U.S. patent application number 14/213617 was filed with the patent office on 2014-10-02 for mitochondrial-derived peptide mots3 regulates metabolism and cell survival.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Laura J. COBB, Pinchas COHEN, Changhan LEE.
Application Number | 20140296139 14/213617 |
Document ID | / |
Family ID | 51537724 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140296139 |
Kind Code |
A1 |
COHEN; Pinchas ; et
al. |
October 2, 2014 |
MITOCHONDRIAL-DERIVED PEPTIDE MOTS3 REGULATES METABOLISM AND CELL
SURVIVAL
Abstract
MOTS3 is a novel polypeptide. Methods of treating diseases such
as diabetes, obesity, fatty liver, and cancer using MOTS3 and
pharmaceutical compositions thereof are disclosed herein.
Inventors: |
COHEN; Pinchas; (Pacific
Palisades, CA) ; LEE; Changhan; (Los Angeles, CA)
; COBB; Laura J.; (Sherman Oaks, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
OAKLAND |
CA |
US |
|
|
Family ID: |
51537724 |
Appl. No.: |
14/213617 |
Filed: |
March 14, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61801474 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
514/4.8 ;
514/19.3; 514/19.4; 514/19.5; 514/19.6; 514/21.4; 514/6.9; 514/7.3;
530/326; 530/387.9 |
Current CPC
Class: |
A61P 3/04 20180101; A61K
9/0019 20130101; A61K 38/1709 20130101; A61P 35/02 20180101; C07K
14/47 20130101; C07K 16/18 20130101; A61P 35/00 20180101; A61P 3/10
20180101; A61P 1/16 20180101 |
Class at
Publication: |
514/4.8 ;
530/326; 530/387.9; 514/21.4; 514/6.9; 514/7.3; 514/19.3; 514/19.4;
514/19.6; 514/19.5 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 16/18 20060101 C07K016/18 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under Grant
Nos. AG034430 and GM090311, awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. An isolated polypeptide comprising 70% sequence identity to SEQ
ID NO:1.
2. The polypeptide of claim 1, comprising 80% sequence identity to
SEQ ID NO:1.
3. The polypeptide of claim 1, comprising 90% sequence identity to
SEQ ID NO:1.
4. The polypeptide of claim 1, comprising the sequence of SEQ ID
NO:1.
5. An isolated antibody that specifically binds to the polypeptide
comprising 70% sequence identity to SEQ ID NO: 1.
6. The antibody of claim 5, wherein the antibody is a monoclonal
antibody.
7. A pharmaceutical composition comprising an isolated polypeptide
comprising 70% sequence identity to SEQ ID NO:1.
8. The composition of claim 7, wherein the polypeptide comprises
80% or 90% sequence identity to SEQ ID NO:1.
9. The composition of claim 7, wherein the polypeptide comprises
the sequence of SEQ ID NO:1.
10. A method of treating diabetes, the method comprising the step
of administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising an
isolated polypeptide comprising 70% sequence identity to SEQ ID
NO:1.
11. The method of claim 10, wherein the polypeptide comprises 80%
or 90% sequence identity to SEQ ID NO:1.
12. The method of claim 10, wherein the polypeptide comprises the
sequence of SEQ ID NO:1.
13. The method of claim 10, wherein the diabetes is type I
diabetes.
14. The method of claim 10, wherein the diabetes is type II
diabetes.
15. The method of claim 10, wherein the subject is a human.
16. A method of treating obesity and/or fatty liver, the method
comprising the step of administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising an isolated polypeptide comprising 70% sequence identity
to SEQ ID NO:1.
17. The method of claim 16, wherein the polypeptide comprises 80%
or 90% sequence identity to SEQ ID NO:1.
18. The method of claim 16, wherein the polypeptide comprises the
sequence of SEQ ID NO:1.
19. The method of claim 16, wherein the subject is a human.
20. A method of treating cancer, the method comprising the step of
administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising an
isolated polypeptide comprising 70% sequence identity to SEQ ID
NO:1.
21. The method of claim 20, wherein the polypeptide comprises 80%
or 90% sequence identity to SEQ ID NO:1.
22. The method of claim 20, wherein the polypeptide comprises the
sequence of SEQ ID NO:1.
23. The method of claim 20, wherein the subject is a human.
24. The method of claim 20, wherein the cancer is a cancer selected
from a group consisting of breast cancer, brain cancer, colon
cancer, melanoma, leukemia (e.g., AML), lymphoma, pancreatic
cancer, prostate cancer, ovarian cancer, lung cancer, and gastric
cancer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Application No. 61/801,474, filed Mar. 15, 2013, which is
incorporated herein by reference in its entirety for all
purposes.
FIELD OF THE INVENTION
[0003] This invention relates to a novel mitochondrial-derived
peptide (MOTS3). This novel peptide can be used in methods of
treating diseases such as diabetes, obesity, fatty liver, and
cancer.
BACKGROUND
[0004] Mitochondria, central to metabolic processes, is involved
energy production, programmed cell death, and reactive oxygen
species (ROS) generation, and is heavily implicated in various
stages of major diseases including cancer, diabetes,
neurodegenerative diseases, and aging; yet its role in the
pathogenesis still remains largely unclear. Traditionally,
mitochondria have been considered as "end-function" organelles,
receiving and processing vast amounts of cellular signals to
regulate energy production and cell death. However, retrograde
signaling, whereby the mitochondria communicate back to the cell,
is a poorly understood biological process.
[0005] Calcium, cytochrome C, and ROS have been considered a
retrograde mitochondrial molecule. Notably, Durieux et al. proposed
that signals originating from mitochondria can regulate the
lifespan of C. elegans in a cell non-autonomous manner, but the
nature of such signals has not been identified in worms (Durieux et
al., Cell 144, 79 (Jan. 7, 2011). The identity of such
mitochondria-derived signals may have been discovered in 2001, when
a 24 amino acid peptide, now known as humanin (HN), was cloned from
a cDNA library constructed from the surviving fraction of an
Alzheimer patient's brain and was mapped to the mitochondrial 16S
rRNA locus (Hashimoto et al., Proc Natl Acad Sci USA 98, 6336 (May
22, 2001). Since then, HN has been shown to be a cytoprotective and
anti-apoptotic factor, partially due to its role as a Bax
antagonist that prevents apoptosis in various cancer cells (Guo et
al., Nature 423, 456 (May 22, 2003) as an IGFBP-3 partner that
antagonizes the apoptotic actions of IGFBP-3 on cancer cells
(Ikonen et al., Proc Natl Acad Sci USA 100, 13042 (Oct. 28, 2003).
More recent work indicates that HN is a wide spectrum survival
factor (Nishimoto et al., Trends Mol Med 10, 102 (March, 2004)),
however, its exact mechanism of action remains unclear.
[0006] Mitochondria-derived humanin shares 92-95% identity with
several nuclear-encoded cDNAs, which represent domains within
larger hypothetical genes, whose expression has not been validated
(Tajima et al., Neurosci Lett 324, 227 (May 24, 2002)). HN
transcripts of mitochondrial origin are present in kidney, testis,
brain, and the gastrointestinal tract. Of note is that humanin is
highly conserved among species (between 90-100% homology),
including lower organisms. The peptide has been demonstrated in
brain and testis and we have shown that it is present in 1-10 ng/ml
concentrations in plasma, CSF and seminal fluid. Novel mutants and
analogs of HN with more potent actions have been described,
including HNG (514G), HNG-F6A (non-IGFBP-3 binding, which we have
recently protected as a possible type-2 diabetes treatment under a
joint AECOM/UCLA patent that has been submitted) and colivelin
(hybrid peptide containing partial sequences of HN and ADNF9).
Humanin and its analogues and derivatives are showing therapeutic
potential for an array of diseases including Alzheimer's disease,
diabetes and kidney failure.
[0007] This report is the first description of a novel open-reading
frame (ORF) in the 12S rRNA. The name of this novel ORF in the 12S
rRNA is Mitochondrial ORF in the Twelve S rRNA 3 (MOTS3). Similar
to HN (Y. Hashimoto et al., Proc Natl Acad Sci USA 98, 6336 (May
22, 2001)), the MOTS3 transcripts are polyadenylated and suggest a
gene within-a-gene structure that is well conserved throughout
species.
[0008] MOTS3 is detected in the liver, heart, testis at the same
molecular weight, but found with a slightly higher molecular weight
in the brain of mice and rats. Its main biological function is
metabolic regulation with strong influence on mitochondrial
respiration and glucose utilization in both cell culture and mice.
MOTS3 has also been tested in various ways in vitro and in vivo to
affect mitochondrial respiration, glucose utilization, insulin
regulation and cellular proliferation/survival.
[0009] Furthermore, MOTS3 is a non-toxic natural peptide derived
from the mitochondria that has a general metabolic regulatory role.
It is the first of its kind that provides strong body weight and
blood glucose regulation as well as activation of AMPK which is a
major drug target for diabetes and cancer via the mTOR pathway. It
is from an entirely novel category of drugs, i.e. mtDNA-derived
signaling peptides that have only recently been described.
Accordingly, MOTS3 and pharmaceutical formulations thereof can be
used to treat various age-related disease with much metabolic
implications such as cancer, diabetes, obesity, and
neurodegenerative diseases are not sufficient to cure the
disease.
[0010] Accordingly, this invention discloses the novel MOTS3 ORFs
and methods of use thereof to treat disease.
BRIEF SUMMARY OF THE INVENTION
[0011] In certain embodiments, this invention comprises an isolated
polypeptide comprising 70% sequence identity to SEQ ID NO:1. In
certain embodiments, the isolated polypeptide comprises an 80%
sequence identity to SEQ ID NO:1. In certain embodiments, the
isolated polypeptide comprises an 90% sequence identity to SEQ ID
NO:1. In certain embodiments, the isolated polypeptide comprises
the sequence of SEQ ID NO:1.
[0012] In certain embodiments, this invention comprises an isolated
antibody that specifically binds to a polypeptide described above.
In certain embodiments, the antibody is a monoclonal antibody.
[0013] In certain embodiments, this invention comprises an isolated
polypeptide comprising 70% sequence identity to SEQ ID NO:1. In
certain embodiments, the polypeptide comprises 80% or 90% sequence
identity to SEQ ID NO:1. In certain embodiments, the polypeptide
comprises the sequence of SEQ ID NO:1.
[0014] In certain embodiments, this invention comprises a method of
treating diabetes, the method comprising the step of administering
to a subject in need thereof a therapeutically effective amount of
a pharmaceutical composition comprising an isolated polypeptide
comprising 70% sequence identity to SEQ ID NO:1. In certain
embodiments, the polypeptide comprises 80% or 90% sequence identity
to SEQ ID NO:1. In certain embodiments, the polypeptide comprises
the sequence of SEQ ID NO:1. In certain embodiments, the diabetes
is type I diabetes. In certain embodiments, the diabetes is type II
diabetes. In certain embodiments, the subject is a human.
[0015] In certain embodiments, this invention comprises a method of
treating obesity and/or fatty liver, the method comprising the step
of administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising an
isolated polypeptide comprising 70% sequence identity to SEQ ID
NO:1. In certain embodiments, the polypeptide comprises 80% or 90%
sequence identity to SEQ ID NO:1. In certain embodiments, the
polypeptide comprises the sequence of SEQ ID NO:1. In certain
embodiments, the subject is a mammal. In certain embodiments, the
subject is a human.
[0016] In certain embodiments, this invention comprises a method of
treating cancer, the method comprising the step of administering to
a subject in need thereof a therapeutically effective amount of a
pharmaceutical composition comprising an isolated polypeptide
comprising 70% sequence identity to SEQ ID NO:1. In certain
embodiments, the polypeptide comprises 80% or 90% sequence identity
to SEQ ID NO:1. In certain embodiments, the polypeptide comprises
the sequence of SEQ ID NO:1. In certain embodiments, the subject is
a human. In certain embodiments, the cancer is a cancer selected
from a group consisting of breast cancer, brain cancer, colon
cancer, melanoma, leukemia (e.g., AML), lymphoma, pancreatic
cancer, prostate cancer, ovarian cancer, lung cancer, and gastric
cancer.
[0017] In some embodiments, this invention comprises a cDNA
molecule that encodes SEQ ID NO:1. In some embodiments, this
invention comprises a cDNA at least 80%, 90% or 100% identical to
SEQ ID NO:2.
[0018] In some embodiments, this invention comprises an expression
vector comprising a nucleic acid sequence that encodes SEQ ID NO:1.
In some embodiments, this invention comprises an expression vector
comprising a cDNA at least 80%, 90% or 100% identical to SEQ ID
NO:2.
[0019] In some embodiments, this invention provides a host cell
transformed or transfected with an expression vector as described
herein in the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A-E shows that MOTS3 is a polyadenylated transcript
from the mitochondrial 12S rRNA (A). MOTS 3 sequence is well
conserved in various organisms (B). Rabbit polyclonal anti-MOTS3
antibody recognizes overexpressed MOTS3 as well as GFP tagged MOTS3
cloned into an expression vector in cells (C). MOTS3 is found in
rat heart, liver, and testis at similar molecular weights but at a
slightly higher molecular weight in the brain (D). MOTS3 is not
detected in Rho-0 cells which do not have mitochondrial DNA (E). CO
I/II (cytochrome oxidase I/II) were used to verify lack of
mitochondrial DNA.
[0021] FIG. 2A-B depicts the effect of MOTS3 on mitochondrial
activity under 10% and 1% FBS culture conditions (A). MTT reduction
by mitochondrial reductase enzymes and oxygen consumption rate
(OCR) and extracellular acidification rate (ECAR; measures
glycolysis by pH changes in the medium made by lactate secretion)
(B).
[0022] FIG. 3 depicts the effect of exogenous MOTS3 treatment on
mitochondrial respiration. Each drug was treated to test the
specific metabolic parameter described at the top of the chart;
i.e. oligomycin-ATP turnover, FCCP-maximum respiratory capacity,
Rotenone-non-ATP respiration.
[0023] FIG. 4A-B depicts the effect of MOTS3 over-expression by
transfection on mitochondrial activity. Various versions of MOTS3
were cloned and expressed in cells.
[0024] FIG. 5 depicts the effect of exogenous MOTS3.
[0025] FIG. 6 depicts the effect of exogenous MOTS3 treatment on
glucose uptake and glycolysis.
[0026] FIG. 7 shows that MOTS3 (M3) significantly induces AMPK
activation compared to the control group (C) following 96 hours of
treatment. This correlates with the sharp decrease in glucose
availability in the medium following 96 hours of treatment in FIG.
6.
[0027] FIG. 8 shows that MOTS3 retards cellular proliferation and
survival in glucose medium, but not in galactose medium which
forces the cells to rely largely on mitochondrial function for
survival.
[0028] FIG. 9 depicts the effect of intracellular expression of
MOTS3 on cellular proliferation. MOTS3 was cloned into expression
vectors and transfected into cells.
[0029] FIG. 10 depicts the effect of exogenous MOTS 3 on cellular
senescence. Senescence was determined by senescence associated
.beta.-galactosidase levels (blue).
[0030] FIG. 11 shows that MOTS3 reduces body weight and liver
mitochondrial respiration capacity. 4 days of MOTS 3 injections
(i.p.; 0.5 and 5.0 mg/kg/day) in mice significantly reduced body
weight but did not alter food intake. Liver mitochondrial
respiration capacity was also reduced at both doses of MOTS3
[0031] FIG. 12 illustrates mice treated with MOTS3 for 4 days show
reduced blood glucose levels (A) and elevated insulin levels
(B).
[0032] FIG. 13a-j describes MOTS-c is a novel peptide encoded in
the mitochondrial genome. a, MOTS-c is encoded within the 12S rRNA
of the mitochondrial genome as a 51 bp open reading frame (ORF).
Use of the mitochondrial genetic code yields tandem start/stop
codons, whereas the cytoplasmic genetic code yields a viable
peptide. b, 3' rapid amplification of cDNA ends (3' RACE) shows
that MOTS-c and humanin transcripts are polyadenylated. c,
Comparison of MOTS-c encoded in mitochondrial DNA (mtDNA) and
peptides encoded in nuclear DNA that have been transferred from
mtDNA (NUMT) through evolution. d-e, HeLa .rho.0 cells, which are
devoid of mitochondrial DNA through prolonged exposure to low doses
ethidium bromide (EtBr), have undetectable levels of: d, 12S rRNA
and MOTS-c transcripts by qRT-PCR, and e, mitochondria-encoded
proteins cytochrome C oxidase I and II (COI/II), as well as MOTS-c,
but normally express the nuclear-encoded GAPDH detected by
immunoblotting. f, MOTS-c (green) and hsp60 (red) immunostaining in
HEK293 cells. Scale bar, 20 .mu.m. g-h, MOTS-c is detected in: g,
various tissues in mice and rats and h, circulation (plasma) in
humans and rodents. i-j, Fasting, a metabolic stress, alters
expression of endogenous MOTS-c in i, tissues, and j, plasma in
mice. Data shown as mean.+-.SEM. Student's t-test. *P<0.05,
**P<0.01, ***P<0.001
[0033] FIG. 14a-i describes MOTS-c modulates gene expression and
regulates metabolism via AICAR and AMPK signaling. a, Principal
component analysis (PCA) on HEK293 cells after 4- and 72-hours of
MOTS-c treatment (10 .mu.M; N=6). b, Parametric analysis of gene
set enrichment (PAGE) further showed time-dependent global gene
expression changes (N=6). c, Venn diagram depicting upregulated
genes (Red) and downregulated genes (Blue) is shown per time point
(N=6). d, Effect of 72 hours of MOTS-c treatment in HEK293 cells
(10 .mu.M; N=6) on various gene sets related to metabolism and
inflammation. e, The effect of MOTS-c on de novo purine
biosynthesis. The pentose phosphate pathway (PPP) and NAD.sup.+
feed into the de novo purine biosynthesis pathway (N=5). Enzymes
altered by 4- and 72-hours of MOTS-c were determined by microarray
analysis (FIG. 18b). GAR: glycinamide ribonucleotide, FGAM:
N-formylglycinamidine ribonucleotide, AIR: aminoimidazole
ribonucleotide, NCAIR: N5-carboxyaminoimidazole ribonucleotide,
SAICAR: N-succinocarboxamide-5-aminoimidazole ribonucleotide.
Colors indicate changes by MOTS-c; Red: upregulation, blue:
downregulation, and gray: unmeasured. f, HEK293 stably
over-expressing MOTS-c have significantly increased AICAR levels
(N=5). g, Metabolic intermediates of the de novo purine
biosynthesis pathway in HEK293 cells stably over-expressing MOTS-c
are shown (N=5). h, AICAR is a potent activator of AMPK. MOTS-c
activates AMPK and its downstream pathways control fatty acid
oxidation (ACC and CPT-1), and glucose uptake (GLUT-4). i, MOTS-c
promotes AMPK and Akt phosphorylation in a time- and dose-dependent
manner. Data shown as mean.+-.SEM. Student's t-test. *P<0.05,
**P<0.01,***P<0.001
[0034] FIG. 15a-k describes MOTS-c coordinates cellular glucose,
mitochondrial, and fatty acid metabolism. Measurements of: a,
glucose and b, lactate in the cell culture medium after MOTS-c
treatment (10 .mu.M; N=6). c, MOTS-c-dependent glycolytic
intermediate changes detected by metabolomics. d, Real-time
glycolytic flux was determined by extracellular acidification rate
(ECAR) (N=6). Glycolysis, glycolytic capacity, and glycolytic
reserve were estimated by challenging cells with glucose,
oligomycin, and 2-deoxyglucose, respectively. e-f, real-time oxygen
consumption rate (OCR) was measured after MOTS-c treatment (N=6).
e, ATP turnover, and maximum respiratory capacity was estimated by
challenging cells with oligomycin and FCCP and f, spare respiratory
capacity, proton leakage, and coupling efficiency was calculated.
g, TCA intermediates after MOTS-c treatment determined by
metabolomics are shown (N=5). h, Cell number was counted after
MOTS-c treatment under glucose or galactose medium. Galactose
forces mammalian cells to rely on mitochondrial metabolism (N=6).
MOTS-c induced changes in intracellular i, acyl-carnitine shuttles,
j, essential fatty acids, and k, the .beta.-oxidation intermediate
myristoyl CoA determined by metabolomics (N=5). Data shown as
mean.+-.SEM. Student's t-test. *P<0.05, **P<0.01,
***P<0.001.
[0035] FIG. 16a-n describes MOTS-c regulates weight, metabolic
homeostasis, and insulin sensitivity in mice. a, Effect of acute
4-day MOTS-c (5 mg/kg/day; IP; BID) treatment on circulating levels
of the major adipokines IL-6 and TNF.alpha. in the outbred CD-1
male mice (N=6). b-f, 8-week old male CD-1 mice fed a high fat diet
(60% by calories) or matched control diet (N=10). MOTS-c was
injected intraperitoneally daily (0.5 mg/kg/day). b, body weight,
c, blood glucose, d, insulin, e, liver H&E staining, f,
skeletal muscle AMPK phosphorylation and GLUT4 levels. g, The
effect of acute MOTS-c treatment (5 mg/kg/day; IP) for 7 days on
intraperitoneal glucose tolerance test (IPGTT) performed on male
C57BL/6 mice (N=7). h-j, Euglycemic-hyperglycemic clamps were
performed on C57BL/6 mice fed a high-fat diet (60% by calories) and
treated with MOTS-c (5 mg/kg/day; IP) for 7 days (N=6-8). h,
glucose infusion rate (GIR), reflecting whole body insulin
sensitivity, i, insulin-stimulated glucose disposal rate (IS-GDR),
primarily reflecting muscle insulin sensitivity, and j, hepatic
glucose production (HGP). k-l, MOTS-c levels in young (4-month) and
aged (32-month) mice (N=3-4) decline in k, skeletal muscle, and 1,
circulation (serum). m, The effect of acute MOTS-c treatment (5
mg/kg/day; IP) for 7 days on insulin-stimulated (60 .mu.U/ml)
2-deoxyglucose uptake into soleus muscles of young (3-month) and
older (12-month) male C57BL/6 mice (N=6). n, proposed model for
MOTS-c action. Data shown as mean.+-.SEM. Student's t-test
*P<0.05, **P<0.01, ***P<0.001.
[0036] FIG. 17a-f describes a, MOTS-c peptide sequence is highly
conserved. b, putative post-translational modifications of MOTS-c.
c, MOTS-c antibodies were generated and tested for specificity
against MOTS-c by immunoblotting. Western blots showing MOTS-c
detection from HEK293 cells transfected with pEV, pMOTS-c,
pMOTS-c-EGFP, pEGFP. d, HEK293 cells were stained with anti-MOTS-c
antibody (green) alone or in the presence of MOTS-c peptide
(blocking) e, MOTS-c (green) and hsp60 (red) immunostaining in
HEK293 cells. Nuclei were stained with Hoechst 33258 (blue). Scale
bar, 20 .mu.m. f, standard-curve of an in-house MOTS-c ELISA, which
was used to measure MOTS-c in plasma.
[0037] FIG. 18a-b describes the effect of MOTS-c on the de novo
purine biosynthesis pathway (N=5). a, MOTS-c treatment (10 .mu.M;
24- and 72-hours post-treatment) regulates the de novo purine
biosynthesis pathway in HEK293 cells. b, MOTS-c treatment (10
.mu.M) alters gene expression of pathways involved in de novo
purine biosynthesis as determined by microarray and Ingenuity
Pathway Analysis (IPA) (N=6). See FIG. 14e for overlay with
metabolomics analysis. Red: upregulation, green: downregulation.
Student's t-test between groups within the same time point.
*P<0.05, **P<0.01,***P<0.001
[0038] FIG. 19a-b describes the effect of MOTS-c on adenylic system
components and nicotinamide adenine dinucleotide (NAD) cycle
components. a, ATP, ADP, AMP, and cyclic AMP (cAMP) levels, and b,
NAD.sup.+ and NADH levels in HEK293 cells stably over-expressing
MOTS-c. EV: HEK293 stably transfected with empty vector. Student's
t-test. *P<0.05, **P<0.01, ***P<0.001
[0039] FIG. 20a-b describes the effect of MOTS-c on the components
of a, purine metabolism and b, cofactors and vitamin metabolism in
HEK293 cells stably over-expressing MOTS-c, determined by
metabolomics (N=5). EV: HEK293 stably transfected with empty
vector.
[0040] FIG. 21a-b describes the effect of MOTS-c on a,
extra-cellular (culture medium) levels of glucose and lactate in
HEK293 cells stably over-expressing MOTS-c (N=6), and b,
intracellular glucose uptake rate, determined by the fluorescent
glucose analog 2-NBDG, in HEK293 cells treated with MOTS-c (10
.mu.M; 72-hours) (N=3). Student's t-test. ***P<0.001
[0041] FIG. 22 describes the effect of MOTS-c on the metabolism of
various sugar substrates in HEK293 cells stably over-expressing
MOTS-c, determined by metabolomics (N=5). EV: HEK293 stably
transfected with empty vector
[0042] FIG. 23a-c describes intermediate metabolites, determined by
metabolomics (N=5), of a, glycolysis and b, the pentose phosphate
pathway (PPP) following 24- or 72-hours of exogenous MOTS-c (10
.mu.M) treatment in HEK293 cells, and c, PPP in HEK293 cells stably
over-expressing MOTS-c. EV: HEK293 stably transfected with empty
vector. Student's t-test between groups within the same time point.
*P<0.05, **P<0.01,***P<0.001
[0043] FIG. 24 describes the effect of exogenous MOTS-c treatment
(10 .mu.M; 24- or 72-hours) on the levels of the tricarboxylic acid
(TCA) cycle intermediates in HEK293 cells, determined by
metabolomics (N=5). Student's t-test between groups within the same
time point. *P<0.05, **P<0.01, ***P<0.001
[0044] FIG. 25 describes the effect of various MOTS-c expression
clones on oxygen consumption rate (OCR) in HEK293 cells (N=12).
MOTS-c-HA: MOTS-c tagged with HA at the N-terminus;
MOTS-c-IRES-GFP: MOTS-c cloned into an expression vector that
independently expresses GFP via the internal ribosome entry site
(IRES); MOTS-c-HA-IRES-GFP: MOTS-c-HA cloned into an expression
vector with IRES-GFP. Student's t-test. *P<0.05, **P<0.01,
***P<0.001
[0045] FIG. 26a-b describes the cellular redox homeostasis was
determined by the state of the glutathione system. Glutathione in
its reduced (GSH) and oxidized (GSSG) form, their ratio (GSH/GSSG),
and total glutathione levels (GSH+GSSG) were determined in a,
HEK293 cells stably over-expressing MOTS-c and b, HEK293 cells
treated with exogenous MOTS-c (10 .mu.M) for 24- or 72-hours (N=5),
EV: HEK293 stably transfected with empty vector. Student's t-test.
*P<0.05, **P<0.01
[0046] FIG. 27a-d describes the effect of MOTS-c on lipid
metabolism. a, MOTS-c regulates lipid metabolism in HEK293 cells
stably over-expressing MOTS-c. Exogenous MOTS-c treatment (10
.mu.M; 24- or 72-hours) regulates the levels of b, the
carnitine-shuttle system members, c, essential fatty acids, and d,
the .beta.-oxidation intermediate myristoyl-CoA. Student's t-test.
*P<0.05, **P<0.01, ***P<0.001
[0047] FIG. 28a-b describes the levels of long-chain fatty acids in
a, HEK293 cells stably over-expressing MOTS-c and b, HEK293 cells
treated with exogenous MOTS-c (10 .mu.M) for 24- or 72-hours,
determined by metabolomics (N=5). EV: HEK293 stably transfected
with empty vector. Student's t-test. *P<0.05, **P<0.01,
***P<0.001
[0048] FIG. 29a-f describes the effect of acute MOTS-c (5
mg/kg/day; IP; BID) treatment for 4 days on a, body weight change
(%), b, food intake, c, blood glucose, and d, adipokines in CD-1
male mice (N=6). e-f, The effect of MOTS-c (0.5 mg/kg/day) on e,
daily food intake and f, total food/caloric intake of mice fed a
high-fat diet (60% by calories) for 8 weeks. Student's t-test
between groups within the same time point. *P<0.05,
**P<0.01
[0049] FIG. 30 describes microarray data on HEK293 cells treated
with MOTS-c (10 .mu.M) for 72-hours (N=6). See FIG. 14d.
[0050] FIG. 31 describes the total number of biochemicals
(metabolites) in HEK293 cells with significant (P.ltoreq.0.05) and
borderline significant (0.05<P<0.1) changes 24- and 72-hours
after exogenous MOTS-c treatment (10 .mu.M) or stably
over-expressing MOTS-c or empty vector (EV) (N=5). Red: increased,
Blue: decreased. Welch's Two Sample t-tests.
[0051] FIG. 32 describes the metabolic parameters and circulating
factors from the euglycemic-hyperinsulinemic clamp study in FIG. 16
h-j. Student's t-test.
[0052] FIG. 33 describes Day 6 fasting blood sugars. ZDF diabetic
rats were followed until their blood sugar was >300 ng/ml, at
which point treatment was initiated with one of two doses of MOTS-c
or insulin, given IV at 8 AM for 6 days. 6 rats per group were
used. Fasting blood sugars were measured after 6-days. ANOVA and
student t-tests were used for analysis and p-values.
[0053] FIG. 34 describes absolute change in blood glucose at Day 6.
In the same experiment described in FIG. 33, absolute changes in
blood glucose are shown. Saline treated ZDF rats exhibit a further
rise of 100 ng/ml in their blood sugar. Rats treated with daily
insulin stabilize their blood sugar at levels similar to baseline,
while MOTS-c treated rats (1 mg per 400 gram rat) show a
substantial reduction of their blood sugar.
[0054] FIG. 35A-B describes MOTS-c stimulates glycolytic flux
evidenced by increased glucose uptake and lactate production.
Extracellular (A) glucose and (B) lactate in the culture medium of
HEK293 cells stably over-expressing MOTS-c (N=6)
[0055] FIG. 36 describes MOTS-c enhances cellular glucose flux in
vitro and acute treatment reduces glucose levels in mice fed a
normal diet. To test insulin-sensitivity, we treated mice with
intraperitoneal injections of MOTS-c (5 mg/kg/day) for 7 days and
then subjected them to a glucose tolerance test (GTT; 1 g/kg
glucose) in male C57BL/6 mice (N=7). We found significantly
enhanced glucose clearance indicative of improved insulin
sensitivity.
[0056] FIG. 37A-E describes testing of the effects of MOTS-c (0.5
mg/kg/day; IP) on metabolism in the context of a high-fat diet (HFD
60% by calories) (N=10). Although MOTS-c treatment had no effect on
body weight when fed a normal diet, it remarkably prevented obesity
in mice fed a HFD (A). (B-C) This difference in body weight was not
attributed to food intake, as caloric intake was identical between
the groups. (D-E) Additionally, MOTS-c treatment prevented
HFD-induced hyperinsulinemia, indicating improved glucose
homeostasis. (F) MOTS-c also reduced HFD-induced fatty liver
determined by the liver H&E staining.
[0057] FIG. 38A-E describes testing of the effects of MOTS-c (5.0
mg/kg/day; IP) on high-fat diet (HFD 60% by calories) in mice from
a different genetic background, C57BL/6 (N=12). (A) Similar to the
CD-1 mice, MOTS-c treatment prevented obesity in C57BL/6 mice fed a
HFD. (B) The reduced weight gain can be partially attributed to
reduced levels of visceral fat. (C) This difference in body weight
was not due to less food intake. Furthermore, MOTS-c treatment
prevented HFD-induced hyperinsulinemia, indicating improved glucose
homeostasis (D-E). ND: normal diet, HFD: high fat diet.
[0058] FIG. 39 shows the effect of MOTS-c on whole body metabolism,
outbred CD-1 male mice fed a normal diet were treated with MOTS-c
(5 mg/kg/day; BID, 4 days) or vehicle control (N=6). (4-day)
treatment with MOTS-c reduced body weight, food and blood glucose
levels. Also, basal levels of circulating IL-6 and TNF.alpha. (A),
implicated in the pathogenesis of obesity and insulin resistance,
were significantly reduced by MOTS-c treatment.
[0059] FIG. 40A-D shows cancer cells are known to have deranged
metabolism to support its rampant growth. MOTS-c is regulator of
metabolic homeostasis, and affects the proliferative capacity of a
malignant cell. Breast cancer cells (4T1) stably over-expressing
MOTS-c show (A) reduced proliferation rate and (B) reduced
respiration. Also, (C) MOTS-c treatment retarded breast cancer
(4T1) proliferation in a subcutaneous allograft mouse model; (D)
MOTS-c treatment was not toxic as reflected by stable body weight
maintenance.
[0060] FIG. 41A-D shows that similar to the breast cancer, MOTS-c
also retards the growth of a prostate cancer cells. (A) MOTS-c
treatment retarded prostate cancer cell (22Rv1) proliferation in
vitro. (B-C) Also, MOTS-c treatment retarded prostate cancer
(22Rv1) proliferation in a subcutaneous allograft mouse model; (D)
MOTS-c treatment was not toxic as reflected by stable body weight
maintenance.
[0061] FIG. 42 describes that MOTS-c treatment retards
proliferation of 4 different prostate cancer cell lines in vitro in
a dose dependent manner. MTT reduction was used to assess cellular
proliferation and relative quantity (RQ) to the control is
presented. Cells were treated for 48-72 hours in optimum growth
conditions.
DEFINITIONS
[0062] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below.
[0063] As used herein, the term "diabetes" refers to the broad
class of disorders characterized by impaired insulin production and
glucose tolerance. Diabetes includes type 1 and type 2 diabetes
(also called juvenile and adult-onset, respectively), gestational
diabetes, prediabetes, insulin resistance, metabolic syndrome, and
impaired glucose tolerance. Common symptoms include frequent
urination, excessive thirst, extreme hunger, unusual weight loss,
increased fatigue, irritability, and blurry vision. Diagnosis of
these individual disorders is described in more detail below.
[0064] Type I diabetes is also known as Insulin Dependent Diabetes
Mellitus (IDDM), Type 1 diabetes, and juvenile diabetes. The terms
are used interchangeably herein. Treatment and diagnosis of the
disease is described in more detail below
[0065] "Pancreatic beta cells," "beta islet cells," and similar
terms refer a population of pancreatic endocrine cells found in the
Islets of Langerhans. Beta islet cells produce and secrete insulin
and amylin into the bloodstream.
[0066] As used herein, "improving cell survival" refers to an
increase in the number of cells that survive a given condition, as
compared to a control, e.g., the number of cells that would survive
the same conditions in the absence of treatment. Conditions can be
in vitro, in vivo, ex vivo, or in situ. Improved cell survival can
be expressed as a comparative value, e.g., twice as many cells
survive if cell survival is improved two-fold. Improved cell
survival can result from a reduction in apoptosis, an increase in
the life-span of the cell, or an improvement of cellular function
and condition. In some embodiments, cell survival is improved by 5,
10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, as compared to control
levels. In some embodiments, cell survival is by two-, three-,
four-, five-, or ten-fold of control levels. Alternatively,
improved cell survival can be expressed as a percentage decrease in
apoptosis. In some embodiments, for example, apoptosis is reduced
by 10, 20, 30, 40, 50, 60, 70, 80, 90 or up to 100%, as compared to
a control sample.
[0067] The term "preventing a disorder" as used herein, is not
intended as an absolute term. Instead, prevention, e.g., of type 1
diabetes, refers to delay of onset, reduced frequency of symptoms,
or reduced severity of symptoms associated with the disorder.
Prevention therefore refers to a broad range of prophylactic
measures that will be understood by those in the art. In some
circumstances, the frequency and severity of symptoms is reduced to
non-pathological levels, e.g., so that the individual does not need
traditional insulin replacement therapy. In some circumstances, the
symptoms of an individual receiving the compositions of the
invention are only 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% as
frequent or severe as symptoms experienced by an untreated
individual with the disorder.
[0068] Similarly, the term "treating a disorder" is not intended to
be an absolute term. In some aspects, the compositions of the
invention seek to reduce the loss of insulin producing cells that
lead to diabetic symptoms. In some circumstances, treatment with
the leads to an improved prognosis or a reduction in the frequency
or severity of symptoms.
[0069] As used herein, the term "an individual in need of treatment
or prevention" refers to an individual that has been diagnosed with
type 1 diabetes, type 2 diabetes, gestational diabetes,
pre-diabetes, insulin resistance, metabolic syndrome, or impaired
glucose tolerance, or one that is at risk of developing any of
these disorders. Individuals in need of treatment also include
those that have suffered an injury, disease, or surgical procedure
affecting the pancreas, or individuals otherwise impaired in their
ability to make insulin. Such individuals can be any mammal, e.g.,
human, dog, cat, horse, pig, sheep, bovine, mouse, rat, rabbit, or
primate.
[0070] As used herein, the term "percentage of sequence identity"
is determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (e.g.,
a polypeptide of the invention), which does not comprise additions
or deletions, for optimal alignment of the two sequences. The
percentage is calculated by determining the number of positions at
which the identical nucleic acid base or amino acid residue occurs
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
[0071] As used herein, the terms "identical" or percent "identity,"
in the context of two or more nucleic acids or polypeptide
sequences, refer to two or more sequences or subsequences that are
the same sequences. Sequences are "substantially identical" if two
sequences have a specified percentage of amino acid residues or
nucleotides that are the same (i.e., 60% identity, optionally 65%,
70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region,
or, when not specified, over the entire sequence), when compared
and aligned for maximum correspondence over a comparison window,
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection,
or across the entire sequence where not indicated. The invention
provides polypeptides or polynucleotides encoding polypeptides that
are substantially identical, or comprising sequences substantially
identical, to the polypeptides exemplified herein (e.g., humanin)
This definition also refers to the complement of a nucleotide test
sequence.
[0072] A "pre-diabetic individual" refers to an adult with a
fasting blood glucose level greater than 110 mg/dl but less than
126 mg/dl or a 2 hour PG reading of greater than 140 mg/dl but less
than 200 mg/dl. A "diabetic individual" refers to an adult with a
fasting blood glucose level greater than 126 mg/dl or a 2 hour PG
reading of greater than 200 mg/dl.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0073] A decade ago, the first mitochondria-derived peptide (MDP),
humanin (HN), was cloned from a cDNA library constructed from the
non-affected fraction of an Alzheimer patient's brain and was
mapped to the mitochondrial 16s rRNA locus. HN has been shown to be
a cytoprotective and anti-apoptotic factor (Bachar, A. R., et al.,
(2010). Humanin is expressed in human vascular walls and has a
cytoprotective effect against oxidized LDL-induced oxidative
stress. Cardiovascular research 88, 360-366; Guo, B., et al.,
(2003). Humanin peptide suppresses apoptosis by interfering with
Bax activation. Nature 423, 456-461; Hashimoto, Y., et al. (2001).
A rescue factor abolishing neuronal cell death by a wide spectrum
of familial Alzheimer's disease genes and Abeta. Proc Natl Acad Sci
USA 98, 6336-6341; Hoang, P. T., et al., (2010). The neurosurvival
factor Humanin inhibits beta-cell apoptosis via signal transducer
and activator of transcription 3 activation and delays and
ameliorates diabetes in nonobese diabetic mice. Metabolism:
clinical and experimental 59, 343-349; and Ikonen, M., et al.
(2003). Interaction between the Alzheimer's survival peptide
humanin and insulin-like growth factor-binding protein 3 regulates
cell survival and apoptosis. Proc Natl Acad Sci USA 100,
13042-13047). There are now over 130 publications describing
various aspects of HN biology.
[0074] Building previous observations, an additional MDP encoded
within the 12S rRNA and have named it MOTS3 (Mitochondrial
Open-reading-frame within the Twelve S rRNA) was discovered in
silico and is disclosed herein (Table 1).
TABLE-US-00001 TABLE 1 Sequence and location of MOTS3 (SEQ ID NO: 1
and SEQ ID NO: 2). Trans- Loca- Name lation tion Region Sequence
#AA MOTS3 Cyto- 1343- 696-746 MRWQEMGYIFYPRKLR 16 plasmic 1393 (SEQ
ID NO: 1) ATGAGGTGGCAAGAAA TGGGCTACATTTTCTA CCCCAGAAAACTACGA TAG
(SEQ ID NO: 2)
[0075] As reported herein, MOTS3 shifts the cellular metabolic
state by modulating mitochondrial function/metabolism and glucose
utilization. This has much potential to be applied to various
diseases in which mitochondria play a major role in either
pathogenesis or maintenance/progression of the disease, such as
cancer, diabetes, fatty liver, neurodegenerative diseases,
hypertension, and aging.
[0076] Furthermore, mitochondrial dysfunction plays a central role
in the pathogenesis of neurodegenerative disorders, including
Alzheimer's disease (AD) as well as hypertension.
[0077] In cancer, mitochondria are central organelles for
biosynthetic precursor production to supply its signature rampant
proliferation, which can be counteracted by MOTS3-dependent
mitochondrial function suppression and reduced cellular
proliferation (Ward and Thompson, (2012) Metabolic reprogramming: a
cancer hallmark even warburg did not anticipate. Cancer cell 21,
297-308).
[0078] Metabolic diseases such as obesity and fatty liver could
also be treated with MOTS3 as it is effective in regulating body
weight and hepatic mitochondrial respiration under the same amount
of food consumption (FIG. 11). In diabetes, drugs that activate
Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK),
such as metformin and AICAR, have been well documented to regulate
glucose homeostatsis and currently used in clinics (Zhang et al.,
(2009) AMPK: an emerging drug target for diabetes and the metabolic
syndrome. Cell metabolism 9, 407-416). Accordingly, MOTS3 is also a
strong activator of AMPK (FIG. 6) as well as a significant
regulator of glucose levels (FIG. 12A) and thus could be used to
treat diabetes.
Diabetes
[0079] Type II diabetes (non-insulin dependent diabetes) is a
common metabolic disorder that is rapidly increasing particularly
in the developed world. It can be characterized by insulin
resistance, insulin deficiency and hyperglycaemia. Factors that are
linked with Type II diabetes include elevated cholesterol, obesity
and hypertension.
[0080] Type II diabetes may not be diagnosed for many years since
symptoms may be sporadic and are certainly milder than those
associated with Type I diabetes. However, elevated blood sugar
levels in untreated Type II diabetes sufferers can lead to
functional impairment of kidneys, eyes and cardiovascular
systems.
[0081] Furthermore, Type II diabetes may be caused by the failure
of beta cells to compensate for insulin resistance. High-caloric
diets and insufficient muscle work seem to be important
environmental factors involved in the pathogenesis of obesity and
Type II diabetes. Environmental factors seem to act via two major
targets. One is the processing of glucose, fatty acids and other
metabolites, as regulated by insulin and other hormones in the
majority of tissues, and the other is beta cell function.
[0082] Obesity has become a major public health problem. Health
conditions caused or exacerbated by obesity include hypertension,
diabetes mellitus, sleep apnea, obesity-related hypoventilation,
back and joint problems, cardiovascular disease, non-alcoholic
fatty liver disease and gastroesophageal reflux disease.
[0083] The body mass index (BMI) (calculated as weight in kilograms
divided by the square of height in meters) is the most commonly
accepted measurement for overweight and/or obesity. A BMI exceeding
25 is considered overweight, while obesity is defined as a BMI of
30 or more, with a BMI of 35 or more considered as serious
comorbidity and a BMI of 40 or more considered morbid obesity.
[0084] Type I diabetes is an autoimmune disease characterized by
the progressive destruction of pancreatic beta cells following
infiltration of the islet by lymphocytes. This results in insulin
deficiency.
[0085] Apoptosis is the primary mode of beta cell death during
development of Type-1 diabetes (O'Brien et al. (1997) Diabetes
46:750-57). IL-1, TNF-alpha and IFN-gamma are released by T cells
and macrophages during this autoimmune response and are important
mediators of beta cell destruction (Eizirik and Mandrup-Poulsen
(2001) Diabetologia 44:2115-2133).
[0086] Insulin-like Growth Factor Binding Protein-3 (IGFBP-3)
induces apoptosis in a manner unrelated to its IGF binding (Rajah
et al. (1997) J Biol. Chem. 272:12181-88). Pro-inflammatory Th1
cytokines increases intranuclear aggregation of endogenous IGFBP-3
and addition of exogenous IGFBP-3 to beta cells induces apoptosis
(Shim et al. (2004) Growth Norm IGF Res. 14:216-25). IGFBP-3 is one
of a number of peptides that includes insulin, leptin, adiponectin,
and resistin, that have been shown to act in the central nervous
system to regulate glucose metabolism (Muse et al. (2007) J Clin
Invest. 117:1670-78; Obici et al. (2002) Nat Med 8:1376-82). Beta
cell loss by apoptosis also occurs after islet graft (Paraskevas et
al. (1999) FEBS Lett. 455:203-8); Tobiasch et al. (2001) J Investig
Med. 49:566-71). Recent studies have demonstrated that isolated
human islets express the pro-apoptotic protein Bax at higher level
than the anti-apoptotic protein Bcl-2 (Thomas et al. (2002)
Transplantation 74:1489).
[0087] Over a million people in the U.S. have Type I diabetes.
According to the American Diabetes Association, the disease causes
thousands of deaths every year and costs more than $20 billion
annually. There is need effective therapeutic or preventative agent
available for Type I diabetes.
[0088] There is a need to develop methods to treat and prevent Type
I and Type II diabetes. Accordingly, in certain embodiments of this
invention, MOTS3 and pharmaceutical compositions thereof can be
used to treat and prevent Type I and Type II diabetes.
Cancer
[0089] Cancer is a serious threat to modern society. Worldwide,
more than 10 million people are diagnosed with cancer every year
and it is estimated that this number will grow to 15 million new
cases every year by 2020. Cancer causes six million deaths every
year or 12% of the deaths worldwide.
[0090] Malignant cancerous growths, due to their unique
characteristics, pose serious challenges for modern medicine. Their
characteristics include uncontrollable cell proliferation resulting
in unregulated growth of malignant tissue, an ability to invade
local and even remote tissues, lack of differentiation, lack of
detectable symptoms and most significantly, the lack of effective
therapy and prevention.
[0091] Cancer can develop in any tissue of any organ at any age.
The etiology of cancer is not clearly defined but mechanisms such
as genetic susceptibility, chromosome breakage disorders, viruses,
environmental factors and immunologic disorders have all been
linked to a malignant cell growth and transformation. Cancer
encompasses a large category of medical conditions, affecting
millions of individuals worldwide. Cancer cells can arise in almost
any organ and/or tissue of the body. Cancer develops when cells in
a part of the body begin to grow or differentiate out of control.
All cancer types begin with the out-of-control growth of abnormal
cells.
[0092] There are many types of cancer, including but not limited
to, brain cancer, colon cancer, melanoma, leukemia (e.g., AML),
lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, lung
cancer, and gastric cancer.
[0093] Currently, some of the main treatments available are
surgery, radiation therapy, and chemotherapy. Surgery is often a
drastic measure and can have serious consequences. For example, all
treatments for ovarian cancer may result in infertility. Some
treatments for cervical cancer and bladder cancer may cause
infertility and/or sexual dysfunction. Surgical procedures to treat
pancreatic cancer may result in partial or total removal of the
pancreas and can carry significant risks to the patient. Breast
cancer surgery invariably involves removal of part of or the entire
breast. Some surgical procedures for prostate cancer carry the risk
of urinary incontinence and impotence. The procedures for lung
cancer patients often have significant post-operative pain as the
ribs must be cut through to access and remove the cancerous lung
tissue. In addition, patients who have both lung cancer and another
lung disease, such as emphysema or chronic bronchitis, typically
experience an increase in their shortness of breath following the
surgery. Radiation therapy has the advantage of killing cancer
cells but it also damages non-cancerous tissue at the same time.
Chemotherapy involves the administration of various anti-cancer
drugs to a patient but often is accompanied by adverse side
effects.
[0094] Thus, there remains a need for methods that can treat and
prevent cancer. These methods can provide the basis for
pharmaceutical compositions useful in the prevention and treatment
of cancer in humans and other mammals. Accordingly, in certain
embodiments of this invention, MOTS3 and pharmaceutical
compositions thereof can be used to treat and prevent cancer.
Specifically, as non-limiting examples, MOTS3 and pharmaceutical
compositions thereof can be used to treat and prevent brain cancer,
colon cancer, melanoma, leukemia (e.g., AML), lymphoma, pancreatic
cancer, prostate cancer, ovarian cancer, lung cancer, and gastric
cancer.
Fatty Liver and Obesity
[0095] Fatty liver, also known as Fatty Liver Disease (FLD), is a
reversible condition where vacuoles of triglyceride fats accumulate
in the liver. The process of fat accumulation in the liver cells is
known as steatosis.
[0096] Obesity is a general term for describing having too much
body fat. Obesity occurs when a subject takes in more calories than
he/she can burn, leading to stores of fat. Obesity is caused by
taking in more calories than a subject needs and not getting enough
exercise. Two of the most common methods for assessing obesity are
the Body Mass Index (BMI) and waist circumference. BMI is
calculated using height and weight. A high BMI, i.e., obesity,
increases the risk of Type II diabetes, heart disease, and
stroke.
[0097] Thus, there remains a need for methods that can treat and
prevent fatty liver disease and obesity. Accordingly, in certain
embodiments of this invention, MOTS3 and pharmaceutical
compositions thereof can be used to treat and prevent fatty liver
disease and obesity.
Expression and Purification of Polypeptides
[0098] Naturally-occurring, synthetic, or recombinant polypeptides
of the invention can be purified for use in compositions and
functional assays. Naturally-occurring polypeptides of the
invention can be purified from any source. Recombinant polypeptides
can be purified from any suitable expression system (e.g.,
mammalian, insect, yeast, or bacterial cell culture).
[0099] The peptides of the present invention (i.e., MOTS3 and MOTS3
analogues) may include both modified peptides and synthetic peptide
analogues. In some embodiments the peptide is a peptide 70%, 75%,
80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO:1. In some
embodiments the peptide is a peptide comprising SEQ ID NO:1. In
some embodiments, the peptide is SEQ ID NO:1. Peptides as described
herein may be modified to improve formulation and storage
properties, or to protect labile peptide bonds by incorporating
non-peptidic structures. Peptides of the present invention may be
prepared using methods known in the art. For example, peptides may
be produced by chemical synthesis, e.g., using solid phase
techniques and/or automated peptide synthesizers. In certain
instances, peptides may be synthesized using solid phase strategies
on an automated multiple peptide synthesizer (Abimed AMS 422) using
9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. The peptides can
then be purified by reversed phase-HPLC and lyophilized.
[0100] For recombinant approaches, the present invention includes
isolated nucleic acids encoding the polypeptides disclosed herein,
expression vectors comprising the nucleic acids, and host cells
comprising the expression vectors. More particularly, the invention
provides isolated nucleic acids encoding MOTS3 peptides and MOTS3
peptide analogues having MOTS3 activities, the peptides including,
but not limited to, SEQ ID NO:1. In some embodiments, this
invention comprises a cDNA molecule that encodes a peptide at least
70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO:1.
In some embodiments, this invention comprises a cDNA molecule that
encodes SEQ ID NO:1. In some embodiments, this invention comprises
a cDNA at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% identical
to SEQ ID NO:2.
[0101] When recombinant proteins are expressed by the transformed
bacteria in large amounts, typically after promoter induction,
although expression can be constitutive, the proteins may form
insoluble aggregates. There are several protocols that are suitable
for purification of protein inclusion bodies. For example,
purification of aggregate proteins (hereinafter referred to as
inclusion bodies) typically involves the extraction, separation
and/or purification of inclusion bodies by disruption of bacterial
cells typically, but not limited to, by incubation in a buffer of
about 100-150 .mu.g/ml lysozyme and 0.1% Nonidet P40, a non-ionic
detergent. The cell suspension can be ground using a Polytron
grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the
cells can be sonicated on ice. Alternate methods of lysing bacteria
are described in Ausubel et al. and Sambrook et al., both supra,
and will be apparent to those of skill in the art.
[0102] The cell suspension is generally centrifuged and the pellet
containing the inclusion bodies resuspended in buffer which does
not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl
(pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic
detergent. It may be necessary to repeat the wash step to remove as
much cellular debris as possible. The remaining pellet of inclusion
bodies may be resuspended in an appropriate buffer (e.g., 20 mM
sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers
will be apparent to those of skill in the art.
[0103] Following the washing step, the inclusion bodies are
solubilized by the addition of a solvent that is both a strong
hydrogen acceptor and a strong hydrogen donor (or a combination of
solvents each having one of these properties). The proteins that
formed the inclusion bodies may then be renatured by dilution or
dialysis with a compatible buffer. Suitable solvents include, but
are not limited to, urea (from about 4 M to about 8 M), formamide
(at least about 80%, volume/volume basis), and guanidine
hydrochloride (from about 4 M to about 8 M). Some solvents that are
capable of solubilizing aggregate-forming proteins, such as SDS
(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for
use in this procedure due to the possibility of irreversible
denaturation of the proteins, accompanied by a lack of
immunogenicity and/or activity. Although guanidine hydrochloride
and similar agents are denaturants, this denaturation is not
irreversible and renaturation may occur upon removal (by dialysis,
for example) or dilution of the denaturant, allowing re-formation
of the immunologically and/or biologically active protein of
interest. After solubilization, the protein can be separated from
other bacterial proteins by standard separation techniques.
[0104] Alternatively, it is possible to purify proteins from
bacteria periplasm. Where the protein is exported into the
periplasm of the bacteria, the periplasmic fraction of the bacteria
can be isolated by cold osmotic shock in addition to other methods
known to those of skill in the art (see, Ausubel et al., supra). To
isolate recombinant proteins from the periplasm, the bacterial
cells are centrifuged to form a pellet. The pellet is resuspended
in a buffer containing 20% sucrose. To lyse the cells, the bacteria
are centrifuged and the pellet is resuspended in ice-cold 5 mM
MgSO.sub.4 and kept in an ice bath for approximately 10 minutes.
The cell suspension is centrifuged and the supernatant decanted and
saved. The recombinant proteins present in the supernatant can be
separated from the host proteins by standard separation techniques
well known to those of skill in the art.
[0105] The polypeptides of the invention may be purified to
substantial purity by standard techniques, including selective
precipitation with such substances as ammonium sulfate; column
chromatography, immunopurification methods, and others (see, e.g.,
Scopes, Protein Purification: Principles and Practice (1982); U.S.
Pat. No. 4,673,641; Ausubel et al., Current Protocols in Molecular
Biology (1995 supplement); and Sambrook et al. MOLECULAR CLONING: A
LABORATORY MANUAL, 2nd Ed., (1989)).
[0106] A number of procedures can be employed when polypeptides are
being purified. For example, polypeptides can be purified using ion
exchange or immunoaffinity columns.
[0107] Often as an initial step, and if the protein mixture is
complex, an initial salt fractionation can separate many of the
unwanted host cell proteins (or proteins derived from the cell
culture media) from the recombinant protein of interest. The
preferred salt is ammonium sulfate. Ammonium sulfate precipitates
proteins by effectively reducing the amount of water in the protein
mixture. Proteins then precipitate on the basis of their
solubility. The more hydrophobic a protein is, the more likely it
is to precipitate at lower ammonium sulfate concentrations. A
typical protocol is to add saturated ammonium sulfate to a protein
solution so that the resultant ammonium sulfate concentration is
between 20-30%. This will precipitate the most hydrophobic
proteins. The precipitate is discarded (unless the protein of
interest is hydrophobic) and ammonium sulfate is added to the
supernatant to a concentration known to precipitate the protein of
interest. The precipitate is then solubilized in buffer and the
excess salt removed if necessary, through either dialysis or
diafiltration. Other methods that rely on solubility of proteins,
such as cold ethanol precipitation, are well known to those of
skill in the art and can be used to fractionate complex protein
mixtures.
[0108] Based on a calculated molecular weight, a protein of greater
and lesser size can be isolated using ultrafiltration through
membranes of different pore sizes (for example, Amicon or Millipore
membranes). As a first step, the protein mixture is ultrafiltered
through a membrane with a pore size that has a lower molecular
weight cut-off than the molecular weight of the protein of
interest. The retentate of the ultrafiltration is then
ultrafiltered against a membrane with a molecular cut off greater
than the molecular weight of the protein of interest. The
recombinant protein will pass through the membrane into the
filtrate. The filtrate can then be chromatographed as described
below.
[0109] The proteins of interest can also be separated from other
proteins on the basis of their size, net surface charge,
hydrophobicity and affinity for ligands. In addition, antibodies
raised against proteins can be conjugated to column matrices and
the proteins immunopurified. All of these methods are well known in
the art.
[0110] Immunoaffinity chromatography using antibodies raised to a
variety of affinity tags such as hemagglutinin (HA), FLAG, Xpress,
Myc, hexahistidine, glutathione S transferase (GST) and the like
can be used to purify polypeptides. The His tag will also act as a
chelating agent for certain metals (e.g., Ni) and thus the metals
can also be used to purify His-containing polypeptides. After
purification, the tag is optionally removed by specific proteolytic
cleavage.
[0111] It will be apparent to one of skill that chromatographic
techniques can be performed at any scale and using equipment from
many different manufacturers (e.g., Pharmacia Biotech).
Pharmaceutical Compositions
[0112] The peptides of the present invention can be administered
with a suitable pharmaceutical excipient as necessary. One of skill
will understand that the composition will vary depending on mode of
administration and dosage unit.
[0113] The compositions typically include a conventional
pharmaceutical carrier or excipient and may additionally include
other medicinal agents, carriers, adjuvants, diluents, tissue
permeation enhancers, solubilizers, and the like. Preferably, the
composition will contain about 0.01% to about 90%, about 0.1% to
about 75%, about 0.1% to 50%, or about 0.1% to 10% by weight of a
conjugate of the present invention or a combination thereof, with
the remainder consisting of suitable pharmaceutical carrier and/or
excipients. Appropriate excipients can be tailored to the
particular composition and route of administration by methods well
known in the art. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,
18TH ED., Mack Publishing Co., Easton, Pa. (1990).
[0114] Examples of suitable excipients include, but are not limited
to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, saline, syrup, methylcellulose, ethylcellulose,
hydroxypropylmethylcellulose, and polyacrylic acids such as
Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The
compositions can additionally include lubricating agents such as
talc, magnesium stearate, and mineral oil; wetting agents;
emulsifying agents; suspending agents; preserving agents such as
methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens);
pH adjusting agents such as inorganic and organic acids and bases;
sweetening agents; coloring agents; and flavoring agents. The
compositions may also comprise biodegradable polymer beads,
dextran, and cyclodextrin inclusion complexes.
[0115] For oral administration, the compositions can be in the form
of tablets, lozenges, capsules, emulsions, suspensions, solutions,
syrups, sprays, powders, and sustained-release formulations.
Suitable excipients for oral administration include pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium
carbonate, and the like.
[0116] In some embodiments, the pharmaceutical compositions take
the form of a pill, tablet, or capsule, and thus, the composition
can contain, along with the conjugate or combination of conjugates,
any of the following: a diluent such as lactose, sucrose, dicalcium
phosphate, and the like; a disintegrant such as starch or
derivatives thereof; a lubricant such as magnesium stearate and the
like; and a binder such a starch, gum acacia, polyvinylpyrrolidone,
gelatin, cellulose and derivatives thereof. The conjugates can also
be formulated into a suppository disposed, for example, in a
polyethylene glycol (PEG) carrier.
[0117] Liquid compositions can be prepared by dissolving or
dispersing a conjugate or a combination of conjugates and
optionally one or more pharmaceutically acceptable adjuvants in a
carrier such as, for example, aqueous saline (e.g., 0.9% w/v sodium
chloride), aqueous dextrose, glycerol, ethanol, and the like, to
form a solution or suspension, e.g., for oral, topical, or
intravenous administration. The conjugates of the present invention
can also be formulated into a retention enema.
[0118] For topical administration, the compositions of the present
invention can be in the form of emulsions, lotions, gels, creams,
jellies, solutions, suspensions, ointments, and transdermal
patches. For delivery by inhalation, the composition can be
delivered as a dry powder or in liquid form via a nebulizer. For
parenteral administration, the compositions can be in the form of
sterile injectable solutions and sterile packaged powders.
Preferably, injectable solutions are formulated at a pH of about
4.5 to about 7.5.
[0119] The compositions of the present invention can also be
provided in a lyophilized form. Such compositions may include a
buffer, e.g., bicarbonate, for reconstitution prior to
administration, or the buffer may be included in the lyophilized
composition for reconstitution with, e.g., water. The lyophilized
composition may further comprise a suitable vasoconstrictor, e.g.,
epinephrine. The lyophilized composition can be provided in a
syringe, optionally packaged in combination with the buffer for
reconstitution, such that the reconstituted composition can be
immediately administered to a patient.
[0120] One of ordinary skill in the art understands that the dose
administered will vary depending on a number of factors, including,
but not limited to, the particular peptide composition to be
administered, the mode of administration, the type of application
(e.g., prophylactic, therapeutic, etc.), the age of the patient,
and the physical condition of the patient. Preferably, the smallest
dose and concentration required to produce the desired result
should be used. Dosage can be appropriately adjusted for children,
the elderly, debilitated patients, and patients with cardiac and/or
liver disease. Further guidance can be obtained from studies known
in the art using experimental animal models for evaluating dosage.
The MOTS3 or MOTS3 analog can be formulated without undue
experimentation for administration to a mammal, including humans,
as appropriate for the particular application. Additionally, proper
dosages of the compositions can be determined without undue
experimentation using standard dose-response protocols.
[0121] In some embodiments, the peptide comprising compositions of
the present invention are administered to a subject at a particular
dose of the peptide or are formulated for unit dosage
administration of the peptide to a subject. In some embodiments,
the dose administered to a subject is from about 0.001 to about
1000 mg per day. In some embodiments, the dose administered to a
subject is from about 0.1 to about 500 mg per day. In some
embodiments, the dose administered to a subject is from about 0.5
to about 100 mg per day. In some embodiments, the compositions of
the present invention are formulated for unit dosage
administration, wherein the unit dosage is from about 0.001 to
about 1000 mg per day. In some embodiments, the compositions of the
present invention are formulated for unit dosage administration,
wherein the unit dosage is from about 0.1 to about 500 mg per day.
In some embodiments, the compositions of the present invention are
formulated for unit dosage administration, wherein the unit dosage
is from about 0.5 to about 100 mg per day.
Methods of Administration
[0122] Administration of the peptides of the present invention with
a suitable pharmaceutical excipient as necessary can be carried out
via any of the accepted modes of administration. Thus,
administration can be, for example, intravenous, topical,
subcutaneous, transcutaneous, transdermal, intramuscular, oral,
intra joint, parenteral, intra-arteriole, intradermal,
intraventricular, intracranial, intraperitoneal, intralesional,
intranasal, rectal, vaginal, or by inhalation. Administration can
be targeted directly to pancreatic tissue, e.g., via injection.
[0123] The compositions of the invention may be administered
repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or
the composition may be administered by continuous infusion.
Suitable sites of administration include, but are not limited to,
dermal, mucosal, bronchial, gastrointestinal, anal, vaginal, eye,
and ear. The formulations may take the form of solid, semi-solid,
lyophilized powder, or liquid dosage forms, such as, for example,
tablets, pills, lozenges, capsules, powders, solutions,
suspensions, emulsions, suppositories, retention enemas, creams,
ointments, lotions, gels, aerosols, or the like, preferably in unit
dosage forms suitable for simple administration of precise
dosages.
[0124] The term "unit dosage form" refers to physically discrete
units suitable as unitary dosages for human subjects, each unit
containing a predetermined quantity of active material calculated
to produce the desired onset, tolerability, and/or therapeutic
effects, in association with a suitable pharmaceutical excipient
(e.g., an ampoule). In addition, more concentrated compositions may
be prepared, from which the more dilute unit dosage compositions
may then be produced. The more concentrated compositions thus will
contain substantially more than, e.g., at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more times the amount of a conjugate or a
combination of conjugates.
[0125] Methods for preparing such dosage forms are known to those
skilled in the art (see, for example, REMINGTON'S PHARMACEUTICAL
SCIENCES, 18TH ED., Mack Publishing Co., Easton, Pa. (1990). The
composition to be administered contains a quantity of the peptides
of the invention in a pharmaceutically effective amount for
improving beta islet cell survival. In addition, pharmaceutically
acceptable salts of the peptides of the present invention (e.g.,
acid addition salts) may be prepared and included in the
compositions using standard procedures known to those skilled in
the art of synthetic organic chemistry and described, e.g., by
March, Advanced Organic Chemistry: Reactions, Mechanisms and
Structure, 4th Ed., New York, Wiley-Interscience (1992).
[0126] In another approach, nucleic acids encoding the polypeptides
of the invention are used for transfection of cells in vitro and in
vivo. These nucleic acids can be inserted into any of a number of
well-known vectors for the transfection of target cells and
organisms as described below. The nucleic acids are transfected
into cells, ex vivo or in vivo, through the interaction of the
vector and the target cell. The nucleic acids, under the control of
a promoter, then express a polypeptide of the present invention,
thereby mitigating the effects of a disease associated with reduced
insulin production.
[0127] Such gene therapy procedures have been used to correct
acquired and inherited genetic defects, cancer, and other diseases
in a number of contexts. The ability to express artificial genes in
humans facilitates the prevention and/or cure of many important
human diseases, including many diseases which are not amenable to
treatment by other therapies (for a review of gene therapy
procedures, see Anderson, Science, 256:808-813 (1992); Nabel et
al., TIBTECH, 11:211-217 (1993); Mitani et al., TIBTECH, 11:162-166
(1993); Mulligan, Science, 926-932 (1993); Dillon, TIBTECH,
11:167-175 (1993); Miller, Nature, 357:455-460 (1992); Van Brunt,
Biotechnology, 6(10):1149-1154 (1998); Vigne, Restorative Neurology
and Neuroscience, 8:35-36 (1995); Kremer et al., British Medical
Bulletin, 51(1):31-44 (1995); Haddada et al., in Current Topics in
Microbiology and Immunology (Doerfler & Bohm eds., 1995); and
Yu et al., Gene Therapy, 1:13-26 (1994)).
[0128] For delivery of nucleic acids, viral vectors may be used.
Suitable vectors include, for example, herpes simplex virus vectors
as described in Lilley et al., Curr. Gene Ther., 1(4):339-58
(2001), alphavirus DNA and particle replicons as described in e.g.,
Polo et al., Dev. Biol. (Basel), 104:181-5 (2000), Epstein-Barr
virus (EBV)-based plasmid vectors as described in, e.g., Mazda,
Curr. Gene Ther., 2(3):379-92 (2002), EBV replicon vector systems
as described in e.g., Otomo et al., J. Gene Med., 3(4):345-52
(2001), adenovirus associated viruses from rhesus monkeys as
described in e.g., Gao et al., PNAS USA, 99(18):11854 (2002),
adenoviral and adeno-associated viral vectors as described in e.g.,
Nicklin et al., Curr. Gene Ther., 2(3):273-93 (2002). Other
suitable adeno-associated virus (AAV) vector systems can be readily
constructed using techniques well known in the art (see, e.g., U.S.
Pat. Nos. 5,173,414 and 5,139,941; PCT Publication Nos. WO 92/01070
and WO 93/03769; Lebkowski et al., Mol. Cell. Biol., 8:3988-3996
(1988); Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor
Laboratory Press); Carter, Current Opinion in Biotechnology
3:533-539 (1992); Muzyczka, Current Topics in Microbiol. and
Immunol., 158:97-129 (1992); Kotin, Human Gene Therapy, 5:793-801
(1994); Shelling et al., Gene Therapy, 1:165-169 (1994); and Zhou
et al., J. Exp. Med., 179:1867-1875 (1994)). Additional suitable
vectors include E1B gene-attenuated replicating adenoviruses
described in, e.g., Kim et al., Cancer Gene Ther., 9(9):725-36
(2002) and nonreplicating adenovirus vectors described in e.g.,
Pascual et al., J. Immunol., 160(9):4465-72 (1998) Exemplary
vectors can be constructed as disclosed by Okayama et al., Mol.
Cell. Biol., 3:280 (1983).
[0129] In some embodiments, this invention comprises an expression
vector comprising a nucleic acid sequence that encodes SEQ ID NO:1.
In some embodiments, this invention comprises an expression vector
comprising a nucleic acid sequence that encodes a peptide at least
70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO:1.
In some embodiments, this invention comprises an expression vector
comprising a cDNA at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or
100% identical to SEQ ID NO:2.
[0130] Molecular conjugate vectors, such as the adenovirus chimeric
vectors described in Michael et al., J. Biol. Chem., 268:6866-6869
(1993) and Wagner et al., Proc. Natl. Acad. Sci. USA, 89:6099-6103
(1992), can also be used for gene delivery according to the methods
of the invention.
[0131] In one illustrative embodiment, retroviruses provide a
convenient and effective platform for gene delivery systems. A
selected nucleotide sequence encoding a polypeptide of the
invention is inserted into a vector and packaged in retroviral
particles using techniques known in the art. The recombinant virus
can then be isolated and delivered to a subject. Suitable vectors
include lentiviral vectors as described in e.g., Scherr et al.,
Curr. Gene Ther., 2(1):45-55 (2002). Additional illustrative
retroviral systems have been described (e.g., U.S. Pat. No.
5,219,740; Miller et al., BioTechniques, 7:980-990 (1989); Miller,
Human Gene Therapy, 1:5-14 (1990); Scarpa et al., Virology,
180:849-852 (1991); Burns et al., Proc. Natl. Acad. Sci. USA,
90:8033-8037 (1993); and Boris-Lawrie et al., Curr. Opin. Genet.
Develop., 3:102-109 (1993).
[0132] Other known viral-based delivery systems are described in,
e.g., Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA, 86:317-321
(1989); Flexner et al., Ann. N.Y. Acad. Sci., 569:86-103 (1989);
Flexner et al., Vaccine, 8:17-21 (1990); U.S. Pat. Nos. 4,603,112,
4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB
2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques,
6:616-627 (1988); Rosenfeld et al., Science, 252:431-434 (1991);
Kolls et al., Proc. Natl. Acad. Sci. USA, 91:215-219 (1994);
Kass-Eisler et al., Proc. Natl. Acad. Sci. USA, 90:11498-11502
(1993); Guzman et al., Circulation, 88:2838-2848 (1993); Guzman et
al., Cir. Res., 73:1202-1207 (1993); and Lotze et al., Cancer Gene
Ther., 9(8):692-9 (2002).
Therapeutic and Prophylactic Applications
[0133] In certain aspects, the compositions of the invention are
used for the treatment or prevention of a disease or disorder in a
subject in need thereof. Examples of diseases or disorders suitable
for treatment with the MOTS3 or MOTS3 analogue compositions
described herein include, but are not limited to, treating and
preventing disorders which include but are not limited to Type 1
and Type 2 diabetes, gestational diabetes, pre-diabetes, insulin
resistance, metabolic syndrome, impaired glucose tolerance, cancers
(e.g., breast cancer, brain cancer, colon cancer, melanoma,
leukemia (e.g., AML), lymphoma, pancreatic cancer, prostate cancer,
ovarian cancer, lung cancer, and gastric cancer), obesity, and
fatty liver disease. The compositions of the invention can be used
prophylactically, e.g., for individuals with a genetic
predisposition for diabetes.
[0134] One of skill in the art will appreciate that the MOTS3
peptides and analogues of the invention can be co-administered with
other therapeutic agents for the treatment or prevention of any of
the diseases described herein. Co-administration can be
simultaneous, e.g., in a single pharmaceutical composition or
separate compositions. The compositions of the invention can also
administered separately from the other therapeutic agent(s), e.g.,
on an independent dosing schedule.
EXAMPLES
Example 1
[0135] 3' RACE analysis revealed that MOTS3 is polyadenylated,
similar to HN (FIG. 1A). After initial screening as described
below, MOTS 3 was determined to have potent biological activity,
and its peptide sequence is well conserved in various species (FIG.
1B). We have generated anti-MOTS3 polyclonal rabbit antibodies that
can detect endogenous and overexpressed MOTS3 by cloning the ORF
into an expression vector in cell culture as well GFP-tagged MOTS3
(FIG. 1C). Using these validated antibodies, we can detect MOTS3 in
rat heart, liver, and testis at similar molecular weights and in
the brain at a slightly higher molecular weight (FIG. 1D). The
expression level appears to be highest in the heart, an organ with
one of the highest mitochondrial density. Further, rho-O HeLa
cells, which have been purged of mitochondrial DNA using ethidium
bromide, do not express MOTS3 as well as other well described
mitochondria-encoded proteins such as cytochrome oxidase I and II
(COI and COII), confirming its mitochondrial origin (FIG. 1E).
Example 2
[0136] Exogenous treatment of synthetic MOTS3 causes a
mitochondria-dependent metabolic shift, measured by oxygen
consumption rate (OCR) measured by Seahorse technology as wells as
MTT reduction (under 10% and 1% FBS conditions) (FIG. 2). Both
exogenous MOTS 3 treatments with synthetic peptides (FIG. 3), as
well as the endogenous expression by cloning (FIG. 4) inhibited
mitochondrial activity. Notably, exogenous MOTS3 treatment reduced
cellular ATP levels and mitochondrial activity (MTT), which
simultaneously occurred with increased autophagy (FIG. 5).
[0137] MOTS3 treatment induced an increase in glucose uptake as
measured by residual glucose levels in the culture medium and by
fluorescence-labeled glucose analog uptake (FIG. 6A and FIG. 6B).
As expected, lactate secretion was increased in keep with elevated
glucose consumption (FIG. 6A). Notably, by 96 hours of MOTS3
treatment, when most of the glucose in the medium has been
consumed, AMPK activation is significantly higher in the MOTS3
treated cells (FIG. 7), agreeing with the sharp decrease in
cellular ATP levels and increased autophagy as shown in FIG. 5.
Impaired cellular metabolism induced by exogenous MOTS3 led to
reduced cellular proliferation and survival under glucose medium
but not galactose medium (FIG. 8); galactose forces the cells to
rely on mitochondrial metabolism for survival.
[0138] Intracellular MOTS3 overexpression by transfecting
expression clones also retarded cellular proliferation (FIG. 9).
Interestingly, 8 days of exogenous MOTS 3 treatment induced
cellular senescence as measured by senescence associated
.beta.-galactosidase staining (FIG. 10).
Example 3
[0139] In mice, 4 days of MOTS3 injections (i.p.; 0.5 or 5.0
mg/kg/day) led to significant weight loss without significant
alteration of food intake (FIG. 11). Further, in agreement with our
in vitro studies, liver mitochondrial respiration capacity was
diminished following MOTS3 treatment at both tested doses (FIG.
11). Notably, MOTS3 treatment significantly reduced blood glucose
levels (FIG. 12A) and also had higher insulin levels (FIG. 12B),
suggesting increased glucose uptake similar to that observed in
cell culture (FIG. 6).
Example 4
MOTS-c: A Novel Peptide Encoded within the Mitochondrial Genome
that Prevents Obesity and Regulates Metabolic Homeostasis
Abstract
[0140] The mitochondrial genome is traditionally known to encode
for only 13 proteins, which are translated using a
mitochondria-specific genetic code. This has been recently
challenged with the discovery of humanin, a peptide encoded within
the mitochondrial 16S rRNA. Humanin suggests the possible existence
of unexplored mitochondrial genes that give rise to bioactive
peptides (C. Lee, K. Yen, P. Cohen, Humanin: a harbinger of
mitochondrial-derived peptides? Trends in endocrinology and
metabolism: TEM, (Feb. 7, 2013)). Here we report a novel peptide
encoded within the mitochondrial 12S rRNA, named MOTS-c
(mitochondrial open-reading-frame of the twelve SrRNA c), which was
detected in various tissues and in circulation. MOTS-c acts as a
key regulator of metabolic homeostasis by modulating nucleotide,
glucose, mitochondrial, and fatty acid metabolism. Notably, MOTS-c
caused a >20-fold increase in endogenous AICAR levels, via the
de novo purine biosynthesis pathway, and also activated AMPK
signaling in HEK293 cells and skeletal muscle in mice. MOTS-c
treatment in mice prevented high fat diet-induced weight gain and
insulin resistance. Furthermore, coinciding with age-dependent
insulin resistance in muscle, MOTS-c levels were found to decline
with age in plasma and muscle, and MOTS-c treatment sufficiently
restored insulin sensitivity in older mice. The discovery of
MOTS-c, along with humanin, suggests previously unknown regulatory
roles for mitochondria in coordinating critical cellular processes,
including metabolism. Our findings suggest that
mitochondrial-derived peptide (MDP) including MOTS-c not only
unravels novel mitochondrial biology, but may also provide novel
diagnostic biomarkers and therapeutic targets that can be exploited
to ameliorate metabolic dysfunctions associated with aging and
age-related chronic diseases.
[0141] An in silico search for potential open reading frames (ORFs)
encoding bioactive peptides within the 12S rRNA region of the
mitochondrial DNA (mtDNA) was conducted. MOTS-c was identified as a
51 bp open reading frame (ORF) that translates into a 16 amino acid
peptide (FIG. 13a), which is highly conserved (FIG. 17a). This
peptide is also subject to several putative post-translation
modifications (FIG. 17b). Translation of MOTS-c could theoretically
be either mitochondrial or cytoplasmic, but because the
mitochondrial genetic code yields tandem start/stop codons (FIG.
13a), MOTS-c must undergo cytoplasmic translation, indicating that
its polyadenylated transcript (FIG. 13b) is exported from the
mitochondria by a currently unknown mechanism (Y. Ninomiya, S.
Ichinose, Subcellular distribution of mitochondrial ribosomal RNA
in the mouse oocyte and zygote. PloS one 2, e1241 (2007) and R.
Amikura, M. Kashikawa, A. Nakamura, S. Kobayashi, Presence of
mitochondria-type ribosomes outside mitochondria in germ plasm of
Drosophila embryos. Proceedings of the National Academy of Sciences
of the United States of America 98, 9133 (Jul. 31, 2001)).
[0142] The possibility that MOTS-c could also be of nuclear origin
due to a phenomenon known as nuclear mitochondrial DNA transfer
(NUMT) (M. Ricchetti, F. Tekaia, B. Dujon, Continued colonization
of the human genome by mitochondrial DNA. PLoS biology 2, E273
(September, 2004)) was recognized. Using the NCBI nucleotide basic
local alignment search tool (BLAST), it was found that none of the
NUMTs or their peptide products had complete homology to the
mitochondrial MOTS-c sequence (FIG. 13c). To further confirm its
mitochondrial origin, MOTS-c expression in HeLa cells that have
been selectively depleted of mitochondrial DNA (.rho.0) was
measured. In HeLa-.rho.0 cells, both 12S rRNA and MOTS-c transcript
were undetectable by qRT-PCR (FIG. 13d), and immunoblots using
MOTS-c specific antibodies (FIG. 17c) showed undetectable levels of
mitochondrial-encoded cytochrome oxidase I and II (COI/II) and
MOTS-c, but unaltered expression of nuclear-encoded GAPDH (FIG.
13e). To study its subcellular distribution pattern, HEK293 cells
were immunostained for MOTS-c and observed mitochondrial
colocalization (FIG. 13f; FIG. 17d, 17e). MOTS-c was detected in
various tissues in mice and rats (FIG. 13g), as well as in
circulation in human and rodent plasma (FIG. 13h; FIG. 17f).
Notably, fasting altered the endogenous expression of MOTS-c in
certain metabolically active tissues (FIG. 13i), and in plasma
(FIG. 13j).
[0143] To unravel the biological role of MOTS-c, microarray
analysis on HEK293 cells following 4- and 72-hours of MOTS-c (10
.mu.M) treatment was performed. Principal component analysis (PCA)
showed that MOTS-c promoted a clear global gene expression profile
shift by 72 hours (FIG. 14a). To further highlight differences
between functional pathways modified by MOTS-c, parametric analysis
of gene set enrichment (PAGE) to show that gene expression
significantly altered by 4 hours after MOTS-c treatment, which
became remarkably distinct by 72 hours was employed (FIG. 14b).
There was modest overlap between gene signatures at 4- and
72-hours, suggesting a time-dependent progression in response to
MOTS-c treatment (FIG. 14c). It was found that MOTS-c had a
profound effect on gene expression in pathways involved in cellular
metabolism and inflammation (FIG. 14d; FIG. 30).
[0144] Next, the effect of MOTS-c on global metabolism was studied
using unbiased metabolomic profiling in HEK293 cells either stably
transfected with MOTS-c or empty expression clones (MOTS-c-ST and
MOTS-c-EV cells, respectively) or exogenously treated with
synthetic MOTS-c (10 uM) for 24- and 72-hours. Of the 356 named
metabolites, 194 were found to be significantly altered in
MOTS-c-ST cells and 49 and 177 were significantly altered by 24-
and 72-hours, respectively (FIG. 31). Interestingly, the de novo
purine biosynthetic pathway was found to be significantly altered
(FIG. 14e, FIG. 20a), including a >20-fold increase in
endogenous AICAR (5-aminoimidazole-4-carboxamide ribonucleotide)
concentration 72-hours of exogenous MOTS-c treatment (FIG. 18a).
Consistent with the metabolomics findings, microarray analysis also
showed that MOTS-c altered purine de novo biosynthesis by as early
as 4 hours post-treatment (FIG. 18b). Purines are synthesized from
ribose-5-phosphate (R5P) produced from the pentose phosphate
pathway (PPP), and also by ADP-ribose derived from NAD.sup.+ (FIG.
14e). Accelerated glycolysis/PPP and increased levels of NAD.sup.+
(FIGS. 18a, 19b), but reduced levels of ADP-ribose and R5P in
MOTS-c treated cells was found. These data are further supported by
reduced glutamine and increased pyrophosphate (PPi) levels (FIG.
14g), which indicate that MOTC-s stimulated increased flux of these
pathways in HEK293 cells. The build-up at AICAR consequently
blocked and reduced downstream intermediates such as inosine,
adenylosuccinate, AMP, and also the purine end-products, including
adenine, guanine, and xanthine (FIGS. 14g, 18 and 20a). MOTS-c may
promote a build-up at AICAR by altering folate metabolism. A
significant reduction of 5-methyl-tetrahydrofolate (5Me-THF) was
observed (FIG. 20b), which is required to catalyze the formylation
of AICAR to its downstream intermediate F-AICAR. Indeed previous
reports show that inhibiting folate metabolism by methotrexate
increases intracellular AICAR levels (E. S. Chan, B. N. Cronstein,
Methotrexate--how does it really work? Nature reviews. Rheumatology
6, 175 (March, 2010)). Impaired folate metabolism, especially
5Me-THF, has also been implicated as a mechanism underlying the
activation of AMPK by the anti-diabetic drug metformin (B.
Corominas-Faja et al., Metabolomic fingerprint reveals that
metformin impairs one-carbon metabolism in a manner similar to the
antifolate class of chemotherapy drugs. Aging 4, 480 (July,
2012)).
[0145] AICAR is a potent activator of the master energy regulator
AMPK, shown to stimulate fatty acid oxidation via
phosphorylation-induced inactivation of acetyl-CoA carboxylase
(ACC) alleviating allosteric inhibition of carnitine
palmitoyltransferase 1 (CPT-1), the central transport mechanism of
fatty acids into mitochondria for .beta.-oxidation. (G. Hasko, B.
Cronstein, Regulation of inflammation by adenosine. Frontiers in
immunology 4, 85 (2013)). AICAR-induced AMPK activation enhances
glucose uptake in muscle, in part, by increasing GLUT4
transcription comparable to that achieved by exercise (G. R.
Steinberg, B. E. Kemp, AMPK in Health and Disease. Physiological
reviews 89, 1025 (July, 2009)). 72-hours of MOTS-c treatment (10
.mu.M) led to the phosphorylation of AMPK.alpha. (Thr172) and ACC
(Ser79), and also increased CPT-1 and GLUT4 protein levels (FIG.
14h) in a time and dose-dependent manner (FIG. 14i). Furthermore,
it was found that MOTS-c promotes the phosphorylation of Akt at
Ser-473 (FIG. 14i). Notably AMPK activation was observed under low
AMP levels and high ADP and ATP levels (FIGS. 19a, 20a). Increased
ATP may be attributed to enhanced glycolysis, which is a rapid but
inefficient method of ATP production (P. S. Ward, C. B. Thompson,
Metabolic reprogramming: a cancer hallmark even warburg did not
anticipate. Cancer cell 21, 297 (Mar. 20, 2012)). These findings
suggest that MOTS-c promotes AMP-independent activation of AMPK,
similar to that achieved by salicylate (K. Hashiguchi, Q. M.
Zhang-Akiyama, Establishment of human cell lines lacking
mitochondrial DNA. Methods in molecular biology 554, 383 (2009)),
leptin (T. Ohta et al., Untargeted metabolomic profiling as an
evaluative tool of fenofibrate-induced toxicology in Fischer 344
male rats. Toxicologic pathology 37, 521 (June, 2009)), and
metformin (N. Fujii, N. Jessen, L. J. Goodyear, AMP-activated
protein kinase and the regulation of glucose transport. American
journal of physiology. Endocrinology and metabolism 291, E867
(November, 2006)).
[0146] Next, the effect of MOTS-c on cellular metabolism focusing
on glucose and fatty acid metabolism and cellular respiration was
investigated. It was found that MOTS-c stimulated glycolysis
reflected by increased glucose uptake (FIG. 15a; FIG. 21a, b) and
lactate production (FIG. 15b; FIG. 21a). Additionally,
intracellular glucose levels were decreased, along with other
glycolytic intermediates (FIG. 15c; FIG. 22), further supporting
increased glycolytic flux. MOTS-c also altered the utilization of
other sugar substrates (FIG. 22). To directly test the rate of
glycolysis in real-time, extracellular acidification rate (ECAR)
was measured. MOTS-c-ST cells had a higher rate of basal glycolysis
and showed improved glycolytic response when stimulated with
glucose (FIG. 15d). Also, total glycolytic capacity, estimated by
oligomycin treatment, was higher in these cells (FIG. 15d).
Intracellular lactate levels were identical between MOTS-c-ST and
MOTS-c-EV cells (FIG. 15c), as excess production by MOTS-c-ST cells
was released into the medium (FIG. 15b; FIG. 21a). Consistent with
observations in MOTS-c-ST cells, mass spectrometry analysis
provided further evidence of increased glycolysis in HEK293 cells
within 24 hours of MOTS-c treatment (FIG. 23a). The pentose
phosphate pathway (PPP), an alternative branch of glycolysis,
provides R5P for de novo purine biosynthesis. Reduced levels of PPP
intermediates, including R5P, were observed in MOTS-c-ST cells and
also MOTS-c-EV cells after 72-hours of exogenous MOTS-c treatment
(10 .mu.M)(FIG. 23b, c).
[0147] Next, mitochondrial respiration was measured because
glycolytic end-products are shuttled into the mitochondria for
further energy extraction through oxidative phosphorylation. In
line with increased glycolysis, MOTS-c treatment reduced basal
oxygen consumption rate (OCR) as well as maximum respiratory
capacity in HEK293 cells (FIG. 15e). This is consistent with the
"Crabtree effect", a phenomenon whereby rapidly dividing cells
including: cancer cells, lymphocytes, stem cells, and spermatozoa,
exhibit suppressed respiration in response to high glucose
concentrations (K. H. Ibsen, The Crabtree effect: a review. Cancer
research 21, 829 (August, 1961); T. Wang, C. Marquardt, J. Foker,
Aerobic glycolysis during lymphocyte proliferation. Nature 261, 702
(Jun. 24, 1976); and V. Gogvadze, S. Orrenius, B. Zhivotovsky,
Mitochondria in cancer cells: what is so special about them? Trends
in cell biology 18, 165 (April, 2008)). Decreased electron
transport chain activity was also indicated by reduced proton
leakage (M. Jastroch, A. S. Divakaruni, S. Mookerjee, J. R.
Treberg, M. D. Brand, Mitochondrial proton and electron leaks.
Essays in biochemistry 47, 53 (2010)) (FIG. 15f). Reduced oxidative
capacity was associated with depletion of TCA cycle intermediates
in MOTS-c-ST cells and in HEK293 cells treated with exogenous
MOTS-c (FIG. 15g; FIG. 24). Transient transfection of MOTS-c
expression clones in HEK293 cells also decreased OCR (FIG. 25).
Despite reduced mitochondrial metabolism, MOTS-c did not perturb
cellular redox homeostasis as determined by the ratio of reduced to
oxidized glutathione (GSH/GSSG), however total glutathione levels
were decreased in MOTS-c-ST cells and in HEK293 cells 72-hours
after MOTS-c treatment (FIG. 26). To test whether MOTS-c suppresses
mitochondrial respiration by increasing glycolytic flux or by
targeting mitochondrial metabolism per se, HEK293 cells were
cultured in medium with either glucose or galactose as the main
carbon source. In mammalian cells, galactose is largely metabolized
by the mitochondria, shifting reliance on oxidative phosphorylation
for energy production (L. D. Marroquin, J. Hynes, J. A. Dykens, J.
D. Jamieson, Y. Will, Circumventing the Crabtree effect: replacing
media glucose with galactose increases susceptibility of HepG2
cells to mitochondrial toxicants. Toxicological sciences: an
official journal of the Society of Toxicology 97, 539 (June,
2007)). MOTS-c reduced cellular proliferation under conditions of
abundant glucose, but failed to affect cells cultured in galactose
medium. These data suggest that MOTS-c-induced respiratory
suppression is likely secondary to increased glucose uptake (FIG.
15h).
[0148] Lipid metabolism was also significantly affected by MOTS-c
(FIG. 27a). MOTS-c-ST cells exhibited higher levels of carnitine
shuttles, as well as increased concentrations of carnitine and
deoxycarnitine compared with MOTS-c-EV cells (FIG. 15i). These
findings were also observed, but to a lesser extent, in cells
treated with exogenous MOTS-c (FIG. 27b). Consistent with increased
carnitine shuttles, we found reduced levels of essential fatty
acids in MOTS-c-ST cells (FIG. 15j), as well as in HEK293 cells
treated with MOTS-c (FIG. 27c). After entering the mitochondria,
fatty acids undergo .beta.-oxidation to extract reducing potential
and acetyl-CoA. Myristoyl-CoA, an early .beta.-oxidation
intermediate, was significantly increased in MOTS-c-ST cells (FIG.
15k), as well as HEK293 cells treated exogenously with MOTS-c (10
uM)(FIG. 27d). Furthermore, other long-chain fatty acids were also
significantly reduced, suggesting increased fatty acid utilization
(FIG. 28). Notably, .beta.-oxidation itself is not an aerobic
reaction, and increased fatty acid oxidation has been observed
under reduced respiration during treatment with AICAR (L. D.
Marroquin, J. Hynes, J. A. Dykens, J. D. Jamieson, Y. Will,
Circumventing the Crabtree effect: replacing media glucose with
galactose increases susceptibility of HepG2 cells to mitochondrial
toxicants. Toxicological sciences: an official journal of the
Society of Toxicology 97, 539 (June, 2007)) and metformin (A.
Martin-Montalvo et al., Metformin improves healthspan and lifespan
in mice. Nature communications 4, 2192 (Jul. 31, 2013)).
[0149] Acute treatment of MOTS-c in outbred CD-1 male mice for 4
days (5 mg/kg/day; BID) caused modest reductions in body weight,
food intake, and blood glucose levels (FIG. 29a-c), but
significantly reduced basal levels of plasma IL-6 and TNF.alpha.
(FIG. 16a; FIG. 29d), which are implicated in obesity and insulin
resistance (M. F. Gregor, G. S. Hotamisligil, Inflammatory
mechanisms in obesity. Annual review of immunology 29, 415 (2011)).
Considering our findings in cultured cells, the effects of MOTS-c
on metabolism in the outbred CD-1 mice fed a high-fat diet (HFD;
60% by calories) were tested (L. A. Scrocchi, D. J. Drucker,
Effects of aging and a high fat diet on body weight and glucose
tolerance in glucagon-like peptide-1 receptor -/- mice.
Endocrinology 139, 3127 (July, 1998) and W. L. Breslin, K.
Strohacker, K. C. Carpenter, L. Esposito, B. K. McFarlin, Weight
gain in response to high-fat feeding in CD-1 male mice. Laboratory
animals 44, 231 (July, 2010)). 8-weeks of MOTS-c treatment had no
effect on body weight in mice fed a normal diet, but remarkably
prevented HFD-induced obesity (FIG. 16b). This difference in body
weight was not attributed to food intake, as caloric consumption
was identical between the groups (FIG. 29e-f). High fat feeding
promotes hyperinsulinemia in an attempt to overcome peripheral
insulin resistance to maintain glucose homeostasis (M. Hou et al.,
Protective effect of metformin in CD1 mice placed on a high
carbohydrate-high fat diet. Biochemical and biophysical research
communications 397, 537 (Jul. 2, 2010)). Importantly, MOTS-c
treatment prevented hyperinsulinemia (FIG. 16c, d) and ameliorated
hepatic lipid accumulation in HFD fed mice (FIG. 16e). Notably, in
line with our in vitro studies, MOTS-c promoted AMPK activation and
GLUT4 expression in the muscle of these HFD fed mice (FIG.
16f).
[0150] Next, the effects of MOTS-c on glucose homeostasis in the
commonly studied, obesity prone C57BL/6 mouse strain were tested by
performing a glucose tolerance test (GTT). MOTS-c treated mice
showed rapid glucose clearance, suggesting enhanced insulin
sensitivity (FIG. 16g). Next, hyperinsulinemic-euglycemic clamp
studies were performed to quantify the effects of 7-day treatment
with MOTS-c on whole body insulin sensitivity independent of
changes in body weight that occur with extended treatment
durations. MOTS-c improved whole body insulin sensitivity as
reflected by a .about.30% increase in the exogenous glucose
infusion rate (GIR) required to maintain euglycemia during insulin
stimulation (FIG. 16h). Insulin promotes glucose disposal into
peripheral tissues and suppresses hepatic glucose production (HGP)
to maintain homeostasis during periods of increased glucose
availability (C. R. Kahn, Banting Lecture. Insulin action,
diabetogenes, and the cause of type II diabetes. Diabetes 43, 1066
(August, 1994)). Tritiated glucose was infused during the clamp to
determine the tissue specificity of MOTS-c action on insulin
sensitivity. Although it was observed that MOTS-c treatment
significantly enhanced the insulin-stimulated glucose disposal rate
(IS-GDR) (FIG. 16i), no effect of MOTS-c on liver insulin
sensitivity was detected, as the rate of hepatic glucose production
(HGP) was comparable between the groups (FIG. 16j). Considering
that 70-85% of insulin-stimulated glucose disposal is into skeletal
muscle, MOTS-c actions to enhance insulin sensitivity and glucose
homeostasis may be mediated in this tissue. Indeed this notion was
supported by strong AMPK activation and increased GLUT4 expression
in skeletal muscle of HFD-fed mice following MOTS-c treatment (FIG.
16f). Moreover, MOTS-c also enhanced glucose uptake into soleus
muscle stimulated with a physiological dose of human insulin ex
vivo. Because MOTS-c levels in muscle (FIG. 16k) and in circulation
(FIG. 16I) decline concomitant with the development of insulin
resistance during aging (C. R. Kahn, Banting Lecture. Insulin
action, diabetogenes, and the cause of type II diabetes. Diabetes
43, 1066 (August, 1994)), it was determined if MOTS-c could reverse
age-dependent impairments in insulin action by measuring
insulin-stimulated glucose (2-deoxyglucose) uptake into soleus
muscles of middle-aged (12 months) and young (3 months) male
C57BL/6 mice. Signs of insulin resistance begin to show around 12
months of age in C57BL/6 mice (C. R. Kahn, Banting Lecture. Insulin
action, diabetogenes, and the cause of type II diabetes. Diabetes
43, 1066 (August, 1994)). Indeed muscles from older mice were more
insulin resistant, but 7 days of MOTS-c treatment restored
sensitivity comparable to young animals (FIG. 16m).
Discussion
[0151] Technological advances have unveiled previously unknown
properties of mitochondrial genetics suggesting the existence of
small ORFs in the mitochondrial DNA (T. R. Mercer et al., The human
mitochondrial transcriptome. Cell 146, 645 (Aug. 19, 2011)).
Notably, various bioactive small ORFs in the nuclear genome have
been reported in Drosophila (E. G. Magny et al., Conserved
regulation of cardiac calcium uptake by peptides encoded in small
open reading frames. Science 341, 1116 (Sep. 6, 2013); J. Savard,
H. Marques-Souza, M. Aranda, D. Tautz, A segmentation gene in
tribolium produces a polycistronic mRNA that codes for multiple
conserved peptides. Cell 126, 559 (Aug. 11, 2006); T. Kondo et al.,
Small peptides switch the transcriptional activity of Shavenbaby
during Drosophila embryogenesis. Science 329, 336 (Jul. 16, 2010);
and M. I. Galindo, J. I. Pueyo, S. Fouix, S. A. Bishop, J. P.
Couso, Peptides encoded by short ORFs control development and
define a new eukaryotic gene family. PLoS biology 5, e106 (May,
2007)). There is evidence in the literature discussing the
potential existence of factors encoded within the mitochondrial DNA
(mtDNA). For instance, polyadenylated mitochondrial rRNA clones
were cloned in the early 1980s, as part of a cDNA library
constructed from interferon-induced human myeloblast cells (J.
Villegas, P. Araya, E. Bustos-Obregon, L. O. Burzio, Localization
of the 16S mitochondrial rRNA in the nucleus of mammalian
spermatogenic cells. Molecular human reproduction 8, 977 (November,
2002)), suggesting a strong interferon-induced factor encoded in
the mtDNA. Also, in humans, the 16S rRNA was found localized in the
nucleus of human spermatogenic cells (J. Durieux, S. Wolff, A.
Dillin, The cell-non-autonomous nature of electron transport
chain-mediated longevity. Cell 144, 79 (Jan. 7, 2011)), whereas in
Drosophila, mitochondrial rRNAs have been found in the cytoplasm
where they play a role in germ cell establishment (R. Amikura, M.
Kashikawa, A. Nakamura, S. Kobayashi, Presence of mitochondria-type
ribosomes outside mitochondria in germ plasm of Drosophila embryos.
Proceedings of the National Academy of Sciences of the United
States of America 98, 9133 (Jul. 31, 2001)).
[0152] MOTS-c may be a product of adaptation for effective
bilateral communication between mitochondria and the cell or
distant organs such as skeletal muscle, especially considering that
its translation requires the mammalian genetic code. Herein is
provided sufficient evidence to suggest MOTS-c, and humanin, as a
novel class of inherent mitochondrial signals that regulate global
physiology (C. Lee, K. Yen, P. Cohen, Humanin: a harbinger of
mitochondrial-derived peptides? Trends in endocrinology and
metabolism: TEM, (Feb. 7, 2013)). A recent report, using the
nematode C. elegans, showed that mitochondrial disruption in
neurons causes mitochondrial unfolded-protein response (UPR) in the
intestine and extends lifespan, an effect mediated by an
unidentified circulating signal that allows inter-organ
communication of stress (D. K. Woo, G. S. Shadel, Mitochondrial
stress signals revise an old aging theory. Cell 144, 11 (Jan. 7,
2011) and C. Cheadle, M. P. Vawter, W. J. Freed, K. G. Becker,
Analysis of microarray data using Z score transformation. The
Journal of molecular diagnostics: JMD 5, 73 (May, 2003)).
[0153] Although the specific mechanistic details of MOTS-c action
have yet to be fully identified, its impact on metabolism will
likely have major implications for aging and age-related diseases
including diabetes, cancer, atherosclerosis, and neurodegeneration.
The emerging biology of MDPs and the unique involvement of AICAR
and AMPK in MOTS-c action (FIG. 16n) provide an interesting
opportunity to expand our understanding of the role of mitochondria
in physiological and pathological conditions, as well as
identifying novel diagnostic and therapeutic targets.
Methods
Cell Culture
[0154] HEK293 and HeLa cells were routinely cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) at 37.degree. C. with 5% CO.sub.2. .rho.0 cells, which
are devoid of mitochondrial DNA, were generated by culturing HeLa
cells in low doses of ethidium bromide (EtBr; 100 ng/mL) as
described before (K. Hashiguchi, Q. M. Zhang-Akiyama, Establishment
of human cell lines lacking mitochondrial DNA. Methods in molecular
biology 554, 383 (2009)). MOTS-c-ST cells were generated by
selection in and maintenance in G418 (500 .mu.M; Sigma) in DMEM
with 10% FBS.
Mice
[0155] All animal work was performed in accordance with the
University of Southern California and University of California Los
Angeles Institutional Animal Care and Use Committee. MOTS-c
(Genscript, USA) was injected daily via intraperitoneal injections
in all in vivo experiments. 8-week old CD-1 (ICR) mice were
purchased from Harlan. C57BL/6 mice were purchased from Jackson
Laboratories. Mice were fed a high-fat diet (60% by calories) and
matching control diet purchased from Research Diets (Cat# D12492
and D12450J, respectively) for 8 weeks. Pellets were replaced twice
weekly, and body weight and food consumption were recorded daily
(N=10).
Cloning
[0156] Directional cloning using restriction enzymes EcoRI (5') and
XhoI (3') was performed to construct MOTS-c expression clones.
MOTS-c and MOTS-c-HA nucleotide sequences flanked by the
restriction enzymes were synthesized and 5' phosphorylated (IDT,
USA), hybridized, and ligated with digested pcDNA3.1(+)
(Invitrogen, USA), or pcDNA-IRES GFP (kind gift from Nir Barzilai
at AECOM). All enzymes were purchased from NEB (USA) unless stated
otherwise.
Immunocytochemistry
[0157] HEK293 cells plated on coverslip were fixed with 4%
paraformaldehyde for 15 min at room temperature. After fixation,
the cells were permeablized with 0.2% Triton X-100 in
phosphate-buffered saline (PBS) for 10 minutes at room temperature
and were blocked in PBS containing 0.2% Triton X-100 and 1% bovine
serum albumin (BSA) for 1 hour at room temperature. Cells were then
incubated with rabbit anti-MOTS-c antibody (1:50) and goat
anti-hsp60 antibody (1:100; Santa Cruz Biotechnology, USA) in PBS
containing 0.2% Triton X-100 and 1% BSA at 4.degree. C. overnight.
After three washes with PBS, the cells were further incubated with
Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:200;
Invitrogen, US) and Alexa Fluor 568-conjugated donkey anti-goat IgG
(1:200; Invitrogen) in PBS containing 0.2% Triton X-100 and 1% BSA
for 1 hour at room temperature. Nuclei were stained for 5 minutes
at room temperature in PBS containing Hoechst 33258 (2 mg/ml;
Invitrogen). Specificity of immunostaining was demonstrated by
using MOTS-c antibody after incubation with 69 .mu.g/ml MOTS-c
peptide (Genscript, USA) for 1 hour at room temperature. Coverslips
were mounted with ProLong Gold antifade reagent (Invitrogen) and
observed under an ELYRA PS.1 (Carl Zeiss, Germany) using Laser wide
field. For structured illumination microscopy (SIM) images, Z-stack
sections were collected with five rotations of gratings and images
were was reconstructed using ELYRA PS.1 software.
Immunoassays
[0158] The entire MOTS-c peptide was conjugated to Keyhole limpet
hemocyanin (KLH) and injected into rabbits. IgG purified serum were
used for western blotting and ELISA. Circulating MOTS-c levels were
measured from serum, plasma, and CSF by in-house sandwich ELISA
developed at USC. The custom rabbit anti-MOTS-c polyclonal
anti-sera were produced at YenZym Antibodies, LLC (South San
Francisco, Calif.). IgG subclasses purified with a protein A/G
column chromatography (Pierce, Rockford, Ill.) was used as a
capture antibody. The anti-MOTS-c IgG was labeled with biotin and
used as a detection antibody. To measure endogenous MOTS-c levels,
synthetic MOTS-c (GenScript) was used as a standard within the
range of 25 pg/mL to 6400 pg/mL. Prior to assay, MOTS-c was
extracted in 90% acetonitrile and 10% 1N HCl. Briefly, 200 .mu.L of
extraction reagent was added to 100 .mu.L of plasma, gently mixed
and incubated at room temperature for 30 minutes. The mixture was
centrifuged and the supernatant was removed and dried. The dried
extract was reconstituted with 200 .mu.L of phosphate buffer with
0.5% Tween 20, and then used for assay. 96-well microtiter plates
were coated with capture antibody at 0.5 .mu.g/well and incubated 4
hr at room temperature on a shaker. Standards, controls or
extracted samples and pre-titered detection antibody were added to
the appropriate wells and incubated overnight after 2 washes with
wash buffer and 2 washes with Superblock buffer (Pierce Chemicals,
Rockford, Ill.). Wells were washed 3 times and then added with
streptavidin-HRP and further incubated for 30 minutes at room
temperature. After washes, 200 .mu.L/well of OPD solution (1 mg/mL
in hydrogen peroxide substrate) was added and incubated for 10-20
minutes. The reaction was terminated by the addition of
2NH.sub.2SO.sub.4, and absorbance was measured on a plate
spectrophotometer (Molecular Designs, Sunnyvale, Calif.) at 490 nm.
The intra- and inter-assay coefficient variations (CV) were less
than 10%. Plasma insulin levels were detected using a mouse insulin
assay kit (MSD; Cat# K112BZC-1) and Sector Imager 2400A (MSD,
USA).
[0159] For western blotting, protein samples were prepared in 1%
Triton X-100 with EDTA-free protease and phosphatase inhibitors
(Roche, USA), heated at 95.degree. C. for 5 minutes, ran on 4-20%
gradient tris-glycine gels (TGX; Bio-Rad, USA) and transferred to
PVDF membranes (Bio-Rad) using a turbo semidry transfer system
(Transblot Turbo; Biorad) at 9V for 15-30 minutes. Membranes were
blocked with 5% BSA for 30 minutes and incubated with primary
antibody overnight at 4.degree. C., followed by secondary
HRP-conjugated antibodies for 1 hour at room temperature.
Chemiluminescence was detected and imaged using enhanced ECL
(Immun-Star WesternC; Bio-Rad) and Chemidoc XRS system
(Bio-Rad).
Microarray
[0160] RNA was isolated using the RNeasy kit (Qiagen, Valencia,
Calif.) and then hybridized to BD-103-0603 Illumina Beadchips. Raw
data were subjected to Z-normalization, as previously described (C.
Cheadle, M. P. Vawter, W. J. Freed, K. G. Becker, Analysis of
microarray data using Z score transformation. The Journal of
molecular diagnostics: JMD 5, 73 (May, 2003)). Principal component
analysis, performed on the normalized Z-scores of all of the
detectable probes in the samples, was performed by using the DIANE
6.0 software (http://www.grc.nia.nih.gov/branches/rrb/dna/diane
software.pdf). Significant genes were selected by the z-test
<0.05, false discovery rate <0.30, as well as z-ratio>1.5
in both directions and ANOVA p value <0.05. Parametric analysis
of gene set enrichment (PAGE) was analyzed as previously described
(S. Y. Kim, D. J. Volsky, PAGE: parametric analysis of gene set
enrichment. BMC bioinformatics 6, 144 (2005)). Gene regulatory
network and canonic pathway analysis was performed using Ingenuity
Pathways Analysis (Ingenuity Systems; Redwood City, Calif.)
(N=6).
Metabolomics
[0161] HEK293 cells were treated with MOTS-c (10 .mu.M) or water
(vehicle) for 24- and 72-hours. Also, HEK293 cells that were stably
transfected with a validated MOTS-c expression vector, or empty
vector (control) were used. Cells were cultured in 10-cm dishes in
7-mL of phenol-free DMEM supplemented with 10% FBS. For collection,
cells were washed twice with ice-cold phosphate buffered saline
(PBS) and immediately scraped, centrifuged, and snap frozen after
aspiration supernatant. Frozen pellets were used for metabolomics
analysis (Metabolon). The non-targeted metabolic profiling
instrumentation employed a combination of three independent
platforms: ultrahigh performance liquid chromatography/tandem mass
spectrometry (UHPLC/MS/MS.sup.2) optimized for basic species,
UHPLC/MS/MS.sup.2 optimized for acidic species, and gas
chromatography/mass spectrometry (GC/MS). Samples were processed
essentially as described previously (T. Ohta et al., Untargeted
metabolomic profiling as an evaluative tool of fenofibrate-induced
toxicology in Fischer 344 male rats. Toxicologic pathology 37, 521
(June, 2009) and A. M. Evans, C. D. DeHaven, T. Barrett, M.
Mitchell, E. Milgram, Integrated, nontargeted ultrahigh performance
liquid chromatography/electrospray ionization tandem mass
spectrometry platform for the identification and relative
quantification of the small-molecule complement of biological
systems. Analytical chemistry 81, 6656 (Aug. 15, 2009)). In a
minimum volume of water, cells were lysed using a Covaris adaptive
focused acoustics E-series tissue disrupter/homogenizer. From the
homogenate, a 100 .mu.L aliquot was withdrawn for subsequent mass
spectrometry and 25 .mu.L used for measurement of total protein
(Bradford Assay). Using an automated liquid handler (Hamilton
LabStar, Salt Lake City, Utah), protein was precipitated from
homogenate with methanol that contained four standards to report on
extraction efficiency. The resulting supernatant was split into
equal aliquots for analysis on the three platforms. Aliquots, dried
under nitrogen and vacuum-desiccated, were subsequently either
reconstituted in 50 .mu.L 0.1% formic acid in water (acidic
conditions) or in 50 .mu.L 6.5 mM ammonium bicarbonate in water, pH
8 (basic conditions) for the two UHPLC/MS/MS.sup.2 analyses or
derivatized to a final volume of 50 .mu.L for GC/MS analysis using
equal parts bistrimethyl-silyl-trifluoroacetamide and solvent
mixture acetonitrile:dichloromethane:cyclohexane (5:4:1) with 5%
triethylamine at 60.degree. C. for one hour. In addition, three
types of controls were analyzed in concert with the experimental
samples: aliquots of a sample derived from pooled experimental
sample aliquots served as technical replicates throughout the data
set, extracted water samples served as process blanks, and a
cocktail of standards spiked into every analyzed sample allowed
instrument performance monitoring. Experimental samples and
controls were randomized across platform run days.
[0162] For UHLC/MS/MS.sup.2 analysis, aliquots were separated using
a Waters Acquity UPLC (Waters, Millford, Mass.) and analyzed using
an LTQ mass spectrometer (Thermo Fisher Scientific, Inc., Waltham,
Mass.) which consisted of an electrospray ionization (ESI) source
and linear ion-trap (LIT) mass analyzer. The MS instrument scanned
99-1000 m/z and alternated between MS and MS.sup.2 scans using
dynamic exclusion with approximately 6 scans per second.
Derivatized samples for GC/MS were separated on a 5% phenyldimethyl
silicone column with helium as the carrier gas and a temperature
ramp from 60.degree. C. to 340.degree. C. and then analyzed on a
Thermo-Finnigan Trace DSQ MS (Thermo Fisher Scientific, Inc.)
operated at unit mass resolving power with electron impact
ionization and a 50-750 atomic mass unit scan range.
[0163] Metabolites were identified by automated comparison of the
ion features in the experimental samples to a reference library of
chemical standard entries that included retention time, molecular
weight (m/z), preferred adducts, and in-source fragments as well as
associated MS spectra, and were curated by visual inspection for
quality control using software developed at Metabolon (C. D.
Dehaven, A. M. Evans, H. Dai, K. A. Lawton, Organization of GC/MS
and LC/MS metabolomics data into chemical libraries. Journal of
cheminformatics 2, 9 (2010)).
[0164] For statistical analyses and data display purposes, any
missing values were assumed to be below the limits of detection and
these values were imputed with the compound minimum (minimum value
imputation). In addition, each value was normalized to a total
protein value on a per sample basis. Statistical analysis of
log-transformed data was performed using "R"
(http://cran.r-project.org/), which is a freely available,
open-source software package. Student's t-test was performed to
compare data between experimental groups.
Glucose and Lactate Assay
[0165] Extracellular glucose and lactate from cell culture medium
was measured using glucose and D-lactate assay kits per
manufacturer's instructions (Eton Bioscienes, USA). Intracellular
glucose was determined by microscopy using 2-NBDG (Invitrogen, USA)
per manufacturer's instructions.
Oxygen Consumption and Extracellular Acidification Rate
[0166] Real-time oxygen consumption rates (OCR) were measured using
XF24/96 Extracellular Flux Analyzer (Seahorse Bioscience). ATP
turnover, and maximum respiratory capacity was estimated by
challenging cells with oligomycin and FCCP. Spare respiratory
capacity was determined by `maximum respiratory rate/basal
respiratory rate`, proton leakage by `ATP turnover
OCR-non-mitochondrial OCR (rotenone/antimycin A)`, and coupling
efficiency by `1-(ATP turnover OCR/Basal Respiratory rate)`.
Glycolytic rate was determined using extracellular acidification
rate (ECAR). Cell were stimulated with glucose to determine active
glycolytic rate, and with oligomycin to determine maximum
glycolytic capacity, and with 2-DG to determine glycolytic
capacity.
Ex-Vivo Soleus Muscle Strip Glucose Uptake
[0167] Whole muscle ex-vivo glucose uptake was assessed in mice 12
weeks of age using 2-deoxy glucose (Perkin Elmer) and 60 .mu.l/ml
human insulin (Novo Nordisk Pharmaceutical Industries), with minor
changes to that described previously (V. Ribas et al.,
Myeloid-specific estrogen receptor alpha deficiency impairs
metabolic homeostasis and accelerates atherosclerotic lesion
development. Proc Natl Acad Sci USA 108, 16457 (Sep. 27, 2011) and
C. E. McCurdy, G. D. Cartee, Akt2 is essential for the full effect
of calorie restriction on insulin-stimulated glucose uptake in
skeletal muscle. Diabetes 54, 1349 (May, 2005)). Briefly, soleus
and EDL muscles were carefully excised from anaesthetized animals
and immediately incubated for 30 min in complete Krebs-Henseleit
buffer with or without 60 .mu.U/ml insulin at 35.degree. C. Muscles
were then transferred to the same buffer containing 3
mCi/ml.sup.3H-2-deoxyglucose and 0.053 mCi/ml.sup.14C-mannitol, and
incubated for 20 min prior to snap freezing. Muscles were
homogenized in lysis buffer and counted for radioactivity or
subjected to western blotting. Glucose uptake was standardized to
the non-specific uptake of mannitol and estimated as mmol of
glucose uptake per gram soleus muscle.
Hyperinsulinemic-Euglycemic Clamp Studies
[0168] Dual catheters were surgically placed in the right jugular
vein and glucose clamp studies were performed 3 days post-surgery
as previously described (V. Ribas et al., Myeloid-specific estrogen
receptor alpha deficiency impairs metabolic homeostasis and
accelerates atherosclerotic lesion development. Proc Natl Acad Sci
USA 108, 16457 (Sep. 27, 2011); A. L. Hevener et al.,
Muscle-specific Pparg deletion causes insulin resistance. Nature
medicine 9, 1491 (December, 2003); and A. L. Hevener et al.,
Macrophage PPAR gamma is required for normal skeletal muscle and
hepatic insulin sensitivity and full antidiabetic effects of
thiazolidinediones. J Clin Invest 117, 1658 (June, 2007)). All
animals were fasted for 6 h and the final MOTS-c dose was
administered 4 h prior to the clamp. Animals were studied in the
conscious state. Basal glucose turnover was determined following a
90-minute constant infusion of (5.0 .mu.Ci/h, 0.12 ml/h) of
[3-.sup.3H] D-glucose (Perkin Elmer). After the basal period,
glucose (50% dextrose, Abbott Laboratories) and insulin (8
mU/kg/min), Novo Nordisk Pharmaceutical Industries) plus tracer
(5.0 .mu.Ci/h) infusions were initiated simultaneously, and glucose
levels clamped at euglycemia using a variable glucose infusion rate
(GIR). At steady state the total glucose disposal rate (GDR),
measured by tracer dilution technique, is equal to the sum of the
rate of endogenous or hepatic glucose production (HGP) and the
exogenous (cold) glucose infusion rate (GIR) (A. L. Hevener et al.,
Muscle-specific Pparg deletion causes insulin resistance. Nature
medicine 9,1491 (December, 2003); A. L. Hevener et al., Macrophage
PPAR gamma is required for normal skeletal muscle and hepatic
insulin sensitivity and full antidiabetic effects of
thiazolidinediones. J Clin Invest 117,1658 (June, 2007); and R.
Steele, Influences of glucose loading and of injected insulin on
hepatic glucose output. Ann N Y Acad Sci 82, 420 (Sep. 25, 1959)).
The insulin-stimulated component of the total GDR (IS-GDR) is equal
to the total GDR minus the basal glucose turnover rate.
Glucose Tolerance Test (GTT)
[0169] Blood glucose was measured using a glucometer (Freestyle,
Abbott). 12-week old male C57BL/6 mice were treated with MOTS-c
(0.5 mg/kg/day; IP), or sterile pure water (vehicle), daily for 7
days. Then mice were injected with D-glucose (1 g/kg; IP) and blood
was sampled from the tail at 0, 15, 30, 60, 90, and 120 minutes
post-glucose injection.
[0170] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the compositions, systems
and methods of the disclosure, and are not intended to limit the
scope of what the inventors regard as their disclosure.
Modifications of the above-described modes for carrying out the
disclosure that are obvious to persons of skill in the art are
intended to be within the scope of the following claims. All
patents and publications mentioned in the specification are
indicative of the levels of skill of those skilled in the art to
which the disclosure pertains. All references cited in this
disclosure are incorporated by reference to the same extent as if
each reference had been incorporated by reference in its entirety
individually.
[0171] All headings and section designations are used for clarity
and reference purposes only and are not to be considered limiting
in any way. For example, those of skill in the art will appreciate
the usefulness of combining various aspects from different headings
and sections as appropriate according to the spirit and scope of
the invention described herein.
[0172] All references cited herein are hereby incorporated herein
by reference herein in their entireties and for all purposes to the
same extent as if each individual publication or patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety for all purposes.
[0173] Many modifications and variations of this application can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments and
examples described herein are offered by way of example only, and
the disclosure is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which the
claims are entitled.
Sequence CWU 1
1
15116PRTHomo sapiens 1Met Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr
Pro Arg Lys Leu Arg 1 5 10 15 251DNAHomo sapiens 2atgaggtggc
aagaaatggg ctacattttc taccccagaa aactacgata g 51314PRTCanis lupus
3Met Arg Trp Met Gly Tyr Tyr Arg Thr His Cys Tyr Lys Asn 1 5 10
413PRTDanio rerio 4Met Lys Trp Met Gly Tyr Thr Tyr Arg Tyr Asn Val
Thr 1 5 10 56PRTMus musculus 5Met Lys Trp Met Gly Tyr 1 5
618PRTRattus norvegicus 6Met Lys Arg Lys Met Gly Tyr Ser Arg Thr
Arg Asn Tyr Thr Lys Gly 1 5 10 15 Arg Arg 712PRTPanthera leo 7Met
Arg Trp Glu Ala Met Gly Tyr Ile Phe Tyr Asn 1 5 10 817PRTArtificial
SequenceMOTS-c Ch. 17a 8Met Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr
Pro Arg Lys Phe Tyr 1 5 10 15 Asp 917PRTArtificial SequenceMOTS-c
Ch. 17b 9Met Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr Pro Arg Lys
Phe Tyr 1 5 10 15 Asn 1016PRTArtificial SequenceMOTS-c Ch. 11 10Met
Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr Phe Arg Lys Leu Arg 1 5 10
15 1117PRTArtificial SequenceMOTS-c Ch. 1 11Met Arg Trp Gln Glu Met
Gly Tyr Ile Phe Tyr Thr Gln Lys Ile Leu 1 5 10 15 Leu
1217PRTArtificial SequenceMOTS-c Ch. 4 12Met Arg Trp Gln Glu Met
Gly Tyr Ile Phe Tyr Ile Arg Gln Ile Ser 1 5 10 15 Gln
1317PRTArtificial SequenceMOTS-c Ch. 17c 13Met Arg Trp Gln Glu Met
Gly Tyr Ile Phe Tyr Thr Gln Lys Ile Ser 1 5 10 15 Arg
1417PRTArtificial SequenceMOTS-c Ch. X 14Met Arg Trp Gln Glu Met
Gly Tyr Ile Phe Tyr Val Gln Lys Leu Ser 1 5 10 15 Arg
1555PRTArtificial SequenceMOTS-c Ch. 17d 15Met Arg Trp Gln Glu Met
Gly Tyr Ile Phe Tyr Thr Gln Lys Ile Ser 1 5 10 15 Arg Val Arg Asn
Thr Val Asp Ser Arg Val Pro Pro Lys Pro Ser Phe 20 25 30 Gly Ser
Arg Leu Thr Asn Gln Leu Ile Pro Val Leu Arg Thr Cys Val 35 40 45
Ala Gly Ser Gly Arg Ser Leu 50 55
* * * * *
References