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Clathrin associated protein (AP) binding motifs in AD5 penton

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Clathrin associated protein (AP) binding motifs in AD5 penton

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Content CLATHRIN ASSOCIATED PROTEIN (AP)
BINDING MOTIFS IN ADS PENTON
by
Meghan Kathleen Cirivello
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2003
Copyright 2003 Meghan Kathleen Cirivello
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UMI Number: 1420362
Copyright 2003 by
Cirivello, Meghan Kathleen
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90089-1695
This thesis, written by
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under the direction o f ha r thesis committee, and
approved by all its members, has been presented to and
accepted by the Director o f Graduate and Professional
Programs, in partial fulfillment of the requirements fo r the
degree o f
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Date December 1 7 , 2003
Thesis Committe
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Chair
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Meghan Kathleen Cirivello Laurence H. Kedes
ABSTRACT
CLATHRIN ASSOCIATED PROTEIN (AP)
BINDING MOTIFS IN ADENOVIRUS5 PENTON
Phage display biopanning has identified mutual binding domains
between the fiber and penton, two adenovirus coat proteins. These
domains fit the consensus sequences of sorting signals typically found
on membrane-associated proteins that translocate from the plasma
membrane to endosomes. This study investigates whether the di-leucine
motif serves as a sorting signal within the penton, thus indicating a role
in capsid trafficking. Mutations were made in the wild type penton (PB)
di-leucine motif sequence and investigated to determine their affect on
uptake and trafficking of the protein. Additionally, the affect of PB
mutations on de novo synthesis was studied. The wild-type and mutant
pentons localized to the nucleus after expression. However, experiments
on PB mutant uptake and trafficking revealed that each mutant was
differentially taken up by HeLa cells.
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Dedication
This is for my husband, Jim, for all his love, support, ideas,
time, and lots of patience.
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Acknowledgements
iii
I’d like to thank Lali Medina-Kauwe for all her help, support, encouragement, and
inspiration. Thank you to Michelle MacVeigh for obtaining confocal micrographs
and to the Sarah Hamm-Alvarez lab for assistance with immunostaining procedures
and supplies. I am, also, thankful to the following people for ongoing support and
discussions: Serge Abdishoo, Xinhua Chen, Gene Chung, Norri Kasahara, Larry
Kedes, Terry Saluna, Toshiaki Shichinohe, and Zolten Tokes. A training grant to
Meghan Cirivello from the National Institute o f Health (T32 NCI 9569) supported
this work.
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Table of Contents
1. Dedication ii
2. Acknowledgements iii
3. List of Tables v
4. List of Figures vi
5. Abstract vii
6. Introduction 1
7. Results 4
8. Discussion 16
9. Experimental Procedures 18
10. Alphabetized Bibliography I
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List of Tables
Table 1: Putative Binding and Trafficking Mutants
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vi
List of Figures
Figure 1: Fiber and Penton Proteins Mediate Cell Binding 1
And Entry o f Ad
Figure 2: Effect of Fiber Tail (F20) on 3PO Gene Transfer to HeLa 3
Figure 3: The Fiber Tail Conceals a Putative Di-Leucine (LL) M otif 4
Figure 4: Pentamerization of Penton Mutants 6
Figure 5: Ad-GFP Competition with Wild-Type PB and Mutants 7
Figure 6: Binding Studies Using Ad-GFP 9
Figure 7: Binding and Internalization o f Di-Leucine M otif Mutants 11
Figure 8: Wild-Type and Mutant Penton Scoring 12
Figure 9: Perinuclear vs. Cytoplasmic Localization 13
Figure 10: Intracellular Localization o f Penton Mutants in HeLa 14
Figure 11: Detection of GFP-PB S253D in HeLa Cells 15
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Abstract
vii
Phage display biopanning has identified mutual binding domains between the
fiber and penton, two adenovirus coat proteins. These domains fit the consensus
sequences o f sorting signals typically found on membrane-associated proteins that
translocate from the plasma membrane to endosomes. This study investigates
whether the di-leucine m otif serves as a sorting signal within the penton, thus
indicating a role in capsid trafficking. Mutations were made in the wild type penton
(PB) di-leucine m otif sequence and investigated to determine their affect on uptake
and trafficking of the protein. Additionally, the affect of PB mutations on de novo
synthesis was studied. The wild-type and mutant proteins localized to the nucleus
after expression. However, experiments on PB mutant uptake and trafficking
revealed that each mutant was differentially taken up by HeLa cells.
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Introduction
Adenovirus’ (Ad) entry into the host cell occurs through receptor-mediated
endocytosis (Fitzgerald et al„, 1983). A subgroup of Ad, including the two most
characterized viruses Ad2 and Ad5, requires two receptors for entry into the cell.
Initial attachment to the primary receptor takes place at a coxsackie-adenovirus
receptor (CAR) (Nakano et ah, 2000), while the secondary receptors, a vps or a vP3
integrins, promote internalization (Wickham et ah, 1993). Interaction with these
receptors involves viral proteins projecting from each vertex of the icosahedral
particle (See figure 1).
Figure 1: Fiber and Penton Proteins Mediate
Cell Binding and Entry of Ad
. — (Cell
membrane)
Integrin
3. M em brane
penetration (Penton)
O ' -
(Cell
1 nucleus)
2. Cell entry
(Penton)
CAR
K
1. Cell surface
binding (Fiber)
PENTON :
BASE i
ADENOVIRUS
TYPE 5 (AdS)
(ENLARGED)
Ad initially binds to the cell surface through the interaction o f the fiber protein and CAR. This leads
to the penton protein binding to integrins on the cell surface, followed by entry into the cell cytosol.
Once in the cytosol the viron must exit the membrane vesicle, then make its way to the cell nucleus.
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2
Each projection is made up of a penton base and a fiber molecule. The
penton base consists of a 400 kD coat protein, noncovalently associated with the
fiber, forming a heterodimeric (pentamer) complex (Stewart et al., 1991; Boudin and
Boulanger, 1982). The Ad5 fiber is a trimeric protein projecting from the penton-
base with three domains: the fiber shaft, the tail and the knob. The tail binds to the
penton-base, while the shaft extends from it with a repeating m otif of approximately
15 residues (Green et. a l, 1983). The knob tops the shaft and has been show to be
essential and sufficient for binding of the Ad virus to the host cell (Kirby et al,
1999).
Viral entry into the host cell begins when the Ad 5 fiber binds to CAR,
attaching Ad5 to the cell as represented in figure 1 (Green et. al., 1983). This is
followed by binding of the penton through an Arg-Gly-Asp (RGD) peptide sequence
to a v integrins on the cell surface initiating endocytosis (Wickham et. al., 1993). The
virus enters the cell in an endosome from which the virus particle is released,
allowing the viral DNA to enter the nucleus. The penton appears to facilitate this
process (Seth et. al., 1984; Karayan et. al., 1997).
The role o f the penton in the entry o f Ad5 into the cell makes it an excellent
candidate for a non-viral gene delivery system. Engineered Ad5 penton proteins
with an added lysine tail (PBK10) have been shown to enable delivery of a GFP
reporter gene to HeLa cells (Medina-Kauwe et al., 2001). During further study of
the penton gene delivery system, the full-length fiber was added to the penton
complex in an attempt to enhance gene delivery (Medina-Kauwe et al., 2002). The
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3
addition of the fiber tail alone, however, served to inhibit gene transfer in a
luciferase reporter assay (See figure 2). Leading to the question: Which sequence of
penton does the fiber tail conceal, blocking penton’s ability to internalize?
Figure 2: Effect ofFiber Tail (F20) on 3PO
Gene Transfer to HeLa
f l H M
35.00
001
1
25.00
20,000
15,000*
10.00
5,000
0.2
5,000
Protamine
PBK10 (mg)
( - fiber tail)
2.5 3
PBKlO(mg)
( + fiber tail)
A luciferase reporter gene was condensed using varying amounts o f protamine. The condensed
plasmid was then combined with different amounts o f PBK10 alone (3PO) or with PK10 and equal
amounts o f fiber tail. The fiber tail bound to 3PO inhibits gene transfer.
Phage display biopanning has identified mutual binding domains between the
fiber and penton. These proteins overlap at a possible di-leucine motif involving
residues 254-260 in the penton. (Hong and Boulanger, 1995) These motifs are
recognized sorting signals, typically found on membrane-associated proteins that
translocate from the plasma membrane to the endosomes. The di-leucine motif
directly binds clathrin associated protein (AP) complexes, which recognize sorting
signals within the protein to recruit them to clathrin coated pits and vesicles.
(Kirchhausen et al., 1999, Heilker et al., 1999) Putative di-leucine motifs consist of
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a serine regulated by phosphorylation, followed by 2-4 amino acids (See figure 3).
The next position Is usually polar, followed by two leucines.
Figure 3: The Fiber Tail Conceals a Putative Di-Leucine (LL) Motif
H^SRLSNLL
- (-) = D, E, or
Phospho-Ser
- (Ser) = reg. by
phosphorylation
(2-4) (LL)
Usually
polar
This shows the amino acid sequence o f the putative di-leucine motif identified within the penton
sequence. Characteristics o f typical di-leucine motifs are identified. Di-leucine motifs act as sorting
signals that directly binds clathrin associated protein (AP) complexes. APs recognize sorting signals o f
proteins and are recruited to clathrin coated pits and vesicles.
This study investigates whether the di-leucine m otif serves as a sorting signal
in the penton, thus indicating a role in capsid trafficking. Elucidating the trafficking
of the penton protein separate from the intact vims will aid in development of the
penton protein as a gene delivery system. To answer this question, mutations were
made in the wild-type penton’s di-leucine sequence and investigated to determine
their affect on trafficking of the protein. Furthermore, the affect of the mutations on
de novo synthesis of the penton mutants was studied.
Results
Design of Di-Leucine and Integrin Binding Mutants
The di-leucine motif has key amino acids involved in its regulation. To
determine the role of the di-leucine motif in penton binding and trafficking a series
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5
of mutant proteins were designed. Serine 253 can be regulated by phosphorylation
(See table 1), thus activating or deactivating the motif. The first mutations are
Table 1 : Putative Binding & Trafficking Mutants
NAME OF SITE OF PUTATIVE
MUTANT MUTATION FUNCTION
PBrgd 3 4 0 RGD av integrin binding
S253A 2 5 3 SRLSNLLG AP complex binding
R254A 2 5 3 SRLSNLLG AP complex binding
SR/AA 2 5 3 SRLSNLLG AP complex binding
LL/AA 2 5 3 SRLSNLLG AP complex binding
S253D 2 5 3 SRLSNLLG AP complex binding
directed at this amino acid to form an inactive di-leucine motif. The serine was
mutated first to an alanine, removing m otifs ability to be regulated by
phosphorylation and decreasing the size of the amino acid. The second mutation
made to serine changes the serine to an aspartic acid which will cause the motif to be
constitutively active. The hydrophobic nature of the di-leucines gives these amino
acids the tendency to be in the hydrophobic core of a protein, rather than exposed to
the solvent. Charged residues surrounding the di-leucines help to keep theses amino
acids exposed. To further investigate the role of the charged residues, the arginine
was mutated to an alanine and a double mutant with the serine and arginine both
mutated to alanines was made. The final mutation made was to the di-leucines,
changing them both to alanines. To further aid in the study of binding effects the
penton’s intgrin binding sequence (RGD) was deleted for use as a negative control.
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6
Pentamerization
Each penton base consists of five interacting penton subunits (Neumann et
al, 1988). In order to check that the mutations made had no affect on the penton’s
interaction with itself, a non-denaturing gel (ND-PAGE) was run. It has been s h o w
elsewhere that recombinant penton pentamirization is detectable by ND-PAGE
(Karayan et ah, 1997). As shown in figure 4, all the di-leucine mutants were able to
form pentamers like the wild type penton. This shows that the di-leucine motif does
Figure 4: Pentamerization of Penton Muntants
A. Non-denaturing-PAGE of Wild-type and Mutant Pentons
kD M 1 2 3 4 5
Pentam ers
61 a M onomers
M-protein ladder
1-wild-type penton (PB)
2-S253A
173 3-R254A
4-SR/AA
111 A l f , y > ■ 5-S253D
80 • • . k
6-LL/AA
B. Western Blot H ljjlj ^
LL/AA S253A SR/AA R254A S253D PB PBrgd
Recombinant proteins were analyzed for (a) pentameriazation and (b) fall length protein levels,
(a) 5 pg o f each protein was used, (b) The western blot was performed using Ad5 antibodies.
not interfere with pentamer formation, allowing the penton mutants to structurally
function like the wild type penton. Previously, cell attachment was shown to be
possible for pentamerization-defective mutants, indicating that the RGD sequence
was not involved in pentamer formation (Hong et al., 1999).
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7
Binding and Trafficking of Di-Leucine Motifs
To study binding of the mutant pentons to the a v integrins a competition
assay using Ad5-GFP was carried out. For the first attempt 20 fig, 2 fig, and 0.2 fig
of each protein was applied to HeLa cells in a 100 pi volume. Cells were incubated
1 h at 37°C. Then, vims at MOI (multiplicity of infection) 5 was added in 0.9 ml
media. Cells underwent FACS analysis as described in experimental procedures.
Infection levels only reached 54% of cells for Ad5-GFP vims alone (See figure 5A).
All proteins either had increased the level of infection.
A.
Figure 5: A d-G FP Competition With Wild-Type PB and Mutants
B. Ad-GFP Competition with PB Proteins
< D
O
-A
0s -
Ad-GFP MOI 5 Competition with PB Proteins
s o -
A v—
- ■
/ ■
f .." ' J '
ifcC _________
0 0.2 2 20
ug Proteins
PB v PBrgd
PB S253A ...PB R254A
-s e -P B SR/AA -■*- PB S253D
~n— PBLL/AA
cri-
2 1
19
17
15
13
9
7
20 0 0.2 2
ug Proteins
— PB : PBrgd
..PB S253A • • • • » • • • • PBR254A
- * - P B SR/AA PB S253D
----- PBLUAA
A) HeLa cells were plated 24 h prior to treatment. Indicated protein amounts were applied in 100 ul and
incubated for 1 hr at 37°C. Ad-GFP was applied at an MOI of 5 in 0.9 ml media. B) HeLa cells were treated in
suspension with protein in 100 ul at 4°C for 1 h. Ad-GFP was added in 100 ul and incubation was continued for
1 hr. The Ceils were transferred to 37°C for 30 min, followed by treatment with trypsin before plating in fresh
media.
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For the second experiment cells were treated in suspension and the proteins
were allowed to pre-bind at 4°C for Ih. The virus was then added and the cell
mixture was incubated for another hour at 4°C, followed by incubation at 37°C for 30
min. Cells were next trypsinized and the media changed, preventing excess virus
from entering the cells during continued incubation. Only 15 % o f cells were
infected by Ad5-GFP alone (See figure 5B). All PB mutants were able to decrease
the percentage o f cells infected, however, due to the initial level o f infection the
difference may not be significant. Given that specific binding o f recombinant penton
to a v integrins has been demonstrated previously (Medina-Kauwe et al., 2001;
Karayan et al., 1997), the decrease in the number of cells infected indicates that
residual infection by Ad5 may be due to alternative paths o f cell entry. The next
study was performed using only wild-type PB in an attempt to better understand the
experimental system. Attached cells, rather than suspended cells, were treated for 1
hr at 37°C, followed by treatment with Ad5-GFP for 4 h. Cells were observed under
UV microscopy. Due to low levels of infection, FACS analysis was not performed.
The remaining experiments were performed on cells in suspension.
To check the vims infection activity on the HeLa cells used, cells were
treated at varying MOIs with Ad5-GPP for 48 h. All colonies were near 100%
infection. Time appeared to be the key factor. This experiment was repeated with
incubation with the virus reduced to four hours (See figure 6 A, lOOOul volume).
Even at MOI 25 80% transfection did not occur. In a further attempt to find a middle
point between 100% and 15% infection, cells were treated in a decreased volume.
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9
Cells were treated for 4 h at 37°C in 100, 250, and 500 fil volumes (See figure 6 A).
500 {il achieved the balance of volume and viral load to produce 80% transfection.
A final Ad5-GFP competition with the
A.
Cl"
100
80
60
40
20
0
Figure 6: Binding Studies Using A d-G FP
Ad-GFP Infection vs. Volume
A
\
/
\
-
A,..;
- i
7 :
I 7 :
1
i I ...* / A w .....
100 ul
250 ul
500 ul
1000 ul
5 7 10 15 20 25
MOI o f Infection
B.
_ 50
o
I 40
% 30
s 20
< D
U 10
A
^ 0
Ad-GFP MOI 30 Competition
.. PB
PBrgd
PB S253A
...
PB SR/AA
-®- PB S253D
PB Dileu
0 0.2 2 20
ug Proteins
C. Ad-GFP MOI 15 Competition with PB
I
u
6
4
2
0
0 2.5 5 7.5 10 12.5 15
ug PB protein
D.
U
o '-
Ad-GFP % Infected Cells
5 7 10 15 20 25
MOI o f Infection
A) HeLa cells were incubated at 37°C in suspension with shaking for 4 h at indicated volumes. B) HeLa cells were
incubated at 37°C in suspension with indicated proteins for 1 h. After addition o f Ad-GFP at M O I 30 cells were
incubated for 4 h. C) PB was pre-bound to HeLa cells in suspension for 1 h at 4"C. Ad-GFP virus was added and
cells were incubated at 37°C for 4 h, followed by treatment w ith trypsin. D) Cells were treated as in C) with out
incubation at 4°C.
penton and mutants was attempted. Again, the percentage of cells infected was
below 80% (See figure 6 B). The high MOI of infection may have been cytotoxic to
cells infected. All mutations decreased the percentage of cells infected. The small
differences between each protein made interpretation of results difficult. An
additional study was performed with PB alone at lower MOI and with virus alone at
varying MOI (See figure 6 ,D for experimental details). Incubation of HeLa cells at
4°C further reduced the infection percentage to less than 6 %, while infection with
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10
varying concentrations of Ad5-GFP at 37°C was less than 15% at MOI 25.
Treatment with trypsin to prevent residual vims entry appears to further hinder
reaching 80% infection.
The difficulty of balancing the time frame for Ad5-GFP infection with the
time period of protein attachment and internalization made these experiments far
more complex than a simple competition binding assay. All the experiments using
Ad5-GFP where performed only once, since the system protocol was constantly
being adjusted to reach 80% infection. The purpose o f reaching 80% infection was
to assist in discriminating differences in wild-type penton binding and mutant penton
binding. The results discussed are only representative o f single experiments and
have not been duplicated. Due to the outlined changes in procedure for each
experiment most of the above experiments cannot be compared to one another. No
conclusions were made from these experiments. Due to the virus’s ability to enter
the cell through multiple pathways, the system proved too difficult to control in
relationship to the penton proteins. Immimostaining might overcome this
complexity.
Both binding and trafficking o f the di-leucine mutants were studied using
immunostaining. Immunostaining of initial binding at 4°C showed that wild-type
penton forms punctate binding sites on the cell surface membrane (See figure 7).
Green staining indicates the penton or mutants proteins, while the red staining
indicates weak actin staining and nuclear auto-fluorescence, where the two colors
meet appears yellow. The red staining allows for better visualization o f the cell
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11
nucleus and membrane in order to determine where penton staining localizes. All
mutants showed a more diffuse overall binding with less punctate binding, while the
PBrgd mutant showed no punctate binding to the cell membrane. Once internalized
at 37°C , the wild type penton forms a ring around the nucleus, but does
F igure 7: Binding & Internalization of Di-Leucine Motif Mutants
LL/AA R254A
SR/AA
4°C
Immunostain o f PB and di-Leucine muntations at 4°C to demontrate binding and at 37°C to demonstrate
internalization. A ll mutants show abbarant trafficking compared to wild-type PB.
not enter. All the di-leucine mutants traffick differently than the wild-type,
remaining diffusely in the cytoplasm upon entering the cell. This suggests that the
di-leucine m otif serves as a trafficking signal for the penton.
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12
In order to better quantify the differences in internalization of the wild-type
penton compared to the mutant pentons, cells with internalized penton proteins from
37°C incubation were scored based on cytosolic localization of the penton signal (the
yellow or green stain observed in figure 7). Cells were divided into two
classifications: perinuclear or cytoplasmic. Perinuclear cells had more than 50% of
the penton label concentrated in the inner most third of the cytosol (see figure 8 ).
Figure 8: Wild-Type and Muntant Penton Scoring
Perinuclear
Cytoplasmic
Immunostained cells were placed in two categories based on staining concentration. Cells were
determined to be perinuclear if more than 50% of the fluorescent label was concentrated in the
perinuclear most third of the cytosol, as shown above. Cytoplasmic cells had more than 50% of
the florescent label was concentrated in the outer most two thirds of the cytosol.
Cytoplasmic cells had more than 50% of the penton label concentrated in the outer
most two thirds o f the cytosol. Scores were taken from three independent fields for
each protein from a single experiment. A total of 19 to 50 cells were counted for
each protein used. These scores are reported as the percentage o f total cells scored
(see figure 9). Scoring shows that the wild-type penton protein can be found in
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13
Figure 9: Perinuclear vs. Cytoplasmic Localization
120
100
C O
L 60
o
H
£ 40
j B cyto
20
LL/AA R254A S253A S253D SR/AA PB
-20
Proteins
Cells from the 37°C time point of the immunostaining experiment were scored for perinuclear (pn)
and cytoplasmic (cyto) staining of the penton proteins. Cells were reported as a percentage of total
cells scored using three independent fields of view.
perinuclear area of the cytosol for a majority of the cells, while all the mutants are
trafficked to the cytosol in a majority of the cells. One of the most prominent
differences between the wild-type penton and mutants was the absence of cells with
the mutant protein tightly bound to the nucleus. For the penton samples, 64% of the
perinuclear cells exhibit tight binding to the nuclear membrane, forming a ring
around the nucleus as seen in figure 7. The most significant trafficking mutation
appears to be any mutation made to the serine, controlling the m otifs activation.
De novo Expression of Penton Mutants
To determine whether the di-leucine motif has an effect on penton trafficking
after expression in the cell wild type and mutant penton C-terminal GFP mammalian
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14
Figure 10: Intracellular Localization Of Penton Mutants in HeLa
pEGFP PB PBS253A PBR254A
Combined
SR/AA S253D LL/AA PB rgd
Combined
Light and UV microscopy o f transfected HeLa cells at lOx magnification. De Novo expression
o f wild-type PB remains in the nucleus as do most mutants with the exception o f S253D. These
are representative micrographs taken from at least five experiments.
expression vectors were transfected and overexpressed in HeLa cells. The cells were
observed at 12, 24, 48, and 72 h after transfection. GFP was first detected at 24 h.
The empty pEGFP vector express through out the cytoplasm of the cell (See figure
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15
10). Wild-type penton can be seen localizing to the nucleus. At no time point was
the wild type penton observed in the cytoplasm. All the mutant pentons with the
exception of S253D and LL/AA localize to the nucleus, resembling the wild type
penton. The LL/AA mutant appears to be cytotoxic to the cells, as only rounded up
cells that appear to be dying can be seen to express this protein. Cell rounding was
not observed before the EGFP levels were visible under UV microscopy. At no time
point are normal HeLa cells expressing LL/AA EGFP fusion protein observed.
Mutant S253D expression was undetectable at various levels of transfection either
due to non-expression, misfolding or protein aggregation.
The S253D mutation was examined further to determine if the protein was
expressed at all. First cells were lysed and the lysate was examined by western blot
to look for protein expression. However, the lysate was too dilute to detect even the
wild type penton. Instead, the mutant was studied using immunostaining. Cells
were transfect with the S253D mutant, and then were immunostained to determine if
any protein was expressed. As shown in figure 11, the protein was detected in the
Figure 11: Detection of GFP-PB S253D in HeLa Cells
Combined FITC Light
Transfected and immunostained HeLa cells were viewed using UV microscopy. GFP-PBS253D
was detected in the nucleus of transfected cells.
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16
nucleus like the wild type penton. Therefore, S253D protein is expressed in the
nucleus ofH eLa cells, however, due to misfolding or protein aggregation EGFP fails
to fluoresce.
Discussion
Although studies have shown trafficking o f the entire Ad5 virus, the
trafficking of individual viral proteins and their specific interactions during
endocytosis have not been elucidated. Endocytosis o f Ad5 has been shown to occur
through clathrin-coated pits and clathrin-coated vesicles, followed by transport to a
nuclear pore complex (Meier and Greber, 2003). Still, the steps signaling the
formation of the coated pit/vesicle are unclear. The identification o f a di-leucine
motif in the penton provides a possible explanation for these events. The aberrant
trafficking of the di-leucine mutants indicate that the di-leucine m otif serves as a
signaling sequence for penton entry and positioning inside the cell.
Previously, Ad5 has been shown to bind to a v integrals on the cell surface,
triggering the formation of a coated pit (Meier and Greber, 2003). From these pits
clathrin coated vesicles form. Di-leucine motifs in the cytoplasmic tails of cell
surface receptors are directly recognized by the AP complexes involved in clathrin
pit formation and trafficking (Kirchhausen, 1999). The differential up take of penton
mutants by HeLa cells shows that the di-leucine m otif in the penton provides the
initial endocytosis signal for Ad5. Furthermore, this signal is involved in penton
trafficking within the cytoplasm and, likely, Ad5 trafficking. Also, trafficking of the
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17
wild-type penton suggests that the di-leucine m otif may sort the penton directly to
a nuclear pore, allowing gene transfer without PB translocating into the nucleus. The
initial study involving fiber and penton interaction (figure 2 ) shows that fiber binding
to penton provides additional regulation of the di-leucine motif, and may regulate the
timing at which the di-leucine motif is exposed.
While the di-leucine m otif usually contains two leucines and is surrounded by
polar and/or charged residues, these leucines may be replaced with isoleucine or
methionine (Kirchhausen, 1999). The role o f the polar/charged, surrounding
residues is likely to ensure that the two leucines are exposed on the surface. In Ad5,
the binding of fiber to the penton may further stabilize the di-leucine by concealing it
from solution, as well as, preventing premature interaction with cellular factors. The
penton mutants indicate that the context of the di-luecines are necessary for protein
trafficking. While the LL/AA showed the most severe trafficking problems, all of
the mutants failed to direct the penton to the nucleus like the wild type.
Additionally, the S253D mutation suggests that this signaling m otif must only be
active at the correct time, since this constitutively active protein was not trafficked to
the nucleus.
Overexpression o f the mutants in transfected HeLa cells provided a look at
the role of the di-leucine motif during Ad5 replication. The localization of all
mutants to the nucleus suggests that once the penton has been made in the nucleus it
does not leave. This was expected as Ad5 assembles within the nucleus. The
absence of EGFP fluorescence for the S253D mutant suggests that this mutation
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18
affects c-terminal EGFP folding or protein aggregation blocks the fluorescence
(Waldo et al., 1999; Sacchetti et al., 2001). The cytotoxic nature o f the LL/AA
EGFP fusion protein may be a result o f extra stability (increased protein half-life),
leading to overwhelming protein levels. Cell stress brought on by the combination
of mutant PB protein levels and EGFP levels may have lead to cell death. By the
time the fluorescence was observed, all the cells were already dying, making
determination of a timeline for cell death impossible.
Identification of the di-leucine motif and study o f penton trafficking can
provide greater understanding when using this protein as a gene delivery vehicle.
Further studies o f the protein’s interactions with AP complexes and determining the
quantitative differences in binding can lead to a better engineered delivery system.
Experimental Procedures
Cells and Plasmids
HeLa cells were maintained in DMEM, 10% fetal bovine serum, at 37°C, 5 %
CO2. The mammalian expression vector, pEGFP-Nl was used for de novo synthesis
study.
DNA Constructs
Wild-type penton in pRSET-A bacterial expression plasmid was constructed
as described by Medina-Kauwe et al., 2001. From this construct PB mutants were
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19
constructed using the following primers. For S253A: The 5’ primer was 5’-
GGACTTCACCCACAGCCGCCTGAGCAACTTG-3’. The 3’ primer was 5’-
CAAGTTGCTCAGGCGGGCGTGGGTGAAGTCC-3 ’. For R254A: The 5’ primer
was 5 ’ -GGACTTC ACCC AC AGCCGCCTGAGC AACTTGTTGG-3 ’. The 3’
primer was 55 -CCAACAAGTTGCTCAGGGCGCTGTGGGTGAAGTCC-3 ’. For
SR/AA: The 55 primer was 5’-GGACTTCACCCACGCCGCCCTGAGCAACTT
GTTGG-3The 3’ primer was 5’-CCAACAAGTTGCTCAGGGCGGCGTGGG
TGAAGTCC-3’. For S253D: The 5’ primer was 5 ’-GGACTTC ACCC AC AGC
CGCCTGAGCAACTTG-3’. The 3’ primer was 5’-CAAGTTGCTCAGGCGGT
CGTGGGTGAAGTCC-3 ’. For LL/AA: The 5’ primer was 5 ’-CGCCTGAGCAA
CTTGTTGGGCATCCGCAAG-3’. The 3’ primer was 5’-CTTGCGGATGCCCGC
CGCGTTGCTCAGGCG-3’. Common 5’ and 3’ oligonucleotide primers were used
to amplify both wild-type and mutant penton sequences from the pRSET-A bacterial
expression plasmid (Invitrogen, Carlsbad, CA, USA). The 5’ primer was 5’-
ATCGAAGGATCCATGCGG CGCGCGGCGATGTAT-3 ’. The sequence of the 3’
primer was 5'’-GCATCAGAATTCTCAAAAAGTGCGGCTCGATAG-3 ’. A
Bam Bl restriction site was introduced in the 5‘ primer and an EcoBl restriction site
was introduced in the 3’ primer for in-frame insertion o f both the wild-type and
mutant pentons into the pEGFP-Nl mammalian Expression vector (Clontech, Palo
Alto, CA, USA) between the BglI and EcoRI restriction sites. This vector expresses
recombinant protein as a C-terminally GFP-tagged fusion protein.
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20
Protein Expression and Purification from Bacteria
Overnight cultures of BLR (DE3)pLysS bacterial transformants were
inoculated 1:50 in LB containing 0.1 mg/ml ampicillin, 0.034 mg/ml
chloramphenicol and 0.0125 mg/ml tetracycline. When cultures reached an
absorbance reading o f -0.6 at an optical density wavelength of 600nm (OD 600),
cultures were induced with 1 himIPTG and grown for a further 4 h at 37°C with
shaking. Cultures were harvested and pelleted. Cell pellets were resuspended in
lysis buffer ( 50 him Na-phosphate, pH 8.0, 500 mM NaCl; 5-10 him imidazole, 1 him
phenylmethylsulfonyl fluoride) and lysed by addition o f 0.1% Triton X-100 and one
cycle o f freeze-thawing. Supernatants were recovered, added to Ni-NTA resin
(Qiagen, Valencia, CA, USA) pre-equilibrated in lysis buffer, and incubated for 1 h
on ice. The resin containing bound protein was washed with 10 ml of lysis buffer,
then 6 ml o f a solution of 50 mM Na-phosphate, pH 8.0, 500 mM NaCl; 60 mM
imidazole, and protein was eluted with 2 ml of a solution o f 50 mM Na-phosphate,
pH 8.0, 500 mM NaCl, 400 mM imidazole. Proteins were desalted on YM-10
nominal molecular weight limit spin columns (Millipore, Bedford, MA, USA) and
their concentrations measured using the BioRad protein quantitation assay (Bio Rad
Laboratories, Hercules, CA, USA).
Protein Detection
Denaturing polyacrylamide gel electrophoresis was performed in a
discontinuous gel buffer system. Proteins were electrically transferred on to
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21
nitrocellulose using 39 mM glycine, 48 h im Tris-HCl, 0.0375% SDS, 20%
methanol in a BioRad semi-dry transfer cell set at constant voltage (15 V) for 30
miH. Blots were blocked with 3% milk in phosphate-buffered saline (PBS 137 mM
NaCl, 2.7 mM KC1, 4.3 h im NaaHPCL, 1.4 h im KH2PO4). Anti-His Tag antisera
(Sigma) was used at a 1:3000 dilution in blocking buffer. Anti-Ad5 antisera (Access
Bio Medical, San Diego, CA, USA) was used at a 1: 5000 dilution in blocking
buffer. Antibody-antigen complexes were detected by incubation with horseradish
peroxidase (HRP) conjugated secondary antibodies (Sigma, St Louis, MO, USA),
reaction with chemiluminescence detection reagents (Amersham Pharmacia Biotech,
Piscataway, NJ, USA), and exposure to film (Hyperfilm ECL; Amersham Pharmacia
Biotech).
Pentamerization Assay
Non-denaturing polyacrylamide gel electrophoresis was performed in a
discontinuous gel buffer system to determine mutant pentons ability to pentamerize.
5 pg of each protein was applied to a 3-12% tris-HCl gel. Proteins were detected as
described above.
Penton Ad5-GFP Competition Assay
1 x 105 cells were plated per well and grown 24 h. Cells were either treated
on the plate or lifted using PBS supplemented with Ca+ + and Mg*4 * . Penton proteins
in 100 pi blocking buffer (PBS with 3 % milk) to reduce nonspecific binding were
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22
added to each well. After 1 h incubation a 37°C or 4°C as indicated, media with
Ad5-GFP (kindly provided by Dr. N. Kasahara) to infect at various MOIs was added
to the cells. Cells were then incubated at 37°C for 48 h. After final incubation cells
were lifted, centrifuged, washed three to four times with PBS, resuspended in 0.5 ml
PBS and measured by FACScan. Alternatively, cells media containing Ad5-GFP
was removed at various time points and replaced with plain media for the remainder
of the incubation.
FACS Analysis
5,000 events were counted on a Becton Dickinson FAC Scan analyzer (Becton
Dickinson, Franklin Lakes, NJ, USA) using a 15 mW air-cooled argon laser set at
488 run and recorded with a 530 run emission filter in the FL1 emission channel.
Cell populations are reported as the percentage of total number of fluorescent cells
out of the 5,000 counted.
Cell Binding and Internalization
Cells were treated with penton proteins as described previously. (Medina-
Kauwe et al., 2000) HeLa cells were plated 1 x 105 cells/ well o f a 6 -well dish with a
sterile cover slip placed in the well and grown for two days. Cells were washed two
to three times with PBS, then PBS containing penton proteins and 3% milk, to
reduce nonspecific binding, was added to each well. After 1 h incubation on ice,
cells were centrifuged and washed three to four times with PBS before proceeding to
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23
immunostaining. For internalization studies, following incubation on ice, cells
were incubated for 1 h at 37°C then washed three to four times with PBS prior to
immunostaining.
Immunostaining
HeLa cells were fixed using 4% paraformaldehyde in PBS for 15 min at room
temperature on shaker. Cells were then washed with PBS three times for 5 min
followed by two washes for 15 min. Cells were then washed with 50 mM NH4CI in
PBS for 5 min. Next, cells were washed with PBS three times for 5 min, and then
washed with 0.1% Tx-100 in PBS for 5 min. Again, the cells are washed with PBS
three times for 5 min. The cells were then blocked using 1% BSA in PBS for 10
minutes. Each cover slip was then incubated with the primary Ab. Anti-Ad5 was
used at a concentration o f 1: 500 in blocking buffer up to 80pi Samples were then
incubated at 37°C for 30 minutes. Cells were then washed three times for 5 min,
then two times for 15 min. Secondary abs were made in 80pl blocking buffer, using
GARabbit FITC at 1: 300 and rhodamine phalloiden (RP) at 1:100. Incubate
covered at 37°C for 30 min. After incubation cells were washed with PBS three
times for 5min. Cover slips were then mounted in ProLong Anti-Fade medium
(Molecular Probes, Eugene, Oregon, USA) for viewing using confocal microscopy.
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Confocal Micrscopy
Confocal micrographs were obtained at 60x magnification using a Nikon
Eclipse confocal fluorescence microscope (Nikon Bioscience Confocal Systems,
Melville, NY, USA) equipped with Argon ion and green HeNe lasers. Confocal
images were compiled in Adobe Photoshop 5.0 for Windows 98 (Adobe Systems
Inc, Mountain View, CA).
Cell Transfection
HeLa cells were grown to -90% confluency in 6 -well dishes. Cells were
rinsed with serum-free DMEM. Penton-GFP mammalian expression plasmids were
mixed with lipfectamin 2000 (Invitrogen) in serum-free DMEM, then applied to cells
for 30 min. Cells were rinsed and incubated in serum-free DMEM for 4 h. After
incubation media was replaced with DMEM, 10% fetal bovine serum. Cells were
visualized using UV microscopy.
UV microscopy
An Olympus (Lake Success, NY, USA) IMT-2 inverted microscope fitted
with a GFP filter was used to visualized EGFP-positive cells. Photomicrographs
were taken at lOx magnification.
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Bibliography
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Asset Metadata

Creator Cirivello, Meghan Kathleen (author)

Core Title Clathrin associated protein (AP) binding motifs in AD5 penton

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

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

Tag biology, genetics,biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Kedes, Laurence H. (committee chair), Hamm-Alvarez, Sarah (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-310547

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Legacy Identifier 1420362.pdf

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Document Type Thesis

Rights Cirivello, Meghan Kathleen

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 au...

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