psma3
by a Psma3-specific antibody (A-17, Santa Cruz Biotechnology) and probed by an
ameloblastin antibody (M-300, Santa Cruz Biotechnology) in Western blot.
Abbreviations: IP, immunoprecipitated; WB, Western blot; co-IP: co-
immunoprecipitation; Ab., antibody.

40

Figure 9

41

Figure 9. Expression of ameloblastin fusion proteins in yeast cells (a) and
identification of the ameloblastin binding site for proteasome subunit alpha type 3
(Psma3) (b). Panel (a), Western blot detection of GAL4 BD-ameloblastin fusion protein
in yeast cell lysate using GAL4 BD protein-specific antibody (15-6E10A7, Abcam).
Total cell lysate prepared from untransformed yeast was used as the negative control
(Lane 1); asterisks indicate the corresponding bands for the GAL4 BD protein (Lane 2),
GAL4 BD-FL fusion protein (Lane 3), GAL4 BD-Np fusion protein (Lane 4), and GAL4
BD-Cp fusion (Lane 5). Molecular weight markers (in KDa) are given to the left. Panel
(b), co-immunoprecipitation assay using cell lysates (shown in panel c) prepared from
transformed yeast and used to detect the Psma3 binding site on human ameloblastin
(AMBN): Lane 1, co-IP AMBN FL with Psma3; Lane 2, negative control (precipitation
beads alone) of lane 1; Lane 3, co-IP AMBN Np with Psma3; Lane 4, negative control
(precipitation beads alone) of lane 3; Lane 5, co-IP AMBN Cp with Psma3; Lane 6,
negative control (precipitation beads alone) of lane 5. Psma3 GAL4 AD-fusion protein
(arrowhead) was co-immunoprecipitated with either the AMBN FL or the Cp fusion
proteins, showing the interaction is confined to the ameloblastin carboxyl terminus.
Abbreviations: IP, immunoprecipitated; WB, Western blot; co-IP: co-
immunoprecipitation; Ab., antibody.

42

Figure 10

43

Figure 10

44

Figure 10. Digestion of ameloblastin with 20S proteasome in vitro. Panel (a),
Western blot with the ameloblastin C’-terminal antibody (ab116347, Abcam) (lanes 1-4)
or with the N’-terminal antibody (N-18, Santa Cruz Biotechnology) (lanes 5-8): Lanes 1
and 5, recombinant human ameloblastin (rhAMBN) alone in assay buffer (50 mM Tris
buffer, pH 7.0) and the band at ~100 kDa corresponds to the intact rhAMBN protein;
Lanes 2 and 6, purified 20S proteasome alone in assay buffer and no specific band was
detected by either of the ameloblastin antibodies; Lanes 3 and 7, rhAMBN incubated with
purified 20s proteasome in assay buffer at 37°C for 1h with decreased intensity of the
full-length rhAMBN band (~100 kDa); Lanes 4 and 8, rhAMBN incubated with purified
20S proteasome in assay buffer at 37°C for 3h, and the intensity of the full-length
rhAMBN band (~100 kDa) diminished further. Furthermore, with 20S digestion, a band
corresponding to a cleavage product (~43kDa) was detected only by the N’-terminal
antibody (lane 8). Equal amount of rhAMBN was used in each lane. Panel (b), positive
control of recombinant proline-rich homeodomain protein (PRH) demonstrated that the
purified 20S proteasome used in the digestion assay retained its proteolytic activity.
Equal amount of PRH was used in each lane. Panel (c), inhibition of proteasome
digestion of ameloblastin by epoxomicin. Western blot with ameloblastin antibody
specific to the C’-terminus (ab116347) (lanes 1-3) or the N’-terminus (N-18) (lanes 4-6)
was used to detect ameloblastin with or without 50 µM epoxomicin. In the presence of
epoxomicin and the 20S proteasome, the quantity of ameloblastin protein was not
reduced dramatically (lanes 2 and 5). In contrast, without epoxomicin, incubation of
rhAMBN with 20S resulted in diminution of ameloblastin protein quantity (lanes 3 and 6)
with the release of a smaller molecular weight fragment (lane 6). Equal amounts of

45

rhAMBN were used in each lane. Densitometry tracing of 20S proteasome digestion for
rhAMBN and PRH is shown as a percentage of the total substrate mass used in each lane.

46

2.4 Discussion
Enamel is known for its unique mechanical properties that integrate hardness with
remarkable resistance to fracture (Baldassarri et al., 2008; Chun et al., 2010a; Fong et al.,
2003; Imbeni et al., 2005; Simmer et al., 2010; Smith et al., 2005; White et al., 2001).
These unique properties are dependent on the hierarchical structure of enamel imparted
by the protein matrix precursor. Enamel forms in the extracellular space with the
replacement of enamel matrix proteins by carbonated HAP crystallites. The crystallites
within the rods are woven together with the interrod crystallites to form a continuum that
provide optimal materials properties of fracture toughness (Paine et al., 2001; White et al.,
2001; Zhu et al., 2006). The enamel rod is the smallest divisible repeating structural
element of enamel and each rod is fabricated by a single ameloblast cell that synthesizes
and secretes enamel matrix proteins that guide their replacement by the mineral phase.
During enamel formation, protein self-assembly and protein-to-protein interaction serve
to organize the matrix proteins which in turn control HAP crystal habit and patterning. To
date, numerous studies have described the essential roles of enamel matrix proteins
including amelogenin, ameloblastin, and enamelin in the assembly and interactions that
occur during enamel biomineralization (Bartlett et al., 2006; Du et al., 2005; Fang et al.,
2011; Fincham et al., 1995; Fukumoto et al., 2004; Fukumoto and Yamada, 2005;
Moradian-Oldak et al., 2000; Paine et al., 1998; Paine and Snead, 1997; Paine et al., 2000;
Wang et al., 2005). In particular, ameloblastin was reported to serve as an interfacial
anchor between the ameloblast and the extracellular matrix (Beyeler et al., 2010;
Fukumoto et al., 2004). Moreover, ameloblastin proteolytic fragments become enriched
along the cell perimeter that defines the lateral boundary of each enamel rod (Hu and

47

Yamakoshi, 2003; Uchida et al., 1991). Here, I extend my studies to investigate
ameloblastin-protein interactions hypothesized to facilitate redistribution of ameloblastin
fragments responsible for protein patterning within the matrix that establishes the lateral
boundaries of each enamel rod fabricated by an ameloblast cell.
In this work, Psma3 was identified as interacting with ameloblastin in the
developing mouse tooth. I used human ameloblastin protein as the bait in a yeast two-
hybrid (Y2H) assay to screen a mouse ameloblast cDNA library. The amino acid
sequence for ameloblastin is well-conserved among diverse vertebrate species, with
human and mouse ameloblastin sharing extensive homology at the amino acid level
(Vymetal et al., 2008). Due to limitations in obtaining developing human teeth as a
source of mRNAs, I generated an ameloblast cDNA library using post-natal day 3 mouse
molars. At this developmental stage, most ameloblasts are in the secretory stage and the
expression of matrix proteins is robust.
The Y2H assay is a mature molecular tool for detecting protein-protein interactions.
However, false-positive interactions remain a problem for this assay, especially in large-
scale screening such as the cDNA library screening employed here. In order to
considerably decrease the rate of false-positive interactions, I used a stringent yeast strain
coupled with stringent selection conditions and added a high concentration of 3AT (80
mM) to the triple auxotrophic plate (Leu-/Trp-/His-) for yeast colony analysis. After
secondary screening, seven of forty-three candidate colonies were identified as
containing ameloblastin-interacting proteins. I picked these seven colonies based on their
interactions being stronger than the positive control interaction of Krev1 with mutant
RalGDS-m1, while excluding candidates that contained more weakly interacting proteins.

48

DNA nucleotide sequence analysis showed that each of the seven candidates encoded the
Psma3 protein. The most truncated Psma3 protein comprised only amino terminal
residues 1 through 216, supporting the interpretation that the amino-terminal 216-amino-
acid fragment is sufficient for Psma3 interaction with ameloblastin.
Proteasome subunit alpha type 3 is one of the components of proteasome core
structure. The proteasome is more widely known as an important intracellular device for
protein degradation. Organized as a cylindrical complex containing a core structure and
two “cap” subunits, the core is composed of four stacked rings with the two outer rings
composed of seven alpha subunits and the two inner rings composed of seven beta
subunits. The outer rings are responsible for maintaining a pore structure and the inner
rings contain catalytic sites where protein degradation occurs (Coux et al., 1996). To date,
seven different alpha subunits and ten different beta subunits have been reported in
mammals (Elenich et al., 1999). In the mouse, the major Psma3 isoform is 255 amino
acids in length (NCBI #NP_035314.3). The proteasome is widely acknowledged to
localize in the nucleus and cytoplasm in eukaryotes (Peters et al., 1994). However,
several recent investigations reported that a biologically active proteasome core is present
in extracellular space, including the alveolar space (Sixt et al., 2007; Sixt and Peters,
2010), blood plasma (Sixt and Dahlmann, 2008), and cerebrospinal fluid (Mueller et al.,
2012). These data suggest that cells secrete/shed proteasome and/or proteasome subunits
into the extracellular space, wherein the proteasome components may retain function. In
this study, the immunofluorescence data revealed that the distribution pattern of Psma3
on Tomes’ processes is very similar to the pattern of ameloblastin, suggesting they co-
localize and therefore could interact there. Furthermore, the results from co-

49

immunoprecipitation analysis demonstrated that Psma3 interacted with ameloblastin and
that the interaction is sufficiently stable that either protein can pull down the other partner.
These findings lead to the interpretation that Psma3 may interact with ameloblastin at the
Tomes’ process or may be secreted into extracellular space and interact with the newly
secreted ameloblastin in the enamel extracellular matrix.
In this study, I generated the ameloblastin N’-terminal domain and C’-terminal
domain by separating the full-length protein between Arg
222
and Leu
223
according to the
previous experiment evidence (Chun et al., 2010b; Wald et al., 2011). I found that Psma3
co-immunoprecipitated with both full-length and C’-terminal ameloblastin protein,
indicating that the binding site for Psma3 maps to the C’-terminal domain of ameloblastin.
Using confocal analysis of immunostained ameloblasts, I observed that the rod matrix
demonstrated abundant levels of Psma3 and ameloblastin, whereas the interrod matrix
was relatively depleted of both ameloblastin and Psma3. These data support the
interpretation that Psma3 interacts with the C’-terminal domain of ameloblastin at or near
Tomes’ processes during enamel formation.
Several studies have reported that binding to Psma3 results in proteins degradation
by proteasome in an ubiquitin-independent pathway (Bae et al., 2002; Boelens et al.,
2001; Shi et al., 2014; Shu et al., 2003). Moreover, investigators found that proteins
binding to Psma3 can undergo proteasome cleavage to form truncated fragments that
retain biological activity (Bess et al., 2003). Therefore, I hypothesized that the binding of
Psma3 to ameloblastin may assist the proteolytic processing of the C’-terminal domain,
leaving the N’-terminal domain to appear enriched along the perimeter of the rod.

50

In the proteolysis studies, I showed that ameloblastin digestion by the 20S
proteasome in vitro generated a smaller fragment that was detected only by the
ameloblastin N’-terminal antibody. This proteolytic activity was inhibited by addition of
the proteasome specific inhibitor epoxomicin. Thus, it is possible that ameloblastin-
Psma3 interaction facilitates proteasome cleavage of ameloblastin resulting in the N’-
terminal domain being separated from the C’-terminal domain. The liberated N’-terminal
products could accumulate around the enamel rod-to-interrod boundaries with
degradation of the C’-terminal domain (Hu and Yamakoshi, 2003; Uchida et al., 1991).
Redistribution of the ameloblastin N’-terminal domain may be facilitated through its
recently described self-assembly domain defined by N’-terminal amino acid residues 36-
72 (Wald et al., 2013). The C’-terminal domain was previously reported to distribute
evenly throughout newly formed immature enamel rods, becoming less abundant at later
developmental stages of enamel formation (Hu and Yamakoshi, 2003; Murakami et al.,
1997; Uchida et al., 1997). I suggest the ameloblastin C’-terminal domain may be
degraded by the proteasome, or may dissociate from Psma3 and be digested by other
matrix enzymes, such as MMP20 (Iwata et al., 2007). These data suggest that the
interaction between Psma3 and ameloblastin could facilitate separation and redistribution
of ameloblastin fragments in the forming enamel matrix.
The localization of ameloblastin N’-terminal domains at the rod perimeter (e.g., the
interrod) provides patterning to the matrix where it can play a role in establishing the
enamel microstructure by defining the rod and interrod boundaries that contribute the
required materials properties for a lifetime of masticatory function when fully
mineralized. Defined rod boundaries are essential to the creation of a partly interrupted

51

oriented continuum, greatly increasing the toughness and plasticity of enamel over its
fundamental constituent, crystalline hydroxyapatite (White et al., 2001). While the
underlying molecular mechanism is still obscure, my study offers the possible
interpretation that ameloblastin-Psma3 interaction facilitates the redistribution of
ameloblastin domains, resulting in segregation of the rod and the interrod regions to form
the highly patterned enamel structure. Ultimately, further studies will improve
understanding of the unique structure-function relationship imparted by protein-to-protein
interactions and reflected in the final mineral phase, and thus may contribute to
translational application of enamel regeneration.

52

Chapter 3
Altering the Ameloblastin-Psma3 Interaction by Knockdown of Psma3
using Silencing RNAs

3.1 Introduction
Ameloblastin (Snead, 1996), the second most abundant enamel matrix protein, is
crucial for normal enamel rod-interrod microstructure formation (Fukumoto et al., 2004;
Paine et al., 2003; Smith et al., 2009; Wazen et al., 2009). Immunohistochemical studies
have reported that ameloblastin proteolytic fragments localize to different areas within
the forming enamel matrix. In particular, the ameloblastin N-terminal domain is enriched
along the perimeter of each enamel rod, forming a “honeycomb” pattern (Uchida et al.,
1991), which suggests that the redistribution of ameloblastin domains is involved in the
establishment of enamel rod-interrod boundaries. Enamel mineral crystallites form
entirely in the extracellular matrix, so I hypothesized that the ameloblastin redistribution
is controlled either by interactions with proteins within the matrix or with proteins
localized to Tomes’ processes that contact the matrix. Recently, my colleagues and I
reported that proteasome subunit alpha type 3 (Psma3) interacts with ameloblastin during
enamel formation. Moreover, this interaction results in the digestion of ameloblastin by
the proteasome in vitro, generating an N-terminal cleavage fragment of ameloblastin
(Geng et al., 2015). Based on these findings and my in vitro studies, I sought to explore
the effects of ameloblastin-Psma3 interactions on enamel matrix patterning as well as on
final mineral structure in vivo.

53

My strategy was to use RNA interference with short hairpin RNA (shRNA) to
reduce the expression of Psma3, consequently disrupting ameloblastin-Psma3 interaction
during enamel formation. RNA interference is a gene silencing process that is triggered
by double-stranded RNA targeting homologous messenger RNAs for their destruction
(Fire et al., 1998; Montgomery et al., 1998). This mechanism offers a powerful research
tool that can be exploited in cell culture and model organisms to study the function of
specific genes (Daneholt, 2006; Elbashir et al., 2001a; Elbashir et al., 2001b).
Short hairpin RNA (shRNA) is double-stranded RNA with hairpin loop that can
mediate RNA interference to selectively and specifically inhibit target gene expression.
shRNA is produced inside of the target cell from a DNA construct that is delivered by
vectors into the nucleus (Rao et al., 2009). Due to continuous expression by the target cell,
shRNA provides effective and long-lasting suppression of gene expression.
In this study, I elected to use a lentiviral vector to introduce Psma3 gene-specific
shRNAs into target cells by transduction to obtain the shRNA-mediated Psma3
knockdown. Using a mouse ameloblast-like cell line (LS8) as an in vitro model, I show
that lentiviral transduction enables efficient delivery of shRNA without inducing cell
apoptosis. Moreover, I identified the most effective shRNA of those several ones tested
by evaluating the Psma3 expression levels using Western blotting and immunodetection.
I then used the most effective shRNA identified to perform RNA interference in mouse
incisor organs. Immunohistochemistry revealed mosaic elimination of Psma3 in
ameloblast cells of the target incisors. With these data, I provide evidence that applying
RNA interference mediated by lentiviral delivered shRNA in vivo is a feasible method to
investigate the function of the ameloblastin-Psma3 interaction in enamel formation.

54

3.2 Material and Methods
Preparation for lentiviral packaging
The commercially available 293T/17 [HEK 293T/17] cell line (ATCC #CRL-
11268) was used as the host for lentiviral packaging. The 293T cell line is a derivative of
the HEK 293 cell line (Graham et al., 1977), and clone #17 was selected for high titer
lentivirus production. Following the manufacturer’s protocol, the frozen 293T/17 cells
were recovered and grown in DMEM (Corning, Manassas, VA) supplemented with 10%
fetal bovine serum (FBS) (Invitrogen, Grand Island, NY), 100 U/ml penicillin (Sigma, St.
Louis, MO), and 100 µg/ml streptomycin (Sigma) at 37°C in a humidified atmosphere of
5% CO
2
.
One set of lentiviral transfer vectors containing the shRNA expression cassette was
obtained from OriGene (Rockville, MD). This set included four Psma3 gene-specific
shRNA constructs and one scrambled non-effective shRNA construct (Table 3) in
lentiviral tGFP vectors (Fig. 11). These transfer plasmids, as well as the packaging
plasmid pCMV-dR8.2 (Addgene, Cambridge, MA) and the envelope plasmid pCMV-
VSVG (Addgene), were separately amplified in DH5α cells (Invitrogen) and purified
using a Plasmid Mini Kit (Qiagen, Valenica, CA) for producing lentiviral particles.

Production of lentiviral particles
To produce lentiviral particles, a transfer vector containing the shRNA, pCMV-
dR8.2 and pCMV-VSVG was co-transfected into 293T/17 cells by calcium phosphate-
mediated transfection. This procedure was performed according to the protocol provided
with the ProFection Mammalian Transfection System kit (Promega, Madison, WI).

55

Briefly, 293T/17 cells were subcultured on five 100mm plates the day before
transfection. All cultures were incubated under 5% CO
2
in air at 37°C to reach 60~70%
confluence. A 15µg mass of each transfer vector (A-D, control), 15µg of pCMV-dR8.2,
and 5µg of pCMV-VSVG were mixed with 62µl of CaCl
2
in separate 1.5-ml sterile tubes,
and the final volumes were adjusted to 500µl by adding deionized water. The DNA-
CaCl
2
mixture was added dropwise into 500µl of pre-warmed 2X HBS in a second tube
and incubated at room temperature for 30 min. For each transfection, the DNA-CaCl
2
-
HBS solution was added dropwise such that it was evenly distributed over the 293T/17
cells. After incubation at 37° in 5% CO
2
for 16 hours, the transfection solution was
removed and the transfected cells were cultured in fresh growth medium containing 5
mM sodium butyrate (Sigma) for an additional 36 hours. The culture medium was
collected and passed through a 0.45 µm filter to remove cell debris, then stored at -80°C
for future use. The titer of lentivirus in the supernatant was approximately 10
6
~10
7

colonies/ml.

Transduction of lentivirus to LS8 cells
The mouse ameloblast-like cell line (LS8) was cultured in DMEM (CORNING)
supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/ml penicillin
(Sigma), and 100 µg/ml streptomycin (Sigma). Cells were plated at a density of 2×10
5

cells/well in a six-well plate and incubated until 50~60% confluent for transduction. At
the time of transduction, the culture medium was removed. The lentiviral supernatant
(LVA-D, Control) containing polybrene (8µg/ml) (Sigma) was then sequentially added to
each of five wells (1ml/well), while fresh culture medium (1ml) containing polybrene

56

(8µg/ml) was added to the 6th well as a negative control. The cultures were placed in an
incubator at 37°C with 5% CO
2
. After 24 hours, the lentiviral solutions were aspirated
away, and fresh culture medium was applied to each well. The transduced cells were
cultured at 37°C for an additional 72 hours, allowing time for lentiviral gene expression.
The transduction efficiency for each lentivirus was checked by observing and counting
the GFP signals under a florescence microscope (Leica DMI3000 B, Leica Microsystems,
Buffalo Grove, IL) or by counting the tGFP-positive cells using a flow cytometer (BD
Accuri C6, BD Biosciences, San Jose, CA).

Apoptosis analysis of transduced LS8 cells
72 hours post-transduction, LS8 cells were seeded on Lab-Tek II Chamber Slides
(Thermo Fisher Scientific) and grown overnight at 37°C in a humidified atmosphere of
5% CO
2
. The cells were fixed in 4% paraformaldehyde solution and then subjected to an
apoptosis assay using the DeadEnd Colorimetric TUNEL System (Promega). Following
the manufacturer’s protocol, the nuclei of apoptotic cells were stained dark brown, which
was recorded by light microscopy (OLYMPUS DP72). DNase-treated untransduced LS8
cells were included as a positive control.

Assessment of Psma3 knockdown in transduced LS8 cells
Western blotting with immunodetection was performed to detect Psma3 expression
levels in transduced LS8 cells in order to evaluate knockdown effects for each of the four
gene-specific shRNAs, as described in a previous study (Zhou and Snead, 2000). In brief,
cell lysates were prepared from untreated LS8 cells and lentivirally transduced (LVA-D,

57

Control) LS8 cells at 72 hours post-transduction. Protein concentrations were determined
using a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Equal amounts
of proteins (20µg) from each sample were loaded onto 4-12% SDA-PAGE (Invitrogen)
and resolved to size by gel electrophoresis. The proteins were transferred from the gel to
an Immobilon-P membrane (Millipore, Billerica, MA), and analyzed with anti-Psma3
antibody (EPR5455, ab109532, Abcam, Cambridge, MA) and anti-GAPDH antibody
(GT293, GeneTex, Irvine, CA). The relative intensities of the Psma3 protein bands were
quantified using densitometry with ImageJ software (version 1.50c 27, rsb.info.nih.gov).

Transduction of lentivirus to mouse incisors
Mandibular incisors were microdissected from newborn mouse pups (Swiss
Webster). Lentiviral solution LVC, identified as being the most effective shRNA by the
Western blot assay, or a control lentiviral solution was injected into the epithelium of the
incisor organs through a pre-pulled glass micropipette (World Precision Instrument,
Sarasota, FL). The injection site was near the caudal zone of incisor, which contains
differentiated and undifferentiated ameloblasts (Huang et al., 2010; Huang et al., 2008).
The incisors were immersed in the same lentiviral solution plus 8µg/ml polybrene and
cultured at 37°C in a humidified atmosphere of 5% CO
2
. After 24 hours, the lentiviral
solution was replaced with fresh DMEM (CORNING) culture medium supplemented
with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/ml penicillin (Sigma), and 100
µg/ml streptomycin (Sigma). Then the transduced incisors were placed on a precut
Millipore membrane filter (0.8µm pore size, Millipore) on a stainless steel grid that was
overlaid on the center well of the organ culture dish. The culture medium was added into

58

the center well to wet the filter from below. All cultures were maintained at 37° in 5%
CO
2
for 3 days. The culture medium was replaced every day.

Immunohistochemistry
Lentiviral transduced incisor organs that were verified as tGFP-positive under a
fluorescent microscope were selected for immunohistochemical detection of Psma3. The
selected incisor organs were fixed with 4% paraformaldehyde in PBS overnight at 4°C.
Tissue sections 5 µm in thickness were prepared as described in a previous study
(Couwenhoven et al., 1990). Sections were incubated in 3% H
2
O
2
for 10 min to quench
the endogenous peroxidase activity, then blocked with 5% BSA/PBS containing 0.1%
Tween 20 for 1 hour. A rabbit monoclonal anti-Psma3 antibody (EPR5455, ab109532,
Abcam) was used to detect Psma3. After incubation with the primary antibody overnight,
the sections were processed through sequential incubation with biotinylated secondary
antibody, horseradish peroxidase (HRP) labeled streptavidin and AEC chromogen
provided in a Histostain-SP IHC kit (Invitrogen). The presence of the antigen could be
visualized as red coloration when viewed under a light microscope.

59

Table 3
Name
Backbone
plasmid
Reporter
gene
shRNA cassette
Length
Target sequence on Psma3
mRNA
Location
A pGFP-C-shLenti tGFP 29
TAGGTCCAACTTCGGC
TATAACATTCCTC
Nucleotide bases
421~449
B pGFP-C-shLenti tGFP 29
CAAGTTGAATATGCCA
TGAAGGCTGTGGA
Nucleotide bases
191~219
C pGFP-C-shLenti tGFP 29
CAGAGAAATATGCCA
AGGAATCTTTGAAG
Nucleotide bases
831~859
D pGFP-C-shLenti tGFP 29
GTGCGAATGATGGTGC
ACAGCTCTATATG
Nucleotide bases
549~577
control pGFP-C-shLenti tGFP 29 Scrambled None

Table 3: Lentiviral transfer plasmids. The plasmids were constructed by inserting a
synthetic shRNA expression cassettes into the lentiviral vector pGFP-C-shLenti. Each
shRNA cassette consisted of a 7-nucleotide loop and 29-nucleotide stem with a unique
sequence (Fig. 1). Four gene-specific shRNA sequences (A-D) were designed against
different nucleotide base sites in the mouse Psma3 gene locus (NCBI RefSeq:
NM_011184). A scrambled shRNA (control) was used as the negative control. The
expression of the reporter gene tGFP was used to monitor lentiviral packaging and
transduction.

60

Figure 11

Figure 11. Map of shRNA cloning vector pGFP-C-shLenti (OriGene). pGFP-C-
shLenti vector is a third generation lentiviral vector designed for cloning shRNA
expression cassettes. shRNA expression is driven by the U6 polymerase III promoter and
terminated by the immediate downstream TTTTTT termination sequence. tGFP reporter
gene is driven by the CMV promoter. Chloramphenicol serves as the bacterial selection
marker for the vector.

61

3.3 Results
Lentiviral vectors allow efficient gene delivery in LS8 cells
The turbo green fluorescent protein (tGFP) gene was included in the lentiviral
vector and delivered with the shRNA cassette into target cells via transduction.
Therefore, expression of tGFP could be used to measure the transduction efficiency of the
lentivirus. Lentiviral particles were generated for each shRNA clone (Table 3) and
separately transduced into LS8 cells, and 72 hours post-transduction, green fluorescence
showed tGFP expression in the transduced cells. For each transduction, a large fraction of
cells expressed tGFP (Fig. 12a-e, left row). The transduction efficiency was calculated by
flow cytometry (Fig. 12a-e, right row) with efficiency of 92.5% was achieved for control,
93.0% for LVA, 88.5% for LVB, 84.6% for LVC, and 81.2% for LVD. These data
indicate that the lentiviral particles produced in this study were effective at delivering
shRNA to LS8 cells.

Lentiviral transduction does not cause apoptosis of LS8 cells
A terminal-deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)
assay was performed to analyze LS8 cell apoptosis after lentiviral transduction (Fig 13)
with nuclei stained brown indicating apoptotic cells. DNase I-treated untransduced cells
were used as the positive control. DNase I treatment resulted in fragmentation of
chromosomal DNA, presenting nuclei stained brown in the apoptosis analysis. The
staining results showed that very few apoptotic cells were detected in each transduced
LS8 culture. Compared with non-transduced cells, I observed no difference in the
percentage of apoptotic cells in the lentiviral transduced cells. That is, in this study the

62

concentration of the lentivirus used to obtain high transduction efficiency was not cell-
toxic, and the transduction process did not induce cell apoptosis.

Knockdown of Psma3 in LS8 cells by shRNA
To evaluate the gene silencing effect for each shRNA clone, lysates were prepared
from non-transduced and lentivirally transduced LS8 cells to measure the expression
level of Psma3 by Western blotting using a Psma3-specific antibody (EPR5455) (Fig.
14). Compared with the non-transduced cells, no significant knockdown of Psma3 was
detected in the transduced cells expressing the scrambled control shRNA, whereas each
of the four gene-specific shRNAs caused various degrees of reduction in the expression
of Psma3 in LS8 cells. The best knockdown effect was achieved with shRNA clone C
(~50%), and clone B showed moderate activity (~40%), whereas the clones A and D
revealed only weak silencing effects with 16% and 11% efficiency, respectively. These
results showed that shRNA clone C, targeting the coding sequence
CAGAGAAATATGCC AAGGAATCTTTGAAG of the Psma3 mRNA, was the most
efficient construct in this study.

Mosaic elimination of Psma3 in mouse incisor organs
To investigate shRNA-mediated Psma3 silencing in the developing tooth, the most
effective shRNA lentivirus (LVC) was selected to transduce incisor organs dissected
from newborn mouse pups. Control incisor organs were transduced by the scrambled
shRNA lentivirus (Control) or without vector transduction. I locally injected the lentiviral
particles into the epithelium of the incisor organ using pre-pulled glass needles in order to

63

maximize the lentivirus specifically transducing ameloblast cells. The injected incisors
were directly cultured within the lentiviral solution to obtain higher transduction
efficiency. At 3 days after transduction, both LVC-transduced and control-transduced
incisors revealed robust green fluorescence within some areas on the labial surface of
epithelium, which indicated successful viral transduction (Figure 15c, g). No GFP
fluorescence was detected in untreated incisors. Immunohistochemistry for Psma3 was
performed on incisor sections to verify the Psma3 knockdown in ameloblast cells. In
LVC-transduced samples, chimeric Psma3 expression was detected with some
ameloblasts showing no expression of Psma3 next to other ameloblasts expression
Psma3, indicative of a stochastic pattern of transduction (Fig. 15h, i). In contrast, the
control-transduced incisors and untreated incisors showed Psma3 expression in each and
every ameloblast (Fig.15d, e).

64

Figure 12

65

Figure 12. Efficient transduction of lentivirus in LS8 cells. LS8 cells were transduced
with lentiviral solution Control (panel a), LVA (panel b), LVB (panel c), LVC (panel d),
and LVD (panel e). At 72 hours post-exposure, the transduction efficiency for each
lentivirus was analyzed by identifying tGFP expression. The transduced cells were
imaged for green fluorescence, shown in left row of all panels. Transduction efficiencies
were quantified using flow cytometer to calculate the proportion of tGFP-positive cells
and expressed as a percentage in histograms. Right row corresponds to the respective
flow cytometry histograms. Fluorescent images showed that in each transduction
experiment a large fraction of cells were positive for green fluorescence, indicating the
high transduction efficiency of each lentivirus. Histograms showed 92.5% transduction
efficiency for Control in panel (a), 93.0% for LVA in panel (b), 88.5% for LVB in panel
(c), 84.6% for LVC in panel (d), and 81.2% for LVD in panel (e). Scale bar = 50µm and
applied to all.

66

Figure 13

67

Figure 13. Lentiviral transduction does not lead to massive cell death. Each batch of
lentivirus transduced LS8 cells was analyzed using a TUNEL assay for cell apoptosis at
72 hours post-transduction. The nuclei of apoptotic cells were stained dark brown. Panel
(a), DNase I-treated untransduced cells (positive control). Panel (b), untransduced cells
(negative control). Panel (c) control lentivirus transduction. Panel (d), lentivirus LVA
transduction. Panel (e), lentivirus LVB transduction. Panel (f), lentivirus LVC
transduction. Panel (g), lentivirus LVD transduction. The staining results revealed that
very few apoptotic cells were detected among the batches of transduced LS8 cells. Scale
bar = 100µm and applied to all.

68

Figure 14

69

Figure 14. Knockdown of proteasome subunit alpha type 3 (Psma3) in LS8 cells.
LS8 cells were transduced with lentivirus, and total cell lysates were prepared at 72 hours
post-transduction. The Psma3 expression was probed using a Psma3-specific antibody
(EPR5455, Abcam) in Western blot analysis. GAPDH was used as a loading control. The
relative density of the Psma3 band was quantified by densitometry and is shown as a
percentage compared to GAPDH for each lane. Lane 1, untransduced total cell lysate
severed as the standard of Psma3 quantity in LS8 cells, the relative density of Psma3
band was 100%; Lane 2, control lentivirus transduction delivered the scrambled shRNA
into LS8 cells, the relative density of Psma3 band was 99% indicating no significant
reduction of Psma3 proteins; Lane 3, shRNA delivered by LVA transduction produced
16% reduction of Psma3 proteins (the relative density of the band was 84%); Lane 4,
shRNA delivered by LVB transduction produced 38% reduction of Psma3 proteins (the
relative density of the band was 62%); Lane 5, shRNA delivered by LVC transduction
produced 48% reduction of Psma3 proteins (the relative density of the band is 52%);
Lane 6, shRNA delivered by LVD transduction produced 11% reduction of Psma3
proteins (the relative density of the band is 89%). The most efficient gene-specific
shRNA caused almost 50% knockdown of Psma3 in LS8 cells (lane 5).

70

Figure 15

71

Figure 15. Organ culture of mouse incisors transduced with the control lentivirus
(b-e) and most effective shRNA lentivirus (LVC) (f-i). Panel (a), incisor organs were
dissected from newborn mouse pups. Lentiviral solution was injected into the enamel
organ epithelium near the caudal zone of incisor. The transduced incisors were
maintained in organ culture for 72 hours after transduction. Light micrographs (b, f) and
fluorescence images (c, g) were obtained for the whole cultured incisor organs. Panels (b)
and (c), the same incisor from control lentivirus transduction. Panels (f) and (g), the
same incisor from the most effective shRNA transduction. Green fluorescence on the
labial surface of the incisor in panels (c) and (g) indicates the successful viral
transduction in incisor organs. Panels (d) and (e), detection of Psma3 protein expression
by immunohistochemistry in a tissue section from the incisor shown in panel (b) showed
that Psma3 was detected in all ameloblasts. (e) is a higher magnification view of the
boxed area in (d). Panel (h) and (i), detection of Psma3 protein expression by
immunohistochemistry in a tissue section from the incisor shown in panel (f).
Immunoreactivity showed that Psma3 expression was detected in some ameloblasts but
eliminated in some other ameloblasts, producing a chimeric staining pattern. (i) is a
higher magnification view of the boxed area in (h). Scale bars equal to 200 µm in panels
(d) and (h), 50 µm in panels (e) and (i).

72

3.4 Discussion
Disrupting ameloblastin-Psma3 interaction by altering Psma3 expression is an
effective method to investigate the physiological significance of this interaction. In this
study, I used shRNA-mediated RNA interference to reduce or eliminate Psma3
expression to study the resulting long-term effects on enamel formation. RNA
interference is an RNA-dependent gene silencing process, mediated by a small RNA
molecule binding to a specific messenger RNA (mRNA) molecule to induce suppression
of mRNA translation or destruction of the mRNA molecule. Here, shRNA was chosen as
the interference trigger because it is continuously generated by the host cell, which
provides more efficient, durable gene silencing (McAnuff et al., 2007; Rao et al., 2009;
Siolas et al., 2005; Vlassov et al., 2007). I used lentiviral transduction to deliver shRNA
into ameloblast-like LS8 cells and enamel organs of mouse incisors to study the effects to
enamel formation by knockdown of Psma3.
In this study, I produced four lentiviral particles that contained different shRNA
expression sequences targeting four unique sites of Psma3 mRNA, and one control
lentiviral particle that contained a scrambled shRNA cassette as a control (Table 1).
Transduction efficiencies of above 80% in LS8 cells were achieved for all five lentiviral
particles. Based on the Western blotting of transduced LS8 cell lysates, I found that the
shRNA molecule with the sequence CAGAGAAATATGCC AAGGAATCTTTGAAG
was the most effective, producing an approximately 50% reduction of Psma3. The
findings that none of the transductions caused significant LS8 cell apoptosis
demonstrated that the Psma3 knockdown could not be attributed to massive cell death.
As desired, the shRNA selectively and specifically suppressed Psma3 expression in LS8

73

cells. For most genes, an efficient knockdown should be expected to reach 80%~90%
(Gou et al., 2007; Xu et al., 2009). A number of studies have demonstrated that
proteasome inhibition results in lethality to cells, a mechanism has been exploited in
cancer therapy (Goldberg, 2007; Hideshima et al., 2001; Suraweera et al., 2012). Since
Psma3 is one of components of proteasome core structure that contains the protease
active sites, it is possible that overly efficient knockdown of Psma3 expression could lead
to cell death. In the context of this study, I would like to maintain cellular homeostasis in
transduced cells. Therefore, obtaining 50% reduction is considered to be a useful target.
These results with LS8 cells provided the design fundamental for a study of Psma3
alternation in whole incisor organs in vitro.
A major challenge in the investigation of Psma3 knockdown in incisor ameloblasts is
to deliver shRNA into target ameloblasts that are differentiated from inner enamel
epithelial cells. The inner enamel epithelium contains ameloblasts at different
developmental stages and this cell layer is encased by the stratum intermedium cells. This
layered anatomy impedes vector-based shRNA delivery to the target secretory
ameloblasts. Therefore, in addition to culturing the incisor organ in the lentiviral solution,
at the same time I locally injected the lentivirus into the secretory ameloblast zone near
the growing end of the incisor to increase the opportunity to achieve lentivirus transduced
ameloblasts. I found tGFP expression in some ameloblasts, indicating successful
transduction. Interestingly, lentiviral transduced ameloblasts were segregated into
isolated zones by the non-transduced ameloblasts in the incisor. As result, the
immunohistochemistry for Psma3 in the transduced incisor revealed a chimeric staining
pattern. An advantage of this chimeric model is that it allows me to compare the effects

74

of Psma3 knockdown to normal expression in the identical developmental condition of
the same organ. I also set up two parallel controls, namely an incisor transduced with
scrambled shRNA and an untreated incisor maintained in organ culture. All control
incisors showed specific immunostaining for Psma3 in all ameloblasts.
Taken together, these findings demonstrate that shRNA molecules delivered by
lentiviral transduction were stably expressed in ameloblasts and that their presence
caused interference resulting in specific suppressed Psma3 expression. This in vitro organ
culture model provides the foundation for future in vivo studies.

75

Chapter 4
Immunodetection of Psma3 in Exosomes Recovered from
Ameloblast-like LS8 Cell Culture Medium

4.1 Introduction
The proteasome is a multicatalytic proteinase containing a catalytic core structure
and two regulatory complexes. Although it is well known that the major part of
proteasomes is localized within cell cytoplasmic and nuclear compartments (Ahn et al.,
1996; Peters et al., 1994), it is now accepted that proteasome core can also exist in
extracellular spaces, such as blood plasma, alveolar space, and cerebrospinal fluid.
Investigators have demonstrated that proteasome is not only present in extracellular
spaces but also enzymatically intact and active (Mueller et al., 2012; Sixt et al., 2007).
Proteasome core structure is composed of fourteen α subunits and fourteen β
subunits. None of these subunits contain a signal sequence, so proteasome export does
not rely on the classic secretory pathway. So far, a mechanism for proteasome
transportation between cells and the extracellular spaces is still unknown. However,
several studies have reported that the proteasome core found to be attached to the cell
plasma membrane (Hori et al., 1999; Khan and Joseph, 2001; Klare et al., 2007;
Nakagawa et al., 2007; Rivett et al., 1992) and also could be detected at the cell surface
(Bureau et al., 1997; Henry et al., 1996; Morales et al., 2003). These findings suggest that
membrane associated transportation pathway is responsible for proteasome release to the
extracellular environment. In researching the mechanism of proteasome export,

76

Bochmann et al found that a functional proteasome can be exported from T lymphocytes
by way of microparticles (Bochmann et al., 2014). Additionally, the presence of
proteasome subunits in exosomes also suggested a possible pathway for cell releasing
proteasome (Buschow et al., 2010; Carayon et al., 2011; Gonzales et al., 2009; Lai et al.,
2012).
I found that ameloblastin protein interacts with Psma3 protein in the developing
tooth. Ameloblastin is robustly secreted by ameloblasts into extracellular matrix to
participate in enamel matrix organization that is crucial for proper enamel microstructure
formation. Therefore, it is possible that the ameloblastin-Psma3 interaction occurs within
the forming enamel matrix. In this chapter, I report the presence of Psma3 in LS8
exosomes isolated from culture medium, an outcome suggesting that Psma3 could be
from LS8 cells released into the extracellular environment via exosomes. This finding
suggests that Psma3 can be exported from ameloblast cells, which supports our
speculation that ameloblastin interacting with Psma3 in the forming enamel matrix guides
ameloblastin processing and redistribution to segregate the enamel rod and interrod
regions.

77

4.2 Material and Methods
Cell culture
The mouse ameloblast-like cell line, LS8, was cultured in DMEM (CORNING)
supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/ml penicillin
(Sigma), and 100 µg/ml streptomycin (Sigma) at 37°C in a humidified atmosphere of 5%
CO
2
. When the cells grew to 80-90% confluence, the medium was replaced with fresh
DMEM without FBS, but containing 100 U/ml penicillin and 100 µg/ml streptomycin.

Exosome isolation
LS8 cells were maintained in FBS-free medium for 12 hours, and checked
microscopically to ensure they were healthy. The culture medium was then harvested and
centrifuged at 2,000 xg for 30min at 4°C to remove cells and debris. The supernatant was
collected and mixed with a total exosome isolation reagent (Invitrogen) at a volume ratio
of 2:1. The medium/reagent mixture was incubated at 4°C overnight, then recovered by
centrifugation at 10,000 xg for 1 hour at 4°C. The pellet containing exosomes was
resuspended in 1X PBS and kept at 4°C for downstream analysis.

Western blot analysis
After collecting the medium, cells were washed twice in 1X PBS and lysed in ice-
cold RIPA buffer (Santa Cruz Biotechnology, Dallas, TX). The lysate was cleared by
centrifugation at 15,000 xg for 15 min at 4°C. Samples of cell lysate and isolated
exosomes were mixed with 2X SDS loading buffer, boiled for 5 min, and analyzed by
Western blotting with an anti-Psma3 antibody (EPR5455). The immunodetection

78

procedure was followed as described previously (Zhou and Snead, 2000). A sample of
LS8 cell lysate sample was used as the positive control.

4.3 Results
Presence of Psma3 in LS8 cell exosomes
To determine whether Psma3 could be exported out of ameloblast cells in exosome
bodies, I used Western blotting to detect Psma3 in exosomes that were recovered from
conditioned LS8 cell culture medium. A rabbit monoclonal antibody (EPR5455) specific
to Psma3 protein recognized a protein band of 28 kDa (Fig. 16), indicating that detectable
levels of Psma3 were present in LS8 cell exosomes. Psma3 found in exosomes has a
smaller mass than the corresponding protein detected in LS8 cell lysate (~30 KDa).

79

Figure 16

Figure 16. Western blot analysis of Psma3 in exosomes from LS8 cells. The FBS-free
culture medium was collected from confluent LS8 cells after 12 hours incubation and
subjected to total exosome isolation. Psma3 was detected using by a rabbit monoclonal
specific antibody (EPR5455, Abcam) in Western blot analysis. Lane 1, LS8 cell lysate
was used as the positive control for Psma3 detection. The band at ~30 kDa corresponds to
the intracellular Psma3; Lane2, exosome sample recovered from LS8 cell culture
medium; Lane 3, repeated exosome experiment. In both lane 2 and lane3, Psma3 were
detected as a protein band of ~28 kDa in LS8 exosomes. Molecular mass markers (in
kDa) are given at left.

80

4.4 Discussion
Exosomes are cell-derived micro-vesicles with diameter between 30 and 100nm.
Exosomes contain various biological molecules including RNAs and proteins and
transport their cargo to the extracellular space, thereby playing important roles in cell
biology and immunology (Johnstone, 2006; Keller et al., 2006; Li et al., 2006; Raposo
and Stoorvogel, 2013). Furthermore, exosome delivery is a non-classical secretory
pathway for export of proteins across cell membranes.
Extracellular proteasomes have been reported in several studies (Mueller et al., 2012;
Sixt et al., 2007; Sixt and Dahlmann, 2008; Sixt and Peters, 2010). Although the
mechanism of proteasome export remains ambiguous, the presence of proteasome
recovered from exosomes which retain enzymatically active suggested that exosomes are
used as a delivery vehicle for proteasome release into the extracellular environment (Lai
et al., 2012).
Inspired by these findings in other studies, I examined Psma3 by Western blot
analysis using exosomes samples recovered from LS8 cell culture medium. My result
showed that Psma3 was present in the LS8 exosomes, which suggest that LS8 cells could
extrude Psma3 through the exosome compartment. This study implies that Psma3 may be
released into ameloblast extracellular space and interact with ameloblastin to facilitate the
separation and redistribution of ameloblastin domains in the enamel extracellular matrix
during amelogenesis. Psma3 protein detected in the exosome compartment was smaller
compared to that found in LS8 cell lysate. One of possible reasons for this variation in
relative size is that ameloblasts may produce a relative smaller Psma3 protein, which is

81

destined to be exported out of the cell through the exosome compartment. This smaller
protein may result from alternative splicing of a RNA precursor or from alternative
translational start sites in the Psma3 mRNA. Another possibility is that a certain type of
post-translational modification of Psma3, resulting in cleavage of the protein, may take
place during the export of Psma3 by exosomes. I speculate that Psma3 is anchored to
membrane by hydrophobic residues located its N’-terminus that become entrapped in
membranes of the exosomes. This short peptide may remain with the exosome,
dissociating itself from the Psma3 molecule when it is released into the extracellular
space (Fig. 17). Further study is necessary to confirm if the detected Psma3 band from
exosomes found in this study is collinear with the Psma3 recovered from the lysate.

82

Figure 17

Figure 17. Schematic of the possible mechanism of Psma3 export from ameloblast
into the enamel extracellular matrix. Psma3 hydrophobic residues bind to the
membrane of multivesicular body (MVB) in cytoplasm of ameloblast. MVB bud inward
to entrap Psma3 into a segregated internal vesicle. The MVB fuse with the cell membrane
to release the internal vesicles into the enamel extracellular matrix as exosomes. It is like
when Psma3 is released from the exosome in the enamel matrix, the membrane anchor
peptide is dissociated from the entire Psma3 molecule, resulting in the smaller
extracellular Psma3.

83

Chapter 5
Summary

Enamel is composed of hydroxyapatite (HAP) crystallites fabricated by ameloblast
cells to form a highly organized rod-interrod woven structure among the enamel rods and
interrods. The arrangement of rod and interrod crystallites contributes to enamel’s unique
material properties, including wear resistance and fracture toughness (Baldassarri et al.,
2008; Boyde, 1987, 1997; White et al., 2001). Several indentation studies have reported
crack deflection by enamel rods, suggesting that the boundary zone between rod and
interrod serves to diffuse damage to avoid catastrophic enamel fracture (Hassan et al.,
1981; White et al., 2005; Xu et al., 1998). Therefore, investigating the mechanism of rod
and interrod demarcation allows us to better understand the enamel structure-function
relationship on the nanoscale.
Enamel crystallites form through a complex biomineralization process within an
organic extracellular matrix (Robinson et al., 1981). During enamel formation, protein
self-assembly and protein-to-protein interaction serve to organize the matrix, which in
turn controls HAP crystal habit and pattern. To date, investigators have used loss-of-
function knock out (Fukumoto et al., 2004; Wazen et al., 2009) and gain-of-function
transgenic mouse models (Paine et al., 2003) to demonstrate that ameloblastin plays a
critical role in maintaining ameloblast differentiation and proper enamel formation. In
particular, ameloblastin proteolytic fragments have been reported to become enriched
around the perimeter of ameloblasts in forming enamel matrix (Uchida et al., 1991),
which is considered to participate in establishing enamel rod-to-interrod boundaries.

84

However, the mechanism of ameloblastin redistribution and how this redistribution
contributes to enamel rod-interrod pattern formation is still unknown.
Based on the hypothesis that ameloblastin redistribution is controlled by protein
interactions during enamel formation, a yeast two-hybrid assay was first performed to
screen an ameloblast cDNA library using ameloblastin as the bait. Proteasome subunit
alpha type 3 (Psma3) was identified as interacting with ameloblastin. This interaction was
confirmed to be sufficiently stable in the developing tooth using a series of co-
immunoprecipitation assays. Immunofluorescence data revealed co-localization of Psma3
and ameloblastin at the ameloblast secretory end (Tomes’ process), suggesting that
Psma3 interacts with ameloblastin at or near Tomes’ process during enamel formation.
To determine the domain at which Psma3 binds with ameloblastin, Psma3 protein as well
as full-length ameloblastin protein and its N-terminal and C-terminal domains were
generated in yeast using protein engineering and subjected to co-immunoprecipitation
analysis. The results demonstrated that Psma3 interacts with the C-terminal domain of
ameloblastin.
Binding to Psma3 can result in protein digestion by proteasomes, and the cleavage
fragments may retain biological activity to within the enamel matrix. To investigate the
potential biological function of ameloblastin-Psma3 interaction, I performed proteolysis
studies using recombinant proteins. The results showed that ameloblastin was digested by
the 20S proteasome in vitro, generating an N-terminal fragment. This proteolytic activity
was inhibited by a proteasome-specific inhibitor, epoxomicin. According to these
findings, I propose that ameloblastin-Psma3 interaction could facilitate the ameloblastin
N-terminal domain being separated from the C-terminal domain within the forming

85

enamel matrix. I hypothesize that the liberated N-terminal domain then undergoes
redistribution to accumulate around the enamel rod-interrod boundaries, contributing to
the formation of enamel microstructure.
To further investigate the physiological significance of ameloblastin-Psma3
interaction, I knocked down Psma3 using shRNA-mediated interference to disrupt the
protein interaction. Lentiviral vectors, which are able to efficiently transduce almost any
cell type, were utilized for shRNA delivery. In cell culture studies, lentiviral transduction
supported high transduction efficiency of above 80% in ameloblast-like LS8 cells.
Furthermore, lentiviral transduction did not cause significant LS8 cell apoptosis. Using
Western blot analysis, I identified the most effective shRNA molecule among those
tested, which produced nearly 50% knockdown of Psma3 in LS8 cells. Psma3 is one of
the essential subunits that contribute to the formation of proteasome core structure.
Overly efficient knockdown of Psma3 could eliminate proteasomes entirely, resulting in
lethality. Therefore, to maintain cellular homeostasis in transduced cells, 50% reduction
of Psma3 may be ideal. These results support the feasibility of using a lentivirus to
deliver shRNAs and thereby silence Psma3.
To examine the Psma3 knockdown in ameloblasts, whole incisor organs dissected
from newborn mouse pups were transduced by culturing them in lentiviral solution. At
the same time, I locally injected the lentivirus into the secretory ameloblast zone in the
incisors to increase the probability of ameloblast transduction. In transduced incisors, the
gene-specific shRNA caused a mosaic elimination of Psma3 in ameloblasts, revealing a
chimeric pattern of Psma3 protein distribution upon immunohistochemical staining. In
contrast, the scrambled shRNA did not alter Psma3 expression, which was detected in

86

each and every ameloblast in immunostaining experiments. These findings indicate that
the shRNA molecules delivered by lentiviral transduction were stably expressed in
ameloblasts and that their presence caused interference resulting in specific knockdown
of Psma3 proteins.
Although extracellular proteasomes have been reported in several investigations
(Mueller et al., 2012; Sixt et al., 2007; Sixt and Dahlmann, 2008; Sixt and Peters, 2010),
proteasomes or proteasome subunits have yet to be found in enamel matrix. Inspired by
the finding that exosomes contain proteasome subunits (Buschow et al., 2010; Carayon et
al., 2011; Gonzales et al., 2009; Lai et al., 2012), I performed an experiment using
exosomes to examine extracellular Psma3 in LS8 cell culture. The presence of Psma3 in
exosomes recovered from LS8 cell culture medium implied that Psma3 could be
transported into the extracellular space surrounding ameloblasts via association with
exosomes. Therefore, it is possible that Psma3 interacts with ameloblastin within the
forming enamel matrix to control ameloblastin redistribution.
Taken together, my studies lead to a possible interpretation that ameloblastin-
Psma3 interaction facilitates the redistribution of ameloblastin in the forming enamel
matrix, resulting in segregation of the rod and the interrod regions to form the highly
patterned enamel structure. My findings may help to elucidate the function of
ameloblastin in enamel microstructure formation on the protein-to-protein level.
The goal of this research project was to explore the effects of ameloblastin-Psma3
interactions on enamel microstructure formation in vivo. In future studies, I intend to
knockdown Psma3 in ameloblasts of developing teeth, in vivo by injecting the tooth
bearing jaws of young mouse pups and maintaining the injected tooth in the host animal

87

for several days, allowing formation of enamel under conditions of interfering RNA-
induced reduction of Psma3 protein expression. I am currently engaged in injecting
lentiviral particles carrying the most effective shRNA into newborn mouse pups. The
injection site corresponds to the growth center of the mouse mandibular incisor. The
shRNA will be transferred into ameloblasts by locally injected lentivirus via the
transduction process and will mediate Psma3 knockdown. I will analyze the enamel
microstructure from injected incisors using SEM to ascertain the role of ameloblastin-
Psma3 interaction in enamel hierarchical structure formation.
An expected outcome of in vivo study is that shRNA knockdown of Psma3 in
ameloblasts results in enamel structural defects. Here, I might observe that the formed
enamel crystallites are not organized in a typical rod-interrod pattern. Based on the in
vitro organ culture model, the transduction of lentivirus resulted in the chimeric
knockdown of Psma3 in cultured incisor ameloblasts, in which one cohort of ameloblasts
showed elimination of Psma3, while other cohorts of unaffected ameloblasts are
interspersed between and among the Psma3 knockdown cohort of ameloblasts (Fig. 15h,
i). I assume that this chimeric knockdown pattern of Psma3 expression will also occur in
ameloblasts in vivo. Therefore, incisors from lentivirus injected mouse with transduced
shRNA construct will also show the knock-down phenotype of aprismatic enamel
adjacent to normal enamel.
Proteolytic processing of the enamel matrix proteins is required for proper enamel
formation (Simmer and Hu, 2002). Different proteinases are secreted by ameloblasts into
the enamel matrix to cleave and degrade proteins. One specific matrix protein can be
digested by several different enzymes that may collaborate or compensate to each other.

88

Even though ameloblastin digestion by proteasome members is blocked by disrupting the
ameloblastin-Psma3 interaction, ameloblastin can still be cleaved by other matrix
enzymes, such as matrix metalloproteinase (MMP20), to form the N’-terminal domain
and the C’-terminal domain. Because of the cooperation and compensation of matrix
proteinases during matrix organization, altering the ameloblastin-Psma3 interaction may
not be sufficient to influence the redistribution of ameloblastin domains without
inhibiting these other proteinase pathways. It is possible that knockdown of Psma3 in
vivo will not be associate with a structural imperfection in the formed enamel. However,
the absence of an enamel phenotype is not sufficient to negate the role of the
ameloblastin-Psma3 interactions in patterning enamel matrix. It may be possible to use
proteinase inhibitors in organ culture that affect kallikrein 4 and MMP20 to reduce these
other proteinases so that the impact of the Psma3 silencing is more apparent.
Clarifying the underlying mechanism of enamel architecture formation will
improve the understanding of enamel’s unique structure-function relationship on the
nanoscale, which may contribute to translational applications in enamel regeneration and
enhance dental caries prevention in the future.

89

References
Ahn, K., Erlander, M., Leturcq, D., Peterson, P.A., Fruh, K., and Yang, Y. (1996). In vivo
characterization of the proteasome regulator PA28. The Journal of biological chemistry
271, 18237-18242.
Ausubel, F. (2005). Current Protocols in Molecular Biology (John Wiley and Sons).
Bae, M.H., Jeong, C.H., Kim, S.H., Bae, M.K., Jeong, J.W., Ahn, M.Y., Bae, S.K., Kim,
N.D., Kim, C.W., Kim, K.R., et al. (2002). Regulation of Egr-1 by association with the
proteasome component C8. Biochimica et biophysica acta 1592, 163-167.
Baldassarri, M., Margolis, H.C., and Beniash, E. (2008). Compositional determinants of
mechanical properties of enamel. Journal of dental research 87, 645-649.
Bartlett, J.D., Beniash, E., Lee, D.H., and Smith, C.E. (2004). Decreased mineral content
in MMP-20 null mouse enamel is prominent during the maturation stage. J Dent Res 83,
909-913.
Bartlett, J.D., Ganss, B., Goldberg, M., Moradian-Oldak, J., Paine, M.L., Snead, M.L.,
Wen, X., White, S.N., and Zhou, Y.L. (2006). 3. Protein-protein interactions of the
developing enamel matrix. Current topics in developmental biology 74, 57-115.
Bess, K.L., Swingler, T.E., Rivett, A.J., Gaston, K., and Jayaraman, P.S. (2003). The
transcriptional repressor protein PRH interacts with the proteasome. The Biochemical
journal 374, 667-675.
Beyeler, M., Schild, C., Lutz, R., Chiquet, M., and Trueb, B. (2010). Identification of a
fibronectin interaction site in the extracellular matrix protein ameloblastin. Experimental
cell research 316, 1202-1212.
Bochmann, I., Ebstein, F., Lehmann, A., Wohlschlaeger, J., Sixt, S.U., Kloetzel, P.M.,
and Dahlmann, B. (2014). T lymphocytes export proteasomes by way of microparticles:
a possible mechanism for generation of extracellular proteasomes. Journal of cellular
and molecular medicine 18, 59-68.
Boelens, W.C., Croes, Y., and de Jong, W.W. (2001). Interaction between alphaB-
crystallin and the human 20S proteasomal subunit C8/alpha7. Biochimica et biophysica
acta 1544, 311-319.
Boyde, A. (1979). Carbonate concentration, crystal centers, core dissolution, caries,
cross striations, circadian rhythms, and compositional contrast in the SEM. J Dent Res
58, 981-983.
Boyde, A. (1987). A 3-D model of enamel development at the scale of one inch to the
micron. Advances in dental research 1, 135-140.

90

Boyde, A. (1997). Microstructure of enamel. Ciba Foundation symposium 205, 18-27;
discussion 27-31.
Bureau, J.P., Olink-Coux, M., Brouard, N., Bayle-Julien, S., Huesca, M., Herzberg, M.,
and Scherrer, K. (1997). Characterization of prosomes in human lymphocyte
subpopulations and their presence as surface antigens. Experimental cell research 231,
50-60.
Buschow, S.I., van Balkom, B.W., Aalberts, M., Heck, A.J., Wauben, M., and Stoorvogel,
W. (2010). MHC class II-associated proteins in B-cell exosomes and potential functional
implications for exosome biogenesis. Immunology and cell biology 88, 851-856.
Carayon, K., Chaoui, K., Ronzier, E., Lazar, I., Bertrand-Michel, J., Roques, V., Balor,
S., Terce, F., Lopez, A., Salome, L., et al. (2011). Proteolipidic composition of exosomes
changes during reticulocyte maturation. The Journal of biological chemistry 286, 34426-
34439.
Cerny, R., Slaby, I., Hammarstrom, L., and Wurtz, T. (1996). A novel gene expressed in
rat ameloblasts codes for proteins with cell binding domains. Journal of bone and
mineral research : the official journal of the American Society for Bone and Mineral
Research 11, 883-891.
Chun, Y.H., Lu, Y., Hu, Y., Krebsbach, P.H., Yamada, Y., Hu, J.C., and Simmer, J.P.
(2010a). Transgenic rescue of enamel phenotype in Ambn null mice. Journal of dental
research 89, 1414-1420.
Chun, Y.H., Yamakoshi, Y., Yamakoshi, F., Fukae, M., Hu, J.C., Bartlett, J.D., and
Simmer, J.P. (2010b). Cleavage site specificity of MMP-20 for secretory-stage
ameloblastin. Journal of dental research 89, 785-790.
Couwenhoven, R.I., Luo, W., and Snead, M.L. (1990). Co-localization of EGF transcripts
and peptides by combined immunohistochemistry and in situ hybridization. The journal
of histochemistry and cytochemistry : official journal of the Histochemistry Society 38,
1853-1857.
Coux, O., Tanaka, K., and Goldberg, A.L. (1996). Structure and functions of the 20S and
26S proteasomes. Annual review of biochemistry 65, 801-847.
Daneholt, B. (2006). RNA INTERFERENCE (Nobel prize. org.: "The 2006 Nobel Prize in
Physiology or Medicine - Advanced Information").
Du, C., Falini, G., Fermani, S., Abbott, C., and Moradian-Oldak, J. (2005).
Supramolecular assembly of amelogenin nanospheres into birefringent microribbons.
Science 307, 1450-1454.
Eastoe, J.E. (1979). Enamel protein chemistry--past, present and future. J Dent Res 58,
753-764.

91

Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T.
(2001a). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature 411, 494-498.
Elbashir, S.M., Lendeckel, W., and Tuschl, T. (2001b). RNA interference is mediated by
21- and 22-nucleotide RNAs. Genes & development 15, 188-200.
Elenich, L.A., Nandi, D., Kent, A.E., McCluskey, T.S., Cruz, M., Iyer, M.N., Woodward,
E.C., Conn, C.W., Ochoa, A.L., Ginsburg, D.B., et al. (1999). The complete primary
structure of mouse 20S proteasomes. Immunogenetics 49, 835-842.
Fang, P.A., Conway, J.F., Margolis, H.C., Simmer, J.P., and Beniash, E. (2011).
Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the
nanoscale. Proceedings of the National Academy of Sciences of the United States of
America 108, 14097-14102.
Fields, S., and Song, O. (1989). A novel genetic system to detect protein-protein
interactions. Nature 340, 245-246.
Fincham, A.G., Moradian-Oldak, J., Diekwisch, T.G., Lyaruu, D.M., Wright, J.T., Bringas,
P., Jr., and Slavkin, H.C. (1995). Evidence for amelogenin "nanospheres" as functional
components of secretory-stage enamel matrix. Journal of structural biology 115, 50-59.
Fincham, A.G., Moradian-Oldak, J., and Simmer, J.P. (1999). The structural biology of
the developing dental enamel matrix. J Struct Biol 126, 270-299.
Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998).
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis
elegans. Nature 391, 806-811.
Fong, C.D., Slaby, I., and Hammarstrom, L. (1996). Amelin: an enamel-related protein,
transcribed in the cells of epithelial root sheath. Journal of bone and mineral research :
the official journal of the American Society for Bone and Mineral Research 11, 892-898.
Fong, H., White, S.N., Paine, M.L., Luo, W., Snead, M.L., and Sarikaya, M. (2003).
Enamel structure properties controlled by engineered proteins in transgenic mice.
Journal of bone and mineral research : the official journal of the American Society for
Bone and Mineral Research 18, 2052-2059.
Fukumoto, S., Kiba, T., Hall, B., Iehara, N., Nakamura, T., Longenecker, G., Krebsbach,
P.H., Nanci, A., Kulkarni, A.B., and Yamada, Y. (2004). Ameloblastin is a cell adhesion
molecule required for maintaining the differentiation state of ameloblasts. The Journal of
cell biology 167, 973-983.
Fukumoto, S., and Yamada, Y. (2005). Review: extracellular matrix regulates tooth
morphogenesis. Connective tissue research 46, 220-226.

92

Geng, S., White, S.N., Paine, M.L., and Snead, M.L. (2015). Protein Interaction between
Ameloblastin and Proteasome Subunit alpha Type 3 Can Facilitate Redistribution of
Ameloblastin Domains within Forming Enamel. The Journal of biological chemistry 290,
20661-20673.
Gibson, C.W., Yuan, Z.A., Hall, B., Longenecker, G., Chen, E., Thyagarajan, T.,
Sreenath, T., Wright, J.T., Decker, S., Piddington, R., et al. (2001). Amelogenin-deficient
mice display an amelogenesis imperfecta phenotype. The Journal of biological chemistry
276, 31871-31875.
Goldberg, A.L. (2007). Functions of the proteasome: from protein degradation and
immune surveillance to cancer therapy. Biochemical Society transactions 35, 12-17.
Gonzales, P.A., Pisitkun, T., Hoffert, J.D., Tchapyjnikov, D., Star, R.A., Kleta, R., Wang,
N.S., and Knepper, M.A. (2009). Large-scale proteomics and phosphoproteomics of
urinary exosomes. Journal of the American Society of Nephrology : JASN 20, 363-379.
Gou, D., Weng, T., Wang, Y., Wang, Z., Zhang, H., Gao, L., Chen, Z., Wang, P., and
Liu, L. (2007). A novel approach for the construction of multiple shRNA expression
vectors. The journal of gene medicine 9, 751-763.
Graham, F.L., Smiley, J., Russell, W.C., and Nairn, R. (1977). Characteristics of a
human cell line transformed by DNA from human adenovirus type 5. The Journal of
general virology 36, 59-74.
Hassan, R., Caputo, A.A., and Bunshah, R.F. (1981). Fracture toughness of human
enamel. Journal of dental research 60, 820-827.
Henry, L., Baz, A., Chateau, M.T., Scherrer, K., and Bureau, J.P. (1996). Changes in the
amount and distribution of prosomal subunits during the differentiation of U937 myeloid
cells: high expression of p23K. Cell proliferation 29, 589-607.
Hideshima, T., Richardson, P., Chauhan, D., Palombella, V.J., Elliott, P.J., Adams, J.,
and Anderson, K.C. (2001). The proteasome inhibitor PS-341 inhibits growth, induces
apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer
research 61, 3071-3076.
Hori, H., Nembai, T., Miyata, Y., Hayashi, T., Ueno, K., and Koide, T. (1999). Isolation
and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver
microsomes. Journal of biochemistry 126, 722-730.
Hu, C.C., Fukae, M., Uchida, T., Qian, Q., Zhang, C.H., Ryu, O.H., Tanabe, T.,
Yamakoshi, Y., Murakami, C., Dohi, N., et al. (1997). Sheathlin: cloning,
cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath
proteins. Journal of dental research 76, 648-657.

93

Hu, J.C., and Yamakoshi, Y. (2003). Enamelin and autosomal-dominant amelogenesis
imperfecta. Critical reviews in oral biology and medicine : an official publication of the
American Association of Oral Biologists 14, 387-398.
Huang, Z., Newcomb, C.J., Bringas, P., Jr., Stupp, S.I., and Snead, M.L. (2010).
Biological synthesis of tooth enamel instructed by an artificial matrix. Biomaterials 31,
9202-9211.
Huang, Z., Sargeant, T.D., Hulvat, J.F., Mata, A., Bringas, P., Jr., Koh, C.Y., Stupp, S.I.,
and Snead, M.L. (2008). Bioactive nanofibers instruct cells to proliferate and differentiate
during enamel regeneration. Journal of bone and mineral research : the official journal of
the American Society for Bone and Mineral Research 23, 1995-2006.
Imbeni, V., Kruzic, J.J., Marshall, G.W., Marshall, S.J., and Ritchie, R.O. (2005). The
dentin-enamel junction and the fracture of human teeth. Nature materials 4, 229-232.
Iwasaki, K., Bajenova, E., Somogyi-Ganss, E., Miller, M., Nguyen, V., Nourkeyhani, H.,
Gao, Y., Wendel, M., and Ganss, B. (2005). Amelotin--a Novel Secreted, Ameloblast-
specific Protein. J Dent Res 84, 1127-1132.
Iwata, T., Yamakoshi, Y., Hu, J.C., Ishikawa, I., Bartlett, J.D., Krebsbach, P.H., and
Simmer, J.P. (2007). Processing of ameloblastin by MMP-20. Journal of dental research
86, 153-157.
Johnstone, R.M. (2006). Exosomes biological significance: A concise review. Blood
cells, molecules & diseases 36, 315-321.
Keller, S., Sanderson, M.P., Stoeck, A., and Altevogt, P. (2006). Exosomes: from
biogenesis and secretion to biological function. Immunology letters 107, 102-108.
Khan, M.T., and Joseph, S.K. (2001). Characterization of membrane-associated
proteasomes in WB rat liver epithelial cells. Archives of biochemistry and biophysics
385, 99-107.
Klare, N., Seeger, M., Janek, K., Jungblut, P.R., and Dahlmann, B. (2007). Intermediate-
type 20 S proteasomes in HeLa cells: "asymmetric" subunit composition, diversity and
adaptation. Journal of molecular biology 373, 1-10.
Kobayashi, K., Yamakoshi, Y., Hu, J.C.C., Gomi, K., Arai, T., Fukae, M., Krebsbach,
P.H., and Simmer, J.P. (2007). Splicing Determines the Glycosylation State of
Ameloblastin. Journal of dental research 86, 962-967.
Krebsbach, P.H., Lee, S.K., Matsuki, Y., Kozak, C.A., Yamada, K.M., and Yamada, Y.
(1996). Full-length sequence, localization, and chromosomal mapping of ameloblastin. A
novel tooth-specific gene. The Journal of biological chemistry 271, 4431-4435.

94

Lacruz, R.S., Hacia, J.G., Bromage, T.G., Boyde, A., Lei, Y., Xu, Y., Miller, J.D., Paine,
M.L., and Snead, M.L. (2012). The circadian clock modulates enamel development.
Journal of biological rhythms 27, 237-245.
Lacruz, R.S., Smith, C.E., Chen, Y.B., Hubbard, M.J., Hacia, J.G., and Paine, M.L.
(2011). Gene-expression analysis of early- and late-maturation-stage rat enamel organ.
Eur J Oral Sci 119 Suppl 1, 149-157.
Lacruz, R.S., Smith, C.E., Kurtz, I., Hubbard, M.J., and Paine, M.L. (2013). New
paradigms on the transport functions of maturation-stage ameloblasts. J Dent Res 92,
122-129.
Lai, R.C., Tan, S.S., Teh, B.J., Sze, S.K., Arslan, F., de Kleijn, D.P., Choo, A., and Lim,
S.K. (2012). Proteolytic Potential of the MSC Exosome Proteome: Implications for an
Exosome-Mediated Delivery of Therapeutic Proteasome. International journal of
proteomics 2012, 971907.
Landy, A. (1989). Dynamic, structural, and regulatory aspects of lambda site-specific
recombination. Annual review of biochemistry 58, 913-949.
Lee, S.K., Kim, S.M., Lee, Y.J., Yamada, K.M., Yamada, Y., and Chi, J.G. (2003). The
structure of the rat ameloblastin gene and its expression in amelogenesis. Molecules
and cells 15, 216-225.
Li, B., and Fields, S. (1993). Identification of mutations in p53 that affect its binding to
SV40 large T antigen by using the yeast two-hybrid system. FASEB journal : official
publication of the Federation of American Societies for Experimental Biology 7, 957-963.
Li, X.B., Zhang, Z.R., Schluesener, H.J., and Xu, S.Q. (2006). Role of exosomes in
immune regulation. Journal of cellular and molecular medicine 10, 364-375.
McAnuff, M.A., Rettig, G.R., and Rice, K.G. (2007). Potency of siRNA versus shRNA
mediated knockdown in vivo. Journal of pharmaceutical sciences 96, 2922-2930.
Moffatt, P., Smith, C.E., St-Arnaud, R., Simmons, D., Wright, J.T., and Nanci, A. (2006).
Cloning of rat amelotin and localization of the protein to the basal lamina of maturation
stage ameloblasts and junctional epithelium. Biochem J 399, 37-46.
Montgomery, M.K., Xu, S., and Fire, A. (1998). RNA as a target of double-stranded
RNA-mediated genetic interference in Caenorhabditis elegans. Proceedings of the
National Academy of Sciences of the United States of America 95, 15502-15507.
Moradian-Oldak, J., and Goldberg, M. (2005). Amelogenin supra-molecular assembly in
vitro compared with the architecture of the forming enamel matrix. Cells, tissues, organs
181, 202-218.
Moradian-Oldak, J., Paine, M.L., Lei, Y.P., Fincham, A.G., and Snead, M.L. (2000). Self-
assembly properties of recombinant engineered amelogenin proteins analyzed by

95

dynamic light scattering and atomic force microscopy. Journal of structural biology 131,
27-37.
Morales, P., Kong, M., Pizarro, E., and Pasten, C. (2003). Participation of the sperm
proteasome in human fertilization. Human reproduction 18, 1010-1017.
Mueller, O., Anlasik, T., Wiedemann, J., Thomassen, J., Wohlschlaeger, J., Hagel, V.,
Keyvani, K., Schwieger, I., Dahlmann, B., Sure, U., et al. (2012). Circulating extracellular
proteasome in the cerebrospinal fluid: a study on concentration and proteolytic activity.
Journal of molecular neuroscience : MN 46, 509-515.
Murakami, C., Dohi, N., Fukae, M., Tanabe, T., Yamakoshi, Y., Wakida, K., Satoda, T.,
Takahashi, O., Shimizu, M., Ryu, O.H., et al. (1997). Immunochemical and
immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related
proteins in the porcine tooth germ. Histochemistry and cell biology 107, 485-494.
Nakagawa, T., Shirane, M., Iemura, S., Natsume, T., and Nakayama, K.I. (2007).
Anchoring of the 26S proteasome to the organellar membrane by FKBP38. Genes to
cells : devoted to molecular & cellular mechanisms 12, 709-719.
Nanci, A., Zalzal, S., Lavoie, P., Kunikata, M., Chen, W., Krebsbach, P.H., Yamada, Y.,
Hammarstrom, L., Simmer, J.P., Fincham, A.G., et al. (1998). Comparative
immunochemical analyses of the developmental expression and distribution of
ameloblastin and amelogenin in rat incisors. The journal of histochemistry and
cytochemistry : official journal of the Histochemistry Society 46, 911-934.
Paine, C.T., Paine, M.L., and Snead, M.L. (1998). Identification of tuftelin- and
amelogenin-interacting proteins using the yeast two-hybrid system. Connective tissue
research 38, 257-267;discussion 295-303.
Paine, M.L., Lei, Y.P., Dickerson, K., and Snead, M.L. (2002). Altered amelogenin self-
assembly based on mutations observed in human X-linked amelogenesis imperfecta
(AIH1). The Journal of biological chemistry 277, 17112-17116.
Paine, M.L., and Snead, M.L. (1997). Protein interactions during assembly of the enamel
organic extracellular matrix. Journal of bone and mineral research : the official journal of
the American Society for Bone and Mineral Research 12, 221-227.
Paine, M.L., Snead, M.L., Wang, H.J., Abuladze, N., Pushkin, A., Liu, W., Kao, L.Y.,
Wall, S.M., Kim, Y.H., and Kurtz, I. (2008). Role of NBCe1 and AE2 in secretory
ameloblasts. J Dent Res 87, 391-395.
Paine, M.L., Wang, H.J., Luo, W., Krebsbach, P.H., and Snead, M.L. (2003). A
transgenic animal model resembling amelogenesis imperfecta related to ameloblastin
overexpression. The Journal of biological chemistry 278, 19447-19452.

96

Paine, M.L., White, S.N., Luo, W., Fong, H., Sarikaya, M., and Snead, M.L. (2001).
Regulated gene expression dictates enamel structure and tooth function. Matrix biology :
journal of the International Society for Matrix Biology 20, 273-292.
Paine, M.L., Zhu, D.H., Luo, W., Bringas, P., Jr., Goldberg, M., White, S.N., Lei, Y.P.,
Sarikaya, M., Fong, H.K., and Snead, M.L. (2000). Enamel biomineralization defects
result from alterations to amelogenin self-assembly. Journal of structural biology 132,
191-200.
Peters, J.M., Franke, W.W., and Kleinschmidt, J.A. (1994). Distinct 19 S and 20 S
subcomplexes of the 26 S proteasome and their distribution in the nucleus and the
cytoplasm. The Journal of biological chemistry 269, 7709-7718.
Rao, D.D., Vorhies, J.S., Senzer, N., and Nemunaitis, J. (2009). siRNA vs. shRNA:
similarities and differences. Advanced drug delivery reviews 61, 746-759.
Raposo, G., and Stoorvogel, W. (2013). Extracellular vesicles: exosomes, microvesicles,
and friends. The Journal of cell biology 200, 373-383.
Ravindranath, R.M., Moradian-Oldak, J., and Fincham, A.G. (1999). Tyrosyl motif in
amelogenins binds N-acetyl-D-glucosamine. The Journal of biological chemistry 274,
2464-2471.
Rivett, A.J., Palmer, A., and Knecht, E. (1992). Electron microscopic localization of the
multicatalytic proteinase complex in rat liver and in cultured cells. The journal of
histochemistry and cytochemistry : official journal of the Histochemistry Society 40,
1165-1172.
Robinson, C., Briggs, H.D., and Atkinson, P.J. (1981). Histology of enamel organ and
chemical composition of adjacent enamel in rat incisors. Calcified tissue international 33,
513-520.
Shi, Z., Li, Z., Li, Z.J., Cheng, K., Du, Y., Fu, H., and Khuri, F.R. (2014). Cables1
controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.
Oncogene 0.
Shu, F., Guo, S., Dang, Y., Qi, M., Zhou, G., Guo, Z., Zhang, Y., Wu, C., Zhao, S., and
Yu, L. (2003). Human aurora-B binds to a proteasome alpha-subunit HC8 and
undergoes degradation in a proteasome-dependent manner. Molecular and cellular
biochemistry 254, 157-162.
Simmer, J.P., and Fincham, A.G. (1995). Molecular mechanisms of dental enamel
formation. Crit Rev Oral Biol Med 6, 84-108.
Simmer, J.P., and Hu, J.C. (2002). Expression, structure, and function of enamel
proteinases. Connective tissue research 43, 441-449.

97

Simmer, J.P., Papagerakis, P., Smith, C.E., Fisher, D.C., Rountrey, A.N., Zheng, L., and
Hu, J.C. (2010). Regulation of dental enamel shape and hardness. Journal of dental
research 89, 1024-1038.
Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P.S., Paddison, P.J., Hannon, G.J.,
and Cleary, M.A. (2005). Synthetic shRNAs as potent RNAi triggers. Nature
biotechnology 23, 227-231.
Sixt, S.U., Beiderlinden, M., Jennissen, H.P., and Peters, J. (2007). Extracellular
proteasome in the human alveolar space: a new housekeeping enzyme? American
journal of physiology Lung cellular and molecular physiology 292, L1280-1288.
Sixt, S.U., and Dahlmann, B. (2008). Extracellular, circulating proteasomes and ubiquitin
- incidence and relevance. Biochimica et biophysica acta 1782, 817-823.
Sixt, S.U., and Peters, J. (2010). Extracellular alveolar proteasome: possible role in lung
injury and repair. Proceedings of the American Thoracic Society 7, 91-96.
Smith, C.E. (1998). Cellular and chemical events during enamel maturation. Crit Rev
Oral Biol Med 9, 128-161.
Smith, C.E., Chen, W.Y., Issid, M., and Fazel, A. (1995). Enamel matrix protein turnover
during amelogenesis: basic biochemical properties of short-lived sulfated enamel
proteins. Calcif Tissue Int 57, 133-144.
Smith, C.E., Chong, D.L., Bartlett, J.D., and Margolis, H.C. (2005). Mineral acquisition
rates in developing enamel on maxillary and mandibular incisors of rats and mice:
implications to extracellular acid loading as apatite crystals mature. Journal of bone and
mineral research : the official journal of the American Society for Bone and Mineral
Research 20, 240-249.
Smith, C.E., and Nanci, A. (1995). Overview of morphological changes in enamel organ
cells associated with major events in amelogenesis. The International journal of
developmental biology 39, 153-161.
Smith, C.E., Wazen, R., Hu, Y., Zalzal, S.F., Nanci, A., Simmer, J.P., and Hu, J.C.
(2009). Consequences for enamel development and mineralization resulting from loss of
function of ameloblastin or enamelin. European journal of oral sciences 117, 485-497.
Snead, M.L. (1996). Enamel biology logodaedaly: getting to the root of the problem, or
"who's on first...". Journal of bone and mineral research : the official journal of the
American Society for Bone and Mineral Research 11, 899-904.
Snead, M.L. (2015). Biomineralization of a self-assembled-, soft-matrix precursor:
Enamel. Jom 67, 788-795.

98

Suraweera, A., Munch, C., Hanssum, A., and Bertolotti, A. (2012). Failure of amino acid
homeostasis causes cell death following proteasome inhibition. Molecular cell 48, 242-
253.
Termine, J.D., Torchia, D.A., and Conn, K.M. (1979). Enamel matrix: structural proteins.
J Dent Res 58, 773-781.
Tomes, J. (1849). On the structure of the dental tissues of the order Rodentia. Phil Trans
140, 529-567.
Uchida, T., Murakami, C., Wakida, K., Satoda, T., Dohi, N., and Takahashi, O. (1997).
Synthesis, Secretion, Degradation, and Fate of Ameloblastin During the Matrix
Formation Stage of the Rat Incisor as Shown by Immunocytochemistry and
Immunochemistry Using Region-specific Antibodies. Journal of Histochemistry &
Cytochemistry 45, 1329-1340.
Uchida, T., Tanabe, T., Fukae, M., Shimizu, M., Yamada, M., Miake, K., and Kobayashi,
S. (1991). Immunochemical and immunohistochemical studies, using antisera against
porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on
immature enamel of the pig and rat. Histochemistry 96, 129-138.
Vlassov, A.V., Korba, B., Farrar, K., Mukerjee, S., Seyhan, A.A., Ilves, H., Kaspar, R.L.,
Leake, D., Kazakov, S.A., and Johnston, B.H. (2007). shRNAs targeting hepatitis C:
effects of sequence and structural features, and comparision with siRNA.
Oligonucleotides 17, 223-236.
Vymetal, J., Slaby, I., Spahr, A., Vondrasek, J., and Lyngstadaas, S.P. (2008).
Bioinformatic analysis and molecular modelling of human ameloblastin suggest a two-
domain intrinsically unstructured calcium-binding protein. European journal of oral
sciences 116, 124-134.
Wald, T., Bednarova, L., Osicka, R., Pachl, P., Sulc, M., Lyngstadaas, S.P., Slaby, I.,
and Vondrasek, J. (2011). Biophysical characterization of recombinant human
ameloblastin. European journal of oral sciences 119 Suppl 1, 261-269.
Wald, T., Osickova, A., Sulc, M., Benada, O., Semeradtova, A., Rezabkova, L., Veverka,
V., Bednarova, L., Maly, J., Macek, P., et al. (2013). Intrinsically disordered enamel
matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal
segment encoded by exon 5. The Journal of biological chemistry 288, 22333-22345.
Wang, H., Tannukit, S., Zhu, D., Snead, M.L., and Paine, M.L. (2005). Enamel matrix
protein interactions. Journal of bone and mineral research : the official journal of the
American Society for Bone and Mineral Research 20, 1032-1040.
Warshawsky, H., and Smith, C.E. (1971). A three-dimensional reconstruction of the rods
in rat maxillary incisor enamel. Anat Rec 169, 585-591.

99

Wazen, R.M., Moffatt, P., Zalzal, S.F., Yamada, Y., and Nanci, A. (2009). A mouse
model expressing a truncated form of ameloblastin exhibits dental and junctional
epithelium defects. Matrix biology : journal of the International Society for Matrix Biology
28, 292-303.
White, S.N., Luo, W., Paine, M.L., Fong, H., Sarikaya, M., and Snead, M.L. (2001).
Biological organization of hydroxyapatite crystallites into a fibrous continuum toughens
and controls anisotropy in human enamel. Journal of dental research 80, 321-326.
White, S.N., Miklus, V.G., Chang, P.P., Caputo, A.A., Fong, H., Sarikaya, M., Luo, W.,
Paine, M.L., and Snead, M.L. (2005). Controlled failure mechanisms toughen the
dentino-enamel junction zone. The Journal of prosthetic dentistry 94, 330-335.
Xu, H.H., Smith, D.T., Jahanmir, S., Romberg, E., Kelly, J.R., Thompson, V.P., and
Rekow, E.D. (1998). Indentation damage and mechanical properties of human enamel
and dentin. Journal of dental research 77, 472-480.
Xu, X.M., Yoo, M.H., Carlson, B.A., Gladyshev, V.N., and Hatfield, D.L. (2009).
Simultaneous knockdown of the expression of two genes using multiple shRNAs and
subsequent knock-in of their expression. Nature protocols 4, 1338-1348.
Yamakoshi, Y., Tanabe, T., Oida, S., Hu, C.C., Simmer, J.P., and Fukae, M. (2001).
Calcium binding of enamel proteins and their derivatives with emphasis on the calcium-
binding domain of porcine sheathlin. Archives of oral biology 46, 1005-1014.
Zhou, Y.L., Lei, Y., and Snead, M.L. (2000). Functional antagonism between Msx2 and
CCAAT/enhancer-binding protein alpha in regulating the mouse amelogenin gene
expression is mediated by protein-protein interaction. The Journal of biological chemistry
275, 29066-29075.
Zhou, Y.L., and Snead, M.L. (2000). Identification of CCAAT/enhancer-binding protein
alpha as a transactivator of the mouse amelogenin gene. The Journal of biological
chemistry 275, 12273-12280.
Zhu, D., Paine, M.L., Luo, W., Bringas, P., Jr., and Snead, M.L. (2006). Altering
biomineralization by protein design. The Journal of biological chemistry 281, 21173-
21182.

Abstract (if available)

Abstract Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Amelogenin-ameloblastin protein interaction and function in dental enamel formation

PDF

Integrative analysis of gene expression and phenotype data

PDF

Chitosan‐amelogenin hydrogel‐saliva interactions: towards optimization of a protocol for enamel repair

PDF

Role of MMP-20 in preventing protein occlusion in enamel apatite crystals: relevance in enamel biomineralization and biomimetics

PDF

Global analysis of the molecular activities defining maturation-stage amelogenesis

PDF

Transcellular calcium transport in amelogenesis

PDF

Biomaterial (Col-Tgel)-induced multinucleated cells formation, polarization, and functions

PDF

Remineralization of deminrealized dentin by amelogenin peptide P26

PDF

Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells

PDF

The function of TGF-beta signaling in palatal and dental epithelium during embryogenesis

PDF

Enamelysin (MMP20) and Kallikrein 4 (KLK4) functions during enamel formation

PDF

Transcriptional co-activation functions of Msx homeodomain proteins by activating Hsf proteins

PDF

Amelogenin peptides for biomimetic remineralization of enamel and dentin

PDF

A Western blot analysis and immunohistochemical study of 4-day old mice teeth over-expressing amelotin during amelogenesis

PDF

The effect of leucine-rich amelogenin peptide on mouse bone density by histological analysis

PDF

Dynamics of amelogenin self-assembly during in vitro proteolysis by Mmp-20

PDF

Determination of mineral density of remineralized enamel and dentin: a QLF study

PDF

Influence of enamel biomineralization on bonding to minimally invasive CAD/CAM restorations

PDF

The role of parathyroid hormone-related protein in regulating neonatal lung development

PDF

Regulation of telomerase gene expression and function in epithelial differentiation and tumorigenesis

Asset Metadata

Creator Geng, Shuhui (author)

Core Title Ameloblastin-protein interactions pattern enamel matrix

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 10/30/2015

Defense Date 10/19/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ameloblastin,enamel matrix patterning,enamel rod-to-interrod organization,OAI-PMH Harvest,proteasome subunit alpha type 3,protein interaction

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Snead, Malcolm L. (committee chair), Frenkel, Baruch (committee member), Moradian-Oldak, Janet (committee member), Paine, Michael L. (committee member)

Creator Email boogeng@gmail.com,sgeng@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-193331

Unique identifier UC11276844

Identifier etd-GengShuhui-3996.pdf (filename),usctheses-c40-193331 (legacy record id)

Legacy Identifier etd-GengShuhui-3996.pdf

Dmrecord 193331

Document Type Dissertation

Format application/pdf (imt)

Rights Geng, Shuhui

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA