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Role of mitochondrially derived peptides in the inhibition of IAPP misfolding

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Role of mitochondrially derived peptides in the inhibition of IAPP misfolding

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Content Role of Mitochondrially Derived
Peptides in the Inhibition
of IAPP misfolding

A Thesis Presented to the Faculty of the University of
Southern California - Graduate School

In Partial Fulfillment of the Requirements for the Degree of
Master of Science Biochemistry and Molecular Biology

By: Fleur Lobo

August 2016

Acknowledgements
I would like to express my sincere gratitude to my advisor Dr. Ralf Langen for your
mentorship and encouragement towards research as well as writing this thesis. I am very
grateful for your advice on both research as well as on my future career goals. Thank you
for taking the time to help me understand how to approach certain problems I encountered
during the Master’s research project.
I would like to especially thank my fellow lab members Alan Okada and Kazuki Teranishi
for taking the time to teach me the techniques, for guiding me on the project as well as your
assistance in some of the experiments.
Thank you to Dr. Pinchas Cohen, Dr. Kelvin Yen and Hemal Mehta at the USC School of
Gerontology, our collaborators on the MDP projects and to Dr. Nitin Pandey for your
assistance and guidance in my first research project in Langen Lab.
I would also like to thank the other thesis committee members Dr. Jian Xu, Dr. Ansgar
Siemer and Dr. Tobias Ulmer and for your valuable suggestions while writing the thesis
and members of Langen , Siemer and Ulmer labs for creating a wonderful atmosphere in
the lab throughout these two years.

Table of Contents
1. Abstract……………………………………………………………………………1
2. Introduction……………………………………………………………………...2-8
a. Amyloid Fibrils, IAPP and its link with Type 2 Diabetes……………………….2
b. Amyloid diseases and Mitochondrial dysfunction……………………………….4
c. Mitochrondrially Derived Peptides………………………………………………5
d. Preliminary work: HNG and its role in the inhibition of
IAPP fibrilization ………………………………………………………………..7
e. Aims of the Project……………………………………………………………….8
3. Results……………………………………………………………………………9-26
a. SHLP2 is stable in 10mM Phosphate buffer pH 7.4 and D/W
at room temperature over 72 hours…………………………………………........9
b. SHLP2 inhibits fibril formation of IAPP using Thioflavin T Fluorescence….....10
c. SHLP2 retains IAPP in a predominantly monomeric form using CW-EPR…….11
d. SHLP2 prevents loss of IAPP monomer at stoichiometric ratios
using CD and TEM………………………………………………………………13
e. SHLP2 can bind to IAPP seeds as demonstrated by a dot blot…………………..19
f. MOTS-c, SHLP6 and SHLP1 are stable in 10mM Phosphate
buffer pH 7.4 and D/W at room temperature over 72 hours ………………………20
g. MOTS-c can inhibit fibril formation of IAPP using Thioflavin T Fluorescence…21
h. SHLP1 and SHLP6 do not inhibit fibril formation using Thioflavin T
fluorescence and SHLP1 increases the amount of fibrilization of IAPP…………24

4. Discussion………………………………………………………………………………27
a. ThT Fluorescence studies of SHLP2,SHLP1,MOTS-c and SHLP6…………………27
b. CW-EPR , CD and TEM analysis of SHLP2 in the
inhibition of IAPP misfolding………………………………………………………..27
c. Dot blot provides evidence that SHLP2 works by binding to misfolded IAPP ……..28
d. Future studies and Conclusion………………………………………………………..28

5. Methods and Materials……………………………………………………………….29-33
a. Materials and Peptide handling……………………………………………………..29
b. Thioflavin T Fluorescence………………………………………………………….30
c.CW-EPR……………………………………………………………………………..31
d. CD…………………………………………………………………………………..31
e. TEM………………………………………………………………………………...32
f. Dot Blot……………………………………………………………………………..33
6. References................................................................................................................36-37

1

1. Abstract:
Humanin, the first Mitochondrially derived peptide to be discovered along with Humanin
S14G (HNG) have been known for their neuro-protection in Alzheimer’s disease, anti-
apoptotic effects and protection against oxidative stress. Previous work in the Langen lab
investigated whether HNG could directly interact with an amyloid protein implicated in
Type 2 Diabetes and play a role in the inhibition of amyloid misfolding. Promising results
revealed that HNG can inhibit IAPP misfolding. In this study another Mitochondrially
derived peptide -SHLP2, was primarily used to investigate its role in the inhibition of IAPP
misfolding. ThT Fluorescence, Continuous Wave- Electron Paramagnetic Resonance,
Circular Dichroism and Transmission Electron Microscopy revealed that SHLP2 is an
inhibitor of IAPP aggregation and works by targeting higher order species of IAPP. Thus,
SHLP2 may have a chaperone like function to modify Type 2 Diabetes by inhibiting IAPP
misfolding. ThT fluorescence was then used to survey three other MDPs-SHLP1, SHLP6
and MOTS-c to find out whether they function in a similar manner to SHLP2.
MOTS-c was found to also inhibit IAPP fibrilization using Thioflavin T fluorescence
whereas SHLP1 and SHLP6 did not inhibit IAPP fibrilization. However, SHLP1 increased
the amount of fibrilization. We can conclude that SHLP2 may be a potential therapeutic for
Type 2 Diabetes. More experiments need to be carried out to confirm whether MOTS-c
works in a similar manner to SHLP2 to inhibit IAPP misfolding or functions via a different
mechanism.

2

2. Introduction:
a. Amyloid Fibrils, IAPP and its link with Type 2 Diabetes
Islet Amyloid Polypeptide (IAPP), a 37-aa residue long peptide can form insoluble amyloid
fibrils which is a typical feature of islets in a majority of Type 2 Diabetes patients. Such
amyloid deposition results in β cell loss (1)(2). The monomers in amyloid fibrils are
assembled into β sheet structures arranged perpendicularly to the fibril axis. The formation
of amyloids is known to be a nucleation-dependent process that first begins with a lag
phase where the nucleation of monomeric species takes place. After this phase, comes the
elongation phase where amyloid fibrils spread and finally, the plateau phase is when the
fibrillar mass is no longer changing. The figure below shows how amyloid fibrils are
formed when a monomeric protein unfolds and misfolds. This process is mostly
intracellular(1). Oligomers are then formed which seems to be the toxic species. When
these oligomers interact with membranes, it could lead to their disruption(1)(3).

Westermark et al. (2011) Physio Rev.
Figure 1- The formation of amyloid fibrils from natively folded protein
The first step in the formation of amyloid fibrils is the unfolding followed by the
misfolding of a natively folded protein. If this process is left to continue, toxic species

3

called oligomers, develop. Oligomers could lead to the formation of fibril growth and these
species can disrupt cell membranes or they themselves can insert into membranes and form
pores (1).

The main characteristic of Type 2 diabetes mellitus is insulin resistance and β cell failure.
As summarized in the figure below by Hoppener et al. (2011), both genetic as well as
environmental factors contribute to the beginning of Type 2 Diabetes characteristics.
Increase in demand for insulin could result in an increase in the production of
amyloidogenic islet amyloid polypeptide. If insulin resistance persists for a long time, then
a loss of β cells and their capacity to produce insulin may result in an increase production
of insulin as well as IAPP by the surviving β cells. This cycle of events if allowed to
continue will increase the amount of IAPP formed and contribute to a reduction in the
capacity of the pancreas to produce enough insulin. Serum glucose levels then increase due
to insulin resistance (2).

Hoppener et al. (2000). N. Engl. J. Med
Figure 2- IAPP and the pathogenicity of Type 2 Diabetes Mellitus.
Certain diabetogenic factors can lead to a beta-cell defect or a reduction in insulin
sensitivity. Insulin resistance then results in the increased production of amyloidogenic islet

4

amyloid polypeptide which is related to a loss of beta- cells. If these cycle of events go on
unchecked, the formation of amyloid increases and the insulin producing capacity of the beta- cells
decreases ultimately resulting in beta-cell failure.

Tokuyama, et al, transfected transgenic mice with Human IAPP and found that when the
transfectants expressed the highest amount of hIAPP, insulin secretion was increased in
response to high levels of glucose but the amount of insulin secretion was less than what
was observed in control mice. Thus, this work provides evidence that overexpression of
IAPP could be partly responsible for impaired insulin secretion(4). Ritzel et al. showed that
oligomers of human IAPP when applied to human islets disrupted cell to cell adherence of
islets and led to impaired insulin secretion and thereby, demonstrates the toxicity of IAPP
oligomers(3).

b. Amyloid diseases and Mitochondrial dysfunction
Mitochondrial Dysfunction has been linked to a number of neurodegenerative diseases such
as Alzheimer’s, Parkinson’s, ALS, Huntington’s Disease (5). Besides these diseases,
mitochondrial dysfunction also contributes to β-cell failure, a characteristic of Type 2
Diabetes. The reactive oxygen species are produced by the mitochondria of β-cells due to
metabolic stress and they affect mitochondrial structure and function and result in β-cell
failure. ROS released by the mitochondria activate a protein that leads to a proton leak
across the mitochondrial inner membrane. The resultant effect is reduced β-cell ATP
synthesis and impaired insulin secretion (6).

5

c. Mitochrondrially Derived Peptides
Hasimoto et al. (2001) discovered Humanin, a short polypeptide that suppresses neuronal
cell death observed in Alzheimer’s disease. HNG a variant of Humanin was also effective
in inhibiting death of neurons in Famalial Alzheimer’s Disease mutants of Amyloid
Precursor Polypeptide(APP), Presenelin 1 and Presenelin 2 . This was the first evidence of
Humanin being a neuro-protective factor. Humanin cDNA was discovered using a cDNA
library in order to search for genes that could protect neurons from apoptosis induced by a
mutant form of APP (7). According to Yen et al, (2013) , “Humanin is encoded by an ORF
within the gene for the 16s ribosomal subunit within the mitochondrial genome” (8).

Yen et al. (2013). J Mol Endocrinol.
Figure 3- Humanin is a Mitochondrially Derived peptide
Humanin is encoded by a gene within the mitochondrial genome (8).

6

Besides being a neuroprotective factor (7), Humanin has the ability to protect cells against
oxidative stress (8) (10)(11). Jia et al, (2013) have shown that synthetic Humanin can
rescue rat germ cells from apoptosis induced by BAX (Bcl2-like protein 4) and
Gonadotropin-releasing hormone agonist (GnRH-A). Its mechanism of action is through
interactions of endogeneous rat Humanin with BAX in the cytoplasm. Thus, BAX is unable
to enter the mitochondria to cause apoptosis (9). Humanin was also found to protect β-cells
from cell death and treat diabetes in the non-obese diabetes (NOD) model. It works by
decreasing cytokine – induced apoptosis in β-cells. Thurs, it improved glucose tolerance in
NOD mice (12).
Cobb et al.(2016) reported that, “ an in silico search for potential sORFs within 16S-
rRNA-encoding short peptides (20–40 amino acids) returned six sequences encoding 20–38
amino-acid-long peptides”. (13). (Summarized in the table below)

Cobb et al. (2016). Aging.
Figure 4- Identification of SHLPs encoded by small open reading frames (sORFs) within
the mitochondrial 16S ribosomal RNA (rRNA) gene.
The table summarizes the names, molecular weight, number of amino acids, location and
sequences of the newly identified Small Humanin like peptides.(13)

7

SHLP2 shows remarkable neuro-protective activity by protection against Aβ1–42 induced
cell death. SHLP2 unlike SHLP3 is also an insulin sensitizer (13).
Lee et al (2015) have identified a short Open Reading frame within the 12 s rRNA of
mitochondria which encodes a 16-amino-acid peptide named MOTS-c (mitochondrial open
reading frame of the 12S rRNA-c) . MOTS-c has been found to be an insulin sensitizer as
its treatment in mice shows that it can prevent insulin resistance (14). This MDP has been
recently linked to extension of lifespan (15). Thus the mitochondrial genome encodes a
number of short peptides with important functions (7)-(15).

d. Preliminary work: HNG and its role in the inhibition of IAPP fibril formation
Work shown in this section was done by Langen lab members- Alan Okada and Kazuki
Teranishi prior to the start of the SHLP2 project. As reported in the graph below, HNG (a
variant of Humanin) is very potent in the inhibition of IAPP fibrilization as nM quantities
are sufficient to bring about this effect.

Courtesy of Alan Okada and Kazuki Teranishi (unpublished data).
Figure 5- Thioflavin T Fluorescence indicates that HNG inhibits fibril formation of
IAPP at sub-stoichiometric concentrations
0
0.5
1
1.5
2
0 5 10 15 20
Normalized ThT
fluorescence
Time in hours
0 M
12.5 nM
16.7 nM
50 nM
250 nM
1 µM

8

12.5 µM IAPP was solubilized in 10mM KPO4 Buffer pH =7.4 and its fibrilization was
monitored using 25 µM of Thioflavin T in the presence or absence of HNG over 18 hours.
SHLP2 Concentrations ranged from 5 µM to 12.5 nM. Representative trace using ThT
fluorescence demonstrates the effect of different concentrations of HNG on IAPP
fibrilization.

Since SHLP2 as similar neuro protective functions like HNG and is also known to have
insulin sensitizing activity (7)(13), it seemed to be a promising candidate to study its role in
the inhibition of IAPP misfolding.

e. Aims of this project
The main aim of this project was to investigate the role of SHLP2 in the inhibition of IAPP
misfolding. The second aim was to identify whether other MDPs (like MOTS-c, SHLP1
and SHLP6) play similar or diverse roles with respect to IAPP aggregation.

9

3. Results:
a. SHLP2 is stable in 10mM Phosphate buffer pH 7.4 and D/W at room temperature
over 72 hours.
From a stock concentration of SHLP2 in D/W (1mg/ml), a 33.31 µM solution of the MDP
was prepared in a)D/W and in b)10 mM KPO4 pH=7.4. Circular Dichroism(CD) spectra
were recorded at the start of the experiment and at the end of 72 hours at room temperature.
It was observed SHLP2 is monomeric in either solution as indicated by the retention of
random coil structure (peak at 202nm) using CD (Figure 1). It is important to note that
there is a slight decrease in the random coil peak of SHLP2 in 10mM KPO4 Buffer pH=7.4
versus D/W at the start of the experiment and this difference is more pronounced at the end
of 72 hours. Overall, we can conclude that SHLP2 is relatively stable over 3 days in either
D/W or 10 mM KPO4 Buffer pH=7.4.

Figure 6- Circular dichroism spectra indicate that SHLP2 in either 10mM Phosphate
buffer pH 7.4 or D/W at room temperature is monomeric over 72 hours.
Circular dichroism spectra of SHLP2 (33.31µM) measured in a) D/W and b) 10 mM KPO4
Buffer pH=7.4 at the beginning of the experiment and at the end of 72 hours.

-15
-10
-5
0
5
θ
raw
(x10^-3*deg cm^2 dmol^-1)

Wavelength in nm
SHLP2 in H20 t=0 hrs
SHLP2 in 10mM KP04 Buffer pH 7.4 t=0 hrs
SHLP2 in H20 t=24 hrs
SHLP2 in 10mM KP04 Buffer pH 7.4 t=72 hrs

10

b. SHLP2 inhibits fibril formation of IAPP using Thioflavin T Fluorescence
In order to determine whether SHLP2 could inhibit IAPP fibrilization, IAPP in 10mM
KPO4 Buffer pH=7.4, in the presence of varying concentrations of SHLP2 was monitored
over 18 hours using 25 µM of Thioflavin T. Figure 2A shows a representative trace of
IAPP fibril inhibition using different concentrations of SHLP2. From Figure 2A-C, it is
observed that SHLP2 can inhibit IAPP fibril formation at closer to equimolar
concentrations. However, with this experiment we cannot conclude whether SHLP2 can
inhibit the fibrilization of IAPP by binding to the monomer or other higher order structure
or IAPP. Also, it is important to rule out the possibility that the inhibition of IAPP
misfolding as detected by ThT Fluorescence is actually due to SHLP2 binding to IAPP or
whether SHLP2 binds to IAPP at its ThT binding site and thus prevents the observation of
ThT Fluorescence which is the read out in this assay. Circular Dichroism and CW-EPR
were carried out subsequently to address this question.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 Normalized ThT Fluorescence
at t=18 hrs
Time in Hours
A

0.25 uM of SHLP2 + 12.5uM of IAPP
IAPP
25 uM of SHLP2 + 12.5uM of IAPP
12.5 uM of SHLP2 + 12.5uM of IAPP
7.14 uM of SHLP2 + 12.5uM of IAPP
5 uM of SHLP2 + 12.5uM of IAPP
1uM of SHLP2 + 12.5uM of IAPP

11

Figure 7- Thioflavin T Fluorescence indicates that SHLP2 inhibits fibril formation of
IAPP closer to stoichiometric concentrations
12.5 µM IAPP was solubilized in 10mM KPO4 Buffer pH =7.4 and its fibrilization was
monitored using 25 µM of Thioflavin T in the presence or absence of SHLP2 over 18
hours. SHLP2 Concentrations ranged from 25 µM to 0.25 µM.
A) Representative trace using ThT fluorescence demonstrates the effect of different
concentrations of SHLP2 on IAPP fibrilization.
B) Normalized ThT fluorescence data at time=18 hours was collected and plotted against
differing concentrations of SHLP2(0µM-25µM). Error bars for Normalized Tht
Fluorescence at the end point indicate +/-1 Standard deviation from a minimum of 3
experiments per condition.
C) Average of t
50
for IAPP under the varying SHLP2 treatment conditions (t
50
= time to half
maximal fluorescence) was calculated and normalized against the t
50
average for IAPP
alone. Values are plotted against the concentration of SHLP2 in µM. N/A indicates the
SHLP2 treatment conditions where the IAPP did not fibrilize.

c. SHLP2 retains IAPP in a predominantly monomeric form using CW- EPR
Site-directed spin labeling (SDSL) and Continuous wave- Electron Paramagnetic
Resonance (EPR) Spectroscopy was used to investigate the mechanism by which SHLP2
could prevent IAPP misfolding. The change in the central line amplitude of an EPR
spectrum is indicative of percentage of free monomer in solution. IAPP33R1 was used in
this study in the presence or absence of equimolar concentrations of SHLP2 to first confirm
the ThT Fluorescence result and also to detect whether SHLP2 interacts with the monomer
or binds to misfolded IAPP species. At the start of the experiment, the central line
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 0.25 1 5 7.14 12.5 25
Normalized ThT
Fluorescence at t=18 hrs
Concentration of SHLP2 in uM

B
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.25 1 5 7.14 12.5 25
t
50
/t
50
IAPP
Concentration of SHLP2 in uM

C

12

amplitude of IAPP 33R1 in the presence of SHLP2 was similar to that observed of
IAPP33R1 alone (data not shown). Since a SHLP2-IAPP monomeric interaction would
result in a change in the central line amplitude between these two conditions, this result
may mean that the target of SHLP2 is not the monomer. The CW-EPR spectra of
IAPP33R1 alone and IAPP33R1 + SHLP2 were monitored over 10 hours and it was
observed that SHLP2 maintains the monomeric form of IAPP as more that 80% of the
initial central line amplitude is retained when SHLP2 is added to IAPP33R1. In contrast,
the kinetics for IAPP33R1 in the absence of SHLP2 indicate that IAPP misfolds and the
central line amplitude drastically decreased compared to the initial value at the end of 10
hours (Figure 8). The steady decrease in central line amplitude observed for the spectra of
IAPP33R1 + SHLP2 indicates that even though SHLP2 retains a majority of the IAPP
population in the naïve form by binding to higher order seeded forms of IAPP, a few
seeded forms of IAPP do exist.

0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Normalized Central Line
Amplitude
Time in Hours
A
IAPP33R1 + SHLP2
IAPP33R1

13

Figure 8- CW-EPR demonstrates that SHLP2 retains the monomeric structure of IAPP
A) MTSL Spin labeled IAPP (IAPP33R1) was incubated at room temperature in 10mM
KPO4 Buffer pH =7.4 in the presence or absence of stoichiometric concentrations of
SHLP2 and its misfolding kinetics monitored for t=11 hours. Normalized central line
amplitude indicates the percentage of IAPP in a monomeric form. B) Normalized central
line amplitude at t=10 hours for the two conditions-a) IAPP33R1 alone and b) IAPP22R1
+ SHLP2.

d. SHLP2 prevents loss of IAPP monomer using CD and TEM
A preliminary experiment was carried out in order to investigate whether SHLP2 can
prevent loss of IAPP monomer using CD. 15µM of IAPP either in the presence or absence
of 15µM SHLP2, and 15µM of SHLP2 alone was incubated in 10 mM KPO4 Buffer
pH=7.4 at room temperature. Weighted Mean Residual Elipticities were calculated for the
mixed sample (containing SHLP2 and IAPP) (Please refer to Materials and Methods for
the calculation of Weighted Mean Residual Elipticities). Mean Residual Elipticities were
calculated for sample containing either SHLP2 or IAPP. 202nm and 218 nm were the
wavelengths selected for this CD kinetics experiment because a negative peak at 202nm is
indicative of a random coil structure whereas a negative peak at 218nm indicates the
presence of a β-sheet structure (ordered form). For the SHLP2 and SHLP2 + IAPP
0
0.2
0.4
0.6
0.8
1
IAPP33R1 IAPP33R1 + SHLP2
Normalized Central Line
Amplitude at t=10 hrs
B
IAPP33R1
IAPP33R1 +
SHLP2

14

samples, the MRE/MRE’ values at 202nm and 218 nm were constant throughout the
experiment (from the start till 18 hours). However, for the IAPP sample alone, at the end
of the experiment (18 hrs), a negative MRE value was observed at 218 nm whereas a MRE
at 202nm closer to zero was observed(Figure 9). Thus, in the absence of SHLP2, the
structure of IAPP at 16-18 hours was ordered.

-9
-8
-7
-6
-5
-4
-3
-2
-1
0
0 5 10 15 20
θ
raw
(x10^-3*deg cm^2 dmol^-1)'

Time in Hours
SHLP2:IAPP (1:1) 202 nm
SHLP2:IAPP (1:1) 218 nm
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
0 5 10 15 20
θ
raw
(x10^-3*deg cm^2 dmol^-1)
Time in Hours
SHLP2 202 nm
SHLP2 218 nm

15

Figure 9- Preliminary experiment demonstrates that SHLP2 retains IAPP in the
monomeric form using CD. 15µM of IAPP either in the presence or absence of 15µM
SHLP2 and 15µM of SHLP2 alone was incubated in 10 mM KPO4 Buffer pH=7.4 at room
temperature. Weighted Mean Residual Ellipticities (MRE’) was calculated at wavelengths of
202nm and 218nm at time points t=0, 0.5,16 and 18 hours. (Please refer to Materials and
methods for calculation of MRE’).

Following the approach of the preliminary CD kinetics experiment (Figure 9) and in line
with the time frame for the observation of IAPP fibrilization using CW-EPR, a CD
experiment was carried out in triplicates to further strengthen the findings of the CW-EPR
experiment. 15µM of IAPP either in the presence or absence of 15µM SHLP2 and 15µM of
SHLP2 alone were incubated in 10 mM KPO4 Buffer pH=7.4 at room temperature and
monitored over a period of 10 hours. The Spectra of IAPP alone and SHLP2 (Figure 10A,
10B) indicate that the structure of these peptides is largely monomeric at the start of the
experiment. These two individial spectra when added together give a closely similar
spectrum to the one observed when stoichiometric concentrations of SHLP2 and IAPP are
added together. This finding argues against the possibility that SHLP2 interacts with
monomeric IAPP . It also confirms that the absence of a change in central line amplitude at
t=0 hours when SHLP2 is added to IAPP in the CW-EPR experiment is indicative that there
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
0 5 10 15 20
θ
raw
(x10^-3*deg cm^2 dmol^-1)

Time in Hours
IAPP 202 nm
IAPP 218 nm

16

is no direct interaction between SHLP2 and the IAPP monomer. At the end of experiment the
spectra of IAPP alone, SHLP2 and SHLP2+IAPP are observed and compared to the spectra
collected at the start. Figure 10C clearly demonstrates the ability of SHLP2 to retain the
monomeric population of IAPP in comparison to the Spectra observed when IAPP is
incubated in 10mM KPO4 Buffer pH=7.4 in the absence of SHLP2 at 10 hours (Figure 10A).
CD Kinetics data displayed as MRE or MRE’ versus time (t=0 hrs to t=10hrs) for each of the
three treatment conditions was taken at two wavelengths-202nm to 218 nm like the previous
experiment. The results obtained were also similar to those onserved in Figure 9 . At t=0
hours IAPP , SHLP2 and IAPP+SHLP2 displayed a negative peak at 202nm (random-coil
structure). While SHLP2, IAPP+SHLP2 retained this peak till the end of the experiment, at
around t=7 hours, IAPP started to lose its negative peak at 202nm and gained a stronger
negative peak at 218nm(Figure 10D-F). This indicated the start of a β-sheet structure. At the
end of the experiment, the peak at 202nm for IAPP alone was closer to zero (Figure 10D).
Thus, the CD data shows that SHLP2 maintains the random-coil structure of IAPP by
targeting its higher ordered structures (pre-formed misfolded seeds).

Transmission Electron Microscopy confirms the findings of the CW-EPR as well as the CD
experiments (Figure 11). Figure 11A shows the presence of IAPP fibrils at the end of the CD
experiment while Figure 11C show that when IAPP is treated with SHLP2, the majority of
the population is in the naïve form (disordered). Even though Figure 11C indicate the
absence of fibrils, there were images that showed some fibrillar structures. One of the
reasons for this observation could be that the approach used to prepare the TEM grids was
selecting for heavier structures or another reason is that even though SHLP2 prevents IAPP
misfolding to a large extent, a few higher order species escape being targeted by SHLP2.

17

It is important to note that the TEM, which is a qualitative approach cannot be used alone to
conclude that SHLP2 can inhibit IAPP aggregation. However, with ThT fluorescence, CD
and CW-EPR, TEM images strengthen the results obtained so far.

-15
-10
-5
0
5
θ
raw
(x10^-3*deg cm^2 dmol^-1)

Wavelength in nm
A
IAPP t=0hrs
IAPP t=10
hrs
-15
-10
-5
0
5
θ
raw
(x10^-3*deg cm^2 dmol^-1)

Wavelength in nm
B
SHLP2 t=0 hrs
SHLP2 t=10 hrs
-15
-10
-5
0
5
θ
raw
(x10^-3*deg cm^2 dmol^-1)'
Wavelength in nm
C
SHLP2 to IAPP t=0
hrs
SHLP2 to IAPP t=10
hrs
-12
-10
-8
-6
-4
-2
0
0 5 10 15
θ
raw
(x10^-3*deg cm^2 dmol^-1)
Time in Hours
D
IAPP 202
nm
IAPP 218nm

18

Figure 10- SHLP2 prevents loss of IAPP monomer at stoichiometric ratios using CD

15µM of IAPP either in the presence or absence of 15µM SHLP2 and 15µM of SHLP2
alone was incubated in 10 mM KPO4 Buffer pH=7.4 at room temperature for 10 hours. A),
B), C) CD Spectra for 15µM of IAPP, 15µM of SHLP2 alone and 15µM of IAPP + 15µM
of SHLP2 were taken across the range of wavelengths from 190nm to 260nm at t=0 hrs and
at t=10 hrs.D),E), F) Mean Residual Elipticities (MRE) or Weighted Mean Residual
Ellipticities (MRE’) were calculated at wavelengths of 202nm and 218nm at 1 hour
intervals from t=0 to t=10 hours. Each data point for MRE or MRE’ is an average of three
experiments.

A B C

Figure 11- SHLP2 prevents loss of IAPP monomer at stoichiometric ratios using TEM
TEM images taken for samples monitored in the CD at t=10 hours.
A) IAPP alone, B) SHLP2 alone, and C) IAPP treated with SHLP2. Scale bars equal 200
nm.

-12
-10
-8
-6
-4
-2
0
0 5 10 15
θ
raw
(x10^-3*deg cm^2 dmol^-1)

Time in Hours
E
SHLP2
202nm
SHLP2
218nm
-12
-10
-8
-6
-4
-2
0
0 5 10 15
θ
raw
(x10^-3*deg cm^2dmol^-1)'
Time in Hours
F
SHLP2 to IAPP (1 to 1)
202nm
SHLP2 to IAPP (1 to 1)
218nm

19

e.SHLP2 can bind to sonicated pre-formed IAPP fibrils
A co-sedimentation assay was performed to observe if there was a direct interaction
between SHLP2 and pre-formed misfolded IAPP. IAPP fibrils were sonicated and then
pelleted through ultra-centrifugation in the absence or presence of SHLP2. The
supernatants and pellets of each of the 3 samples (SHLP2+ sonicated pre-formed IAPP
fibrils, sonicated pre-formed IAPP fibrils and SHLP2 alone) were blotted on a membrane
and an Anti-SHLP2 Antibody as used to probe the blots. It was found that SHLP2 gets
enriched in the pellet with sonicated pre-formed IAPP fibrils. An absence of a blot for
sonicated pre-formed IAPP fibrils alone is indicative that the Anti-SHLP2 Antibody is
SHLP2 specific. SHLP2 does get pelleted in the absence of IAPP but to a very small extent
compared to in the presence of sonicated pre-formed IAPP fibrils (Figure 12).

Figure 12- SHLP2 can bind to higher order forms of IAPP as demonstrated by the dot
blot
Representative dot blot of the supernatant and pellet from three different reactions-a)
SHLP2 + sonicated pre-formed IAPP fibrils, b) SHLP2 alone, c) sonicated pre-formed
IAPP fibrils alone. Blots were probed with α-SHLP2 antibody.

SHLP2 + IAPP SHLP2 IAPP
Supernatant
Pellet

20

f.MOTS-c, SHLP1 and SHLP6 and are stable in 10mM Phosphate buffer pH 7.4 and
D/W at room temperature over 72 hours

From stock concentrations of MOTS-c, SHLP1 and SHLP6 in D/W (1mg/ml), 45.9 µM,
41.48 µM and 41.91µM solutions respectively were prepared in a)D/W and in b)10 mM
KPO4 pH=7.4. Circular Dichroism (CD) spectra were recorded at the start of the experiment
and at the end of 72 hours at room temperature. It was observed MOTS-c, SHLP1 and
SHLP6 are monomeric in either solution as indicated by the retention of random coil
structure (peak at 202nm) using CD (Figure 13). SHLP1 and SHLP6 show no difference in
their random coil peak at 202 nm in either D/W or 10mM KPO4 Buffer pH=7.4 at t=0 hrs or
t=72hrs (Figure 13B, 13C). However, there is a decrease in the random coil peak of MOTS-c
in 10mM KPO4 Buffer pH=7.4 versus D/W at the start of the experiment and this difference
remains the same at the end of 72 hours (Figure 13A). Thus, there is no difference in the
structure of MOTS-c in 10mM KPO4 Buffer pH=7.4 at t=0 hours or t=72hours although the
structure changes with respect to which solution MOTS-c is dissolved in.

-15
-10
-5
0
5
θ
raw
(x10^-3*deg cm^2
dmol^-1)
Wavelength in nm
A
MOTS-c in H20 t=0 hrs
MOTS-c in 10mM KP04 Buffer pH 7.4 t=0 hrs
MOTS-c in H20 t=72 hrs
MOTS-c in 10mM KP04 Buffer pH 7.4 t=72 hrs

21

Figure 13A), B), C)- The structures of MOTS-c, SHLP1 and SHLP6 in 10mM Phosphate
buffer pH 7.4 and D/W are monomeric at room temperature over 72 hours
Circular dichroism spectra of MOTS-c(45.9 µM), SHLP1(41.48 µM) and
SHLP6(41.91µM) measured in D/W and 10 mM KPO4 Buffer pH=7.4 at the beginning of
the experiment and at the end of 72 hours.

g.MOTS-c can inhibit fibril formation of IAPP using Thioflavin T Fluorescence
In order to determine whether another MDP like SHLP2, namely, MOTS-c could inhibit
IAPP fibrilization, IAPP was incubated in 10mM KPO4 Buffer pH=7.4, in the presence of
different MOTS-c concentrations ranging from 25 µM to 0.25 µM. The concentration of
IAPP was kept constant at 12.5 µM . The time frame for this experiment was 18 hours and
the fibrilization kinetics was monitored using 25 µM of Thioflavin T. Figure 14A shows a
representative trace of IAPP fibril inhibition using different concentrations of MOTS-c.
-15
-10
-5
0
5
θ
raw
(x10^-3*deg cm^2
dmol^-1)
Wavelength in nm
B
SHLP1 in H20 t=0 hrs
SHLP1 in 10mM KP04 Buffer pH 7.4 t=0 hrs
SHLP1 in H20 t=72 hrs
SHLP1 in 10mM KP04 Buffer pH 7.4 t=72 hrs
-15
-10
-5
0
5
θ
raw
(x10^-3*deg cm^2
dmol^-1)
Wavelength in nm
C
SHLP6 in H20 t=0 hrs
SHLP6 in 10mM KP04 Buffer pH 7.4 t=0 hrs
SHLP6 in H20 t=72 hrs
SHLP6 in 10mM KP04 Buffer pH 7.4 t=72 hrs

22

From Figure 14A-C, it is observed that MOTS-c can inhibit IAPP fibril formation at close
to stoichiometric concentrations (at 1:1 or 2:1 as the ratio of MOTS-c:IAPP). Compared to
Figure 7A-7C, it is observed that SHLP2 is more potent that MOTS-c as it can work to a
certain extent at sub-stoichiometric concentrations (5 µM). However, with this experiment
we do not have sufficient evidence to conclude whether MOTS-c can inhibit the
fibrilization of IAPP by binding to the monomer or other higher order structure or IAPP.
We must first however, rule out the possibility that the inhibition of IAPP misfolding a
detected by ThT Fluorescence is actually due to MOTS-c binding to IAPP or it is in fact
due to the binding of MOTS-c to ThT binding site on IAPP . To answer this question we
need to employ techniques such as Circular Dichroism and CW-EPR as in the case of
SHLP2 which were useful in addressing the mechanism of IAPP inhibition.

0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20
Normalized ThT Fluorescence
Time in Hours
A
25 uM of Mots-c
25 uM of Mots-c + 12.5 uM of IAPP
0 uM of Mots-c + 12.5uM IAPP
12.5 uM of Mots-c + 12.5 uM of IAPP
5 uM of Mots-c + 12.5 uM of IAPP
1 uM of Mots-c +12.5 uM of IAPP
0.25 uM of Mots-c + 12.5 uM of IAPP

23

Figure 14- MOTS-c can inhibit fibrilization of IAPP at super-stoichiometric or
stoichiometric concentrations using Thioflavin T Fluorescence
12.5 µM IAPP was solubilized in 10mM KPO4 Buffer pH =7.4 and its fibrilization was
monitored using 25 µM of Thioflavin T in the presence or absence of MOTS-c over 18
hours. MOTS-c Concentrations ranged from 25 µM to 0.25 µM. A) Representative trace
using ThT fluorescence demonstrates the effect of different concentrations of MOTS-c on
IAPP fibrilization. B) Normalized ThT fluorescence data at time=18 hours was collected
and plotted against differing concentrations of MOTS-c(0µM-25µM). Error bars for
Normalized Tht Fluorescence at the end point indicate +/-1 Standard deviation from a
minimum of 3 experiments per condition. C) Average of t
50
for IAPP under the different
MOTS-c treatment conditions (t
50
= time to half maximal fluorescence) was calculated and
and normalized against the t
50
average for IAPP alone. Values are plotted against the
concentration of MOTS-c in µM. N/A indicates the MOTS-c treatment conditions where
the IAPP did not fibrilize.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.25 1 5 12.5 25
Normalized ThT Fluorescence at
t=18 hours
Concentration of MOTS-c (uM)
B
0
0.5
1
1.5
2
2.5
0 0.25 1 5 12.5 25
t50/t50IAPP
Concentration of MOTS-c (uM)

24

h. SHLP1 and SHLP6 do not inhibit fibril formation using Thioflavin T fluorescence
and SHLP1 increases the amount of fibrilization of IAPP.
A similar ThT Fluorescent experiment as described for SHLP2 and MOTS-c was designed
to investigate the role of SHLP1 and SHLP6 in the inhibition of IAPP fibril formation.
12.5 µM of IAPP was incubated in 10mM KPO4 Buffer pH=7.4 at room temperature in the
presence of 2 different concentrations of SHLP1 and SHLP6 -25 µM and 0.25 µM for
18hours. In contrast to SHLP2 and MOTS-c, SHLP1 and SHLP6 did not inhibit IAPP
fibrilization at either of the two concentrations (Figure 15A,15B).
The normalized time for half maximal fluorescence for each MDP treatment condition
given by t
50
/t
50IAPP
can be used to determine whether the MDP can increase the rate of
fibrilization or not. Figure 15E,15F indicate that SHLP1 and SHLP6 do not have any
significant effect on the rate of fibrilization of IAPP. However, an important result of this
experiment is that a super stoichiometric concentration of SHLP1 increases the amount of
Fibril formation significantly while the sub-stoichiometric concentration of SHLP1(0.25
µM) does not have this thermodynamic effect (Figure 15C). SHLP6 at 25 µM and at 0.25
µM does not have any effect in changing the amount of fibril formation(Figure 15D).
Circular dichroism experiments would be important to further investigate the ability of
SHLP1 to increase the amount of IAPP fibril formation.

25

0
0.5
1
1.5
2
2.5
3
0 5 10 15 20
Normalized Tht Fluorescence
Time in Hours
A
25 uM of SHLP1
12.5uM of IAPP
25 uM of SHLP1 + 12.5uM of IAPP
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20
Normalized Tht Fluoresence
Time in Hours
12.5 uM of IAPP
0.25 uM of SHLP1 +12.5 uM of IAPP
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20
Normalized Tht Fluorescence
Time in Hours
B
25 uM SHLP6 (Negative Control)
0 uM SHLP6 (IAPP Positive control)
25 uM of SHLP6 + 12.5 uM of IAPP
0.25 uM of SHLP6 + 12.5 uM of IAPP

26

Figure 15-SHLP6 and SHLP1 do not prevent IAPP misfolding using Thioflavin T
fluorescence
12.5 µM IAPP was solubilized in 10mM KPO4 Buffer pH =7.4 and its fibrilization was
monitored using 25 µM of Thioflavin T in the presence or absence of SHLP1 or SHLP6
over 18 hours. SHLP1 and SHLP6 Concentrations were either 25 µM or 0.25 µM. A),B)
Representative trace using ThT fluorescence demonstrates the effect of 2 different
concentrations of SHLP1 or SHLP6 on IAPP fibrilization. C),D)Normalized ThT
fluorescence data at time=18 hours was collected and plotted against differing
concentrations of SHLP1 or SHLP6 (0µM,25µM, 0.25µM). Error bars for Normalized Tht
Fluorescence at the end point indicate +/-1 Standard deviation from a minimum of 3
experiments per condition. E), F)Average of t
50
for IAPP under the different SHLP1 or
SHLP6 treatment conditions (t
50
= time to half maximal fluorescence) was calculated and
and normalized against the t
50
average for IAPP alone. Values are plotted against the
concentration of SHLP1 or SHLP6 in µM.
0
0.5
1
1.5
2
2.5
3
0 25 0.25
Normalized Tht Fluorescence at t=18
hrs
Concentration of SHLP1 (uM)
C
0
0.5
1
1.5
2
2.5
3
0 25 0.25
Normalized Tht Fluorescence at t=18hrs
Concentration of SHLP6 (uM)
D
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 25 0.25
T50/T50IAPP
Concentration of SHLP1 (uM)
E
0
0.2
0.4
0.6
0.8
1
1.2
0 25 0.25
T50/T50 IAPP
Concentration
of
SHLP6
(uM)

F

27

4. Discussion:
a. ThT Fluorescence studies of SHLP2,SHLP1,MOTS-c and SHLP6
The data shown here, from Thioflavin T studies of the peptides indicate that each of these
MDPs have different roles in the inhibition of IAPP misfolding. Through this assay, SHLP2
and MOTS-c demonstrate the ability to inhibit IAPP fibrilization. SHLP2 is more potent
than MOTS-c as it can work at sub-stoichiometric concentrations. MOTS-c on the other
hand, seems to inhibit fibril formation mainly at a super stoichiometric or stoichiometric
concentrations. In constrast to MOTS-c and SHLP2, it is interesting to see that other MDPs
like SHLP6 and SHLP1 have the opposing effect where increasing the concentration of
SHLP1 increases the amount of IAPP fibrilization. SHLP6 does not have any significant
kinetic or thermodynamic effect on IAPP fibrilization at either a high or low concentration
but from the ThT study on SHLP6 we can conclude that SHLP6 does not have the capacity
to inhibit fibril formation unlike SHLP2 and MOTS-c. The role of SHLPs (SHLP1, SHLP6
and SHLP2) with respect to IAPP misfolding elucidate that SHLPs may bind to different
receptors and thus serve different functions(13).
b. CW-EPR, CD and TEM analysis of SHLP2 in the inhibition of IAPP misfolding
Besides confirming the results from the ThT study, data shown here from CD and CW-EPR
suggest a possible mechanism for SHLP2 in the inhibition of IAPP misfolding. The two
main targets of SHLP2 could be either the IAPP monomer or any of its higher order
species. However, the absence of an initial change in the central line amplitude when
SHLP2 is added to MTSL labeled IAPP as monitored by CW-EPR and the absence of a
detectable initial interation between SHLP2 and IAPP by CD is indicative that the target of

28

SHLP2 is something other than the monomer. An important observation is that there is a
slight decrease in random coil structure over time detected by CD when SHLP2 is added to
IAPP and this is aggrement with a steady decrease in the central line amplitude by CW-
EPR (80% retention of initial central line amplitide). Thus, SHLP2 does not work by
completely targeting all the misfolded forms of IAPP but it may bind to a majority of them
in such a way that it prevents a seeded misfolding reaction and thereby keeps the
population largely monomeric. There are still some mifolded forms of IAPP that still exist
even in the presence of SHLP2 as indicated by TEM(data not shown).This mechanism
proposed here is similar to a “cap and contain” mechanism of CCT5 in the inhibition of
mutant HTT reported by Darrow et al (18).

c. Dot Blot provides evidence that SHLP2 works by binding to misfolded IAPP
A key experiment reported here that confirms the binding of SHLP2 to misfolded IAPP,
was the dot blot. SHLP2 pellets very strongly through ultracentrifugation in the presence of
sonicated pre-formed IAPP fibrils and can be detected by a specific antibody for SHLP2.
Thus, from the combination of results from ThT , CD, CW-EPR, EM and Dot blot , we can
conclude that SHLP2 is an inhibitor of IAPP misfolding and targets higher order IAPP
species instead of the monomer.

d. Future studies and Conclusion
Whether MOTS-c works in a similar manner to SHLP2 with respect to the inhibition of

29

IAPP misfolding is yet to be investigated. CD and CW-EPR studied must be carried out in
order to find out a possibile mechanism of action. CD analysis of SHLP1 and SHLP6
would be useful to support their inability to inhibit IAPP fibrilization. ThT analysis of 4
MDPs- SHLP2, SHLP1, SHLP6 and MOTS-c demonstrate how mitochondrially derived
peptides can have diverse functions.
Finally, the results obtained here serve as strong evidence that SHLP2 could be a potential
therapeutic for Type 2 Diabetes due to its ability to inhibit IAPP misfolding.

5. Materials and Methods:
a. Materials and peptide handling- SHLP1, SHLP6 and SHLP2 were obtained from CPC
Scientific (Sunnyvale,CA). MOTS-c was obtained from Genescript (Piscataway, NJ). Wild
type Human IAPP was purchased from Bachem Bioscience Inc. (King of Prussia, PA) and
1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl methanethiosulfonate (MTSL), was
purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). HFIP was
purchased from Sigma-Aldrich. Human IAPP cysteine mutants with alanine substitutions
for the native cysteines at positions 2 and 7 were purchased from Biomer Technology
(Pleasanton, CA). SuperBlock T20 (PBS) Blocking Buffer was obtained from Thermo
Scientific (Rockford, IL).

SHLP1, SHLP6, SHLP2 and MOTS-c handling- The different MDP’s were all treated in a
similar manner. The vials of MDPs contained 1 mg and the 1ml of D/W was added to each
vial. The solution was mixed gently and aliquots (20µl-40 µl) were prepared and placed

30

in the -20˚C freezer until use. Care was taken to see that freshly prepared aliquots of
SHLP2, MOTS-c, SHLP6 and SHLP1 were thawed and then used for the experiments
immediately . IAPP vial were suspended in 1ml of HFIP and then aliquoted so that the
concentration was 1mg/ml. The aliquots either 20µl or 50 µl in volume and were then
lyophilized (IAPP handling procedure was carried out by Alan Okada and Kazuki
Teranishi). IAPP single cysteine mutant was labelled by Dr. Mario Isas (16) and was used
for the EPR experiments. MTSL labeled IAPP (IAPP33R1) was stored at
-
20˚C in HFIP.

b.ThT Fluorescence: Lyophilized IAPP was then dissolved in 10mM KPO4 Buffer
pH=7.4 to yield a 1mg/ml solution and was added to 2mm quartz cuvettes so that the final
reaction in each cuvette contained 12.5 µM of IAPP. From a stock of 5mM Thioflavin T
dissolved in D/W, ThT was added to each reaction mixture so that the final concentration
was 25µM per condition. For the conditions which had only IAPP, an amount of D/W
equivalent to the volume of MDP required, was added. From 1 mg/ml stocks of MDPs, the
MDPs were added to IAPP so that the required concentration was obtained in each reaction
mixture. For SHLP2, 25 µM, 12.5 µM, 7.14 µM, 5 µM, 1 µM and 0.25 µM were the
concentrations used for the experiments while for MOTS-c the same range of
concentrations were used with the exception of 7.14 µM. In SHLP6 and SHLP1 ThT
fluorescence experiments, reactions containing only two concentrations -25 µM and 0.25
µM were prepared. In all the ThT experiments, 25 µM of MDP alone was used as a
negative control. An equal volume of 10 mM KPO4 Buffer pH=7.4 was added instead of
12.5 µM IAPP. The ThT fluorescence for each experiment was monitered using a JASCO
FP-6500 spectrofluorometer at excitation of 450nm and emission wavelength of 482 nm.
The excitation slit width was set at 1 nm , emission slit width was 10 nm The duration of

31

each experiment was 18 hours. t
50
values (where t
50
=time to half maximal fluorescence)
were determined as previously reported (17). The maximum Fluorescence intensity of the
positive control of each individual experiment was used to normalize the fluorescence
intensities obtained by the different conditions in a single ThT experiment.

c. CW-EPR- Cysteine mutants such as IAPP33C was labeled with MTSL (IAPP33R1) by
Dr. Mario Isas as described (16) and stored in HFIP at
_
20˚C. Stock IAPP33R1 was dried
using a constant stream of Nitrogen gas and solubilized to a concentration of ~15 µM in 10
mM phosphate buffer pH 7.4 in the presence or absence of stoichiometric equivalents of
SHLP2. Samples were drawn up into glass capillaries (0.6 mm diameter, VitroCom,
Mountain Lakes, NJ) and sealed at the end. First, the spectra of IAPP33R1 alone was
monitered using CW-EPR by a Bruker EMX Spectrometer (Billerica, MA) for over 11
hours. Then the kinetics of MTSL labeled IAPP was monitered in the presence of the MDP.
Central line amplitude was plotted against duration of the experiment to determine the
effect of MDP on inhibition of IAPP misfolding and the central line amplitudes obtained
throughout the experiment were normalized to the starting central line amplitude.

d. CD- Similar to ThT experiments, lyophilzed IAPP was taken and dissovled in 10mM
KPO4 Buffer pH=7.4 so that the final concentration of IAPP in each reaction is 15 µM.
IAPP was incubated in the presence or absence of 15 µM SHLP2 and 15 µM of SHLP2
without IAPP was prepared as a negative control. The reactions were added to 3 different
2mm quartz cuvettes and full spectra were measured at the start and the end of the
experiment (10 hours) between 195 and 260 nm in a Jasco-815 spectropolarimeter. The
spectra were initially scanned at a speed of 100nm/min and measurements taken every 0.5

32

nm with an average time of 1 second. The spectra were appropriately background
subtracted and the MRE or MRE’ were calculated as follows:
a) Mean Residual Elipticity 𝜃=
!"#$
!"#
, where θ is the mean residual ellipticity expressed
in degrees cm
2
dmol
-1
, θ
raw
is the measured ellipticity in millidegrees, n is the number of
amino acids, C is the molar concentration, and l is the path length in millimeters.
b) Mean residual Ellipticity calculations in mixed CD samples (MRE`) - For CD
experiments where IAPP and SHLP2 were measured together, MRE` values were reported.
𝜃=
!
!"#
( !
!
×!
!
!(!
!
×!
!
))!
,
Where n
1
and C
1
represent the number of amino acids and concentration of one peptide,
respectively, and n
2
and C
2
represent the number of amino acids and concentration of the
second peptide.

Kinetics of the three conditions (IAPP, SHLP2+IAPP, SHLP2) were performed by taking
the elipticity values obtained in the CD at wavelengths of 202nm and 218nm at 1 hour
intervals starting at t=0 hours till t=10 hours. MRE and MRE’ were calculated as shown
above from this value.

e. TEM- At the end of the reactions from the CD experiment, about 10µl of samples were
added to each EM grid and incubated at room temperature for 10 mins. The excess sample
was removed and the grid was stained with 1% Uranyl acetate for 2 mins which was
followed by rinsing with Uranyl acetate and then D/W water. The grids were imaged using
a Jeol-1400 transmission electron microscope at 100kV.

33

f. Dot Blot- A co-sedimentation assay was performed by preparing three different reaction
mixtures of 50 µl each such that the final concentration of SHLP2 or IAPP was 1000ng.
Pre-formed IAPP fibrils were sonicated and used in this experiment.The first reaction
mixture contained SHLP2 and IAPP in the ratio of (1000ng:1000ng), the second contained
only 1000ng of SHLP2 while the third contained 1000ng of IAPP. The reactions in
eppndorfs were then spun using an ultracentrifuge at 55,000rpm at 4˚C for 30mins. After
the spin, 90% of the supernatant was collected and the remaining solution was the pellet
which was then diluted 5 fold using 10mM KPO4 Buffer pH=7.4. 2 µl of the supernatant
and pellet of each reaction was added to a nitrocellulose membrane. The blot was incuabted
at room temperture for half an hour and then blocked using Superblock T20 (PBS)
Blocking Buffer for about 1 hour at room temperature. Anti-SHLP2 Antibody (α-SHLP2;
rabbit), the primary antibody used to probe the blot was dissolved in Superblock buffer so
that the final dilution is 1:500. The time for incubation with primary antibody was 1 hour at
room temperature on a shaker following which the antibody was recovered and the blot was
washed using Tris buffered saline with 0.005% tween 20 (1X TBST), 3 times (5 minutes
each) at room temperature. Donkey Anti-rabit(IRD 680) was the secondary Antibody used
and was dissolved in Superblock Buffer (1:10,000 Li-cor). Three 1X TBST washes were
then carried out for 15 minutes at room temperature.The blot was imaged using Li-cor
Odyssey fluorescent imager .

34

Author Contribution Statement:
Dr. Ralf Langen, Alan Okada, Kazuki Teranishi, and Dr. Pinchas Cohen contributed to the
conception and design of the project; I, Fleur Lobo contributed to the data acquisition and
analysis of the CD, TEM, Dot Blot, ThT Fluorescence for MOTS-c, SHLP2, SHLP1 and
SHLP6, and to the data acquisition of one experiment using CW-EPR with IAPP and
SHLP2. Alan Okada and Kazuki Teranishi contributed to the acquisition and analysis of
EPR data for the IAPP Positive controls and the analysis of the EPR experiment with IAPP
and SHLP2 shown in this thesis. Dr. Ralf Langen, Alan Okada, Kazuki Teranishi and I
contributed to data interpretation.

Abbreviations:
MDP - Mitochondrial-derived peptide
HN - Humanin
HNG - Humanin S14G
SHLP - Small humanin-like peptide
MOTS-c - mitochondrial open reading frame of the 12S rRNA-c
IAPP - islet amyloid polypeptide
T2DM - type 2 diabetes mellitus
ThT - Thioflavin T
MTSL -oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl methanethiosulfonate
SDSL- Site-directed Spin labeling
CW-EPR - Continuous Wave Electron Paramagnetic Resonance
CD - Circular Dichroism

35

TEM -Transmission Electron Microscopy
HFIP - Hexafluoro-2-propanol

36

References:

1. Westermark, P., Anderson, A., Westermark G.T.(2011). Islet amyloid polypeptide,
islet amyloid, and diabetes mellitus.Physio Rev. Jul;91(3):795-826.
doi:10.1152/physrev.00042.2009.

2. Hoppener, J. W. M.,Ahren, B., Lips, C. J. M. (2000). Islet amyloid and type 2
diabetes mellitus. N. Engl. J. Med., 343, 411–419.

3. Ritzel RA, Meier JJ, Lin CY, Veldhuis JD, Butler PC.(2007). Human islet amyloid
polypeptide oligomers disrupt cell coupling, induce apoptosis, and impair insulin
secretion in isolated human islets.Diabetes. Jan;56(1):65-71.

4. Tokuyama, T., Yagui, K., Yamaguchi, T., Huang, C.I., Kuramoto, N., Shimada, F.,
Miyazaki J., Horie, H., Saito, Y., Makino, H., Kanatsuka, A. (1997) .Expression of
human islet amyloid polypeptide/amylin impairs insulin secretion in mouse
pancreatic beta cells. Metabolism. 46(9):1044-51.

5. Johri, A., Beal, M.F. (2012).Mitochondrial dysfunction in neurodegenerative
diseases.J Pharmacol Exp Ther. 342(3):619-30. doi: 10.1124/jpet.112.192138.

6. Ma, A.Z., Zhao, Z., Turk, J.(2012). Mitochondrial Dysfunction and β-Cell Failure in
Type 2 Diabetes Mellitus. Experimental Diabetes Research, vol. 2012,
doi:10.1155/2012/703538

7. Hashimoto Y., Niikura T., Tajima H., et al. (2001). A rescue factor abolishing
neuronal cell death by a wide spectrum of familial Alzheimer’s dis- ease genes and
Aβ. Proc. Natl. Acad. Sci. USA 98, 6336–6341. Correction in: Proc. Natl. Acad. Sci.
USA 98, 12,854.
8. Yen, K., Lee, C., Mehta, H., Cohen, P. (2013). The emerging role of the
mitochondrial-derived peptide humanin in stress resistance. J Mol Endocrinol. Jan
11;50(1):R11-9. doi: 10.1530/JME-12-0203.

9. Jia, Y., Lue, Y.H., Swerdloff, R., Lee, K.W. Cobb, L.J, Cohen, P., Wang, C. (2013).
The cytoprotective peptide humanin is induced and neutralizes Bax after pro-
apoptotic stress in the rat testis.Andrology.Jul;1(4):651-9. doi: 10.1111/j.2047-
2927.2013.00091.x.

10. Voigt, A., Jelinek, H.F.,(2016). Humanin: a mitochondrial signaling peptide as a
biomarker for impaired fasting glucose-related oxidative stress. Physiol Rep. May;
4(9) pii: e12796. doi: 10.14814/phy2.12796.

37

11. Sreekumar, P.G., Ishikawa, K., Spee, C., Mehta, H.H., Wan, J., Yen, K., Cohen, P.,
Kannan, R., Hinton, D.R. (2016). The Mitochondrial-Derived
Peptide Humanin Protects RPE Cells From Oxidative Stress, Senescence, and
Mitochondrial Dysfunction. Invest Ophthalmol Vis Sci.Mar 1;57(3):1238-53. doi:
10.1167/iovs.15-17053.

12. Hoang, P.T., Park, P., Cobb, L.J., Paharkova-Vatchkova, V., Hakimi, M., Cohen, P.,
Lee KW.(2010). The neurosurvival factor Humanin inhibits beta-cell apoptosis via
signal transducer and activator of transcription 3 activation and delays and
ameliorates diabetes in non-obese diabetic mice. Metabolism. 59:343–349.

13. Cobb, L. J., Lee, C., Xiao, J., Yen, K., Wong, R.G., Nakamura, H.K., Mehta,
H.H.,…….,Cohen, P.(2016). Naturally occurring mitochondrial-derived peptides
are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory
markers. Aging. Apr;8(4):796-809. doi: 10.18632/aging.100943

14. Lee, C., Zeng, J., Drew, B.G., Sallam, T., Martin-Montalvo, A., Wan, J., Kim, S.J,
Mehta, H., Hevener, A.L., de Cabo R., Cohen P. (2015). The mitochondrial-derived
peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin
resistance.Cell Metab. Mar 3;21(3):443-54. doi: 10.1016/j.cmet.2015.02.009.

15. Fuku, N., Pareja-Galeano, H., Zempo, H., Alis. R., Arai, Y., Lucia, A.,
Hirose,N.(2015).The mitochondrial-derived peptide MOTS-c: a player in
exceptional longevity? Aging Cell. Dec; 14(6):921-3. doi: 10.1111/acel.12389.

16. Bedrood, S., Li,Y., Isas, J.M., Hegde, B.G., Baxa, U., Haworth, I.S., Langen,
R.(2012). Fibril structure of human islet amyloid polypeptide. J. Biol. Chem. 287,
5235–5241.

17. Jayasinghe, S. A. & Langen, R. (2005). Lipid membranes modulate the structure of
islet amyloid polypeptide. Biochemistry .44, 12113–12119.

18. Darrow, M. C., Sergeeva, O.A., Isas, J.M., Galaz-Montoya, J.G., King. J.A.,
Langen, R., Schmid, M.F., Chiu, W. (2015). Structural Mechanisms of Mutant
Huntingtin Aggregation Suppression by Synthetic Chaperonin-like CCT5 Complex
Explained by Cryo-electron Tomography. J. Biol. Chem.
jbc.M115.65537doi:10.1074/jbc.M115.655373

Abstract (if available)

Abstract Humanin, the first Mitochondrially derived peptide to be discovered along with Humanin S14G (HNG) have been known for their neuro-protection in Alzheimer’s disease, anti-apoptotic effects and protection against oxidative stress. Previous work in the Langen lab investigated whether HNG could directly interact with an amyloid protein implicated in Type 2 Diabetes and play a role in the inhibition of amyloid misfolding. Promising results revealed that HNG can inhibit IAPP misfolding. In this study another Mitochondrially derived peptide -SHLP2, was primarily used to investigate its role in the inhibition of IAPP misfolding. ThT Fluorescence, Continuous Wave-Electron Paramagnetic Resonance, Circular Dichroism and Transmission Electron Microscopy revealed that SHLP2 is an inhibitor of IAPP aggregation and works by targeting higher order species of IAPP. Thus, SHLP2 may have a chaperone like function to modify Type 2 Diabetes by inhibiting IAPP misfolding. ThT fluorescence was then used to survey three other MDPs: SHLP1, SHLP6 and MOTS-c to find out whether they function in a similar manner to SHLP2. MOTS-c was found to also inhibit IAPP fibrilization using Thioflavin T fluorescence whereas SHLP1 and SHLP6 did not inhibit IAPP fibrilization. However, SHLP1 increased the amount of fibrilization. We can conclude that SHLP2 may be a potential therapeutic for Type 2 Diabetes. More experiments need to be carried out to confirm whether MOTS-c works in a similar manner to SHLP2 to inhibit IAPP misfolding or functions via a different mechanism.

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Asset Metadata

Creator Lobo, Fleur (author)

Core Title Role of mitochondrially derived peptides in the inhibition of IAPP misfolding

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 01/07/2017

Defense Date 06/02/2016

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

Tag IAPP,inhibition of misfolding,mitochondrially derived peptides,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Langen, Ralf (committee chair), Siemer, Ansgar (committee member), Ulmer, Tobias (committee member), Xu, Jian (committee member)

Creator Email fleurlobo@gmail.com,flobo@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-262931

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