U.S. patent application number 13/402955 was filed with the patent office on 2012-08-09 for mitochondrial function of prohibitin 2 (phb2).
This patent application is currently assigned to JICHI MEDICAL UNIVERSITY. Invention is credited to Hitoshi Endo, Katsumi Kasashima.
Application Number | 20120202215 13/402955 |
Document ID | / |
Family ID | 38067247 |
Filed Date | 2012-08-09 |
United States Patent
Application |
20120202215 |
Kind Code |
A1 |
Endo; Hitoshi ; et
al. |
August 9, 2012 |
MITOCHONDRIAL FUNCTION OF PROHIBITIN 2 (PHB2)
Abstract
The present invention relates to a PHB2 gene regulator and a
therapeutic drug for mitochondrial-function-related disease
containing the same, for example.
Inventors: |
Endo; Hitoshi;
(Shimotsuke-shi, JP) ; Kasashima; Katsumi;
(Shimotsuke-shi, JP) |
Assignee: |
JICHI MEDICAL UNIVERSITY
Shimotsuke-shi
JP
|
Family ID: |
38067247 |
Appl. No.: |
13/402955 |
Filed: |
February 23, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12094787 |
Mar 4, 2009 |
8153362 |
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PCT/JP2006/323379 |
Nov 16, 2006 |
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13402955 |
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Current U.S.
Class: |
435/6.13 ;
435/29; 435/7.1 |
Current CPC
Class: |
C07K 14/4702 20130101;
A61P 25/16 20180101; G01N 2333/4703 20130101; A61P 3/10 20180101;
C12N 2310/14 20130101; A61P 43/00 20180101; C12N 15/113 20130101;
A61P 3/04 20180101; A61P 25/28 20180101; A61P 9/04 20180101; G01N
33/5079 20130101; A61P 35/00 20180101; A61P 1/04 20180101 |
Class at
Publication: |
435/6.13 ;
435/29; 435/7.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/566 20060101 G01N033/566; C12Q 1/02 20060101
C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 24, 2005 |
JP |
2005 339354 |
Claims
1-24. (canceled)
25. A method for obtaining an agent for regulating a PHB2 protein
function, comprising: contacting cells expressing a PHB2 gene with
a candidate substance by adding the candidate substance to a
culture comprising the cells, wherein the candidate substance is
selected from the group consisting of siRNA having activity to
inhibit PHB2 gene expression; an antagonist or an agonist of a
nuclear receptor selected from the group consisting of ER.alpha.,
PPAR.alpha., and PPAR.gamma.2; and a chimeric protein comprising a
mitochondrial targeting signal and a transmembrane domain of a PHB
1 protein and the C-terminus of the PHB2 protein; and determining
that the candidate substance is the agent for regulating a PHB2
protein function if the candidate substance regulates the PHB2
protein function in the cells expressing the PHB2 gene caused to
come into contact with the candidate substance, when the cells are
compared with cells expressing the PHB2 gene in the absence of the
candidate substance.
26. The method according to claim 25, wherein the agent for
regulating a PHB2 protein function is selected from the group
consisting of an anti-apoptotic agent, an agent for regulating
mitochondrial membrane potential, and an agent for regulating
mitochondrial morphology.
27. The method according to claim 25, wherein the siRNA is: (a)
siRNA consisting of the nucleotide sequence represented by any one
of SEQ ID NOS: 1 to 20; or (b) siRNA having 90% or more sequence
identity with the siRNA of (a) and having activity to inhibit PHB2
gene expression.
28. The method according to claim 25, wherein the antagonist or the
agonist is selected from the group consisting of ICI182,780, DDE,
Tamoxifen Diethylstilbestrol (DES), genistein, nonyl phenol,
bisphenol A, WY-14643, ETYA, Benzafibrate, LY171883, GW6471,
GW9662, Troglitazone, and thiazolidinedione.
29. The method according to claim 25, wherein the chimeric protein
comprises amino acid residues 1 to 50 from the N-terminus of the
human PHB 1 protein and amino acid residues 51 to 299 from the
N-terminus of the human PHB2 protein.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for screening for
a PHB2 gene regulator, for example.
BACKGROUND ART
[0002] Mitochondria conduct many reactions in eukaryotes. In
particular, ATP synthesis via electron transport chain is important
for organisms. Most ATP in cells is supplied by mitochondria. Other
reaction systems in mitochondria relate to the TCA cycle, the heme
synthesis system, the .beta. oxidation cycle for fatty acids, the
cycle for amino acid metabolism, and the like. Moreover, functions
for maintaining Ca homeostasis, an active oxygen production system,
and transport systems for metabolites, ions, proteins, and the like
are present in mitochondria. Hence, mitochondria are intracellular
organelles playing important roles in catabolic action and anabolic
action in eukaryotes.
[0003] One thousand (1000) to 1500 types of Protein are inferred to
be present in human mitochondria. Thirteen (13) types thereof are
proteins that are encoded by mitochondrial DNA and are subunits in
electron transport chains. The other proteins, accounting for about
99%, are encoded by nuclear DNA. These proteins translocate to
mitochondria after protein synthesis in cytoplasm. According to
proteomic analysis, approximately 544 types of protein among the
proteins existing in human mitochondria have been identified
(Reichert A S and Neupert W., "Trends in Genetics," 2004, Vol. 20,
No. 11, p. 556-562). However, many unknown proteins are inferred to
be present.
[0004] As described above, mitochondrial DNA encodes some subunits
of complexes I, III, IV, and V in electron transport chains.
Specifically, mutation in mitochondrial DNA causes dysfunction of
electron transport chains. Examples of diseases relating to
dysfunction of electron transport chains include MELAS, MERRF,
cardiomyopathy, LHON, and Leigh encephalopathy. Nucleotide mutation
in mitochondrial DNA has also been observed in early cancer of the
liver, the prostate gland, the bladder, and the head and neck,
primary lung cancer, and Barrett's esophagus (Verma M. et al.,
"Nature reviews cancer," 2003, Vol. 3, No. 10, p. 789-795).
[0005] Meanwhile, abnormalities in mitochondrial proteins encoded
by nuclear DNA cause many diseases as shown below: for example, (i)
Friedreich's ataxia is caused by an abnormality in frataxin protein
involved in Fe--S protein biosynthesis in mitochondria; (ii) an
abnormality in Deafness dystonia peptide 1 (DDP1), which is a
factor involved in protein translocation to mitochondria, is
involved in Mohr-Tranebjaerg syndrome; (iii) retinal atrophy
exhibiting autosomal dominant inheritance is caused by an
abnormality in an OPA1 protein that causes mitochondrial membrane
fusion; (iv) an abnormality in Mfn2, which is another factor
involved in mitochondrial membrane fusion, causes the development
of Charcot-Marie-Tooth neuropathy type 2; and (v) furthermore, an
abnormality in thymidine phosphorylase of mitochondria causes the
development of MNGIE (mitochondrial neurogastrointestinal
encephalomyopathy) exhibiting autosomal recessive inheritance and
causing serious gastrointestinal symptoms.
[0006] In addition to the above involvement, involvement of
mitochondrial dysfunction in more general diseases has also been
demonstrated. For example, abnormalities in sugar metabolism and
lipid metabolism due to mitochondrial dysfunction cause obesity,
diabetes, and the like. Furthermore, a decreased intracellular ATP
level due to mitochondrial dysfunction is a major factor of the
cause of diseases such as Parkinson's disease and Alzheimer's
disease. In recent years, it has also been reported that in
Alzheimer's disease, amyloid .beta. protein, which is an
accumulated substance, binds intracellularly to an ABAD protein,
which is a mitochondrial protein, so as to interfere with
mitochondrial functions (Lustbader, J. W. et al., "Science," 2004,
Vol. 304, No. 5669, p. 448-452).
[0007] It is known that 0.4% to 4% oxygen to be consumed by
mitochondria becomes active oxygen via electron transport chains.
Such active oxygen is thought to damage DNA, proteins, and the like
so as to cause cell injuries, decreased cell counts, and the like,
thereby promoting hypofunction in cells or aging of individual
organisms.
[0008] Furthermore, mitochondria are involved in apoptosis
induction and the pathway is thought to be associated with cell
growth and malignant transformation (canceration).
[0009] Therefore, it is extremely important to maintain normal
mitochondrial functions and to control the functions successfully,
not only for antiaging, but also for maintenance of the homeostasis
of an individual organism's body.
[0010] Meanwhile, a protein called prohibitin (hereinafter,
referred to as "PHB") has been isolated from a mammal for the first
time as a cell growth-suppressing factor. PHB is a protein that is
highly conserved in organisms from yeast to mammals. It is known
that in the PHB protein, 2 types of protein (PHB 1 and PHB2, having
primary amino acid structures analogous to each other) are present,
form a complex, and are localized in the mitochondrial inner
membrane. Yeast PHB proteins have been revealed to exert
chaperone-like functions responsible for cell cycle control and
stabilization of newly synthesized mitochondrial proteins (Berger,
K. H. and Yaffe, M. P., "Mol Cell Biol.," 1998, Vol. 18, No. 7, p.
4043-4052; Nijtmans, L. G. et al., "EMBO J.," 2000, Vol. 19, No.
11, p. 2444-2451; and Piper P. W. and Bringloe, D., "Meth Ageing
Dev.," 2002, Vol. 123, No. 4, p. 287-295). Moreover, in
Caenorhabditis elegans, involvement of PHB 1 in aging and early
development has been reported (Artal-Sanz M. et al., "J Biol.
Chem.," 2003, Vol. 278, No. 34, p. 32091-32099).
[0011] Meanwhile, in mammals, various functions of PHB1 and PHB2,
such as transcriptional control, have been suggested; however,
their physiological functions in mitochondria have not yet been
revealed (Delage-Mourroux R. et al., "J Biol. Chem.," 2000, Vol.
275, No. 46, p. 35848-35856; and Sun L. et al., "J Cell Sci.,"
2004, Vol. 117, p. 3021-3029).
DISCLOSURE OF THE INVENTION
[0012] As described above, it is extremely important to maintain
normal mitochondrial functions and control the functions
successfully, not only for antiaging, but also for maintenance of
the homeostasis of an individual organism's body.
[0013] In view of the above circumstances, an object of the present
invention is to provide, for example, a method for screening for an
agent for regulating mitochondrial functions.
[0014] As a result of intensive studies to achieve the above
object, the present inventors have discovered that human PHB2
protein in mitochondria has an anti-apoptotic effect and functions
to generate mitochondrial membrane potential and to maintain
mitochondrial morphology. Thus, the present inventors have
completed the present invention.
[0015] The present invention encompasses the following (1) to
(24).
(1) A method for screening for a PHB2 gene regulator, comprising
the steps of: causing cells expressing a PHB2 gene to come into
contact with a candidate substance under culture; and determining
that the candidate substance is a PHB2 gene regulator if PHB2 gene
expression or a PHB2 protein function is regulated in the
PHB2-gene-expressing cells caused to come into contact with the
candidate substance, when the cells are compared with cells
expressing the PHB2 gene in the absence of the candidate substance.
(2) The method according to (1), in which the PHB2 protein function
is selected from the group consisting of an anti-apoptotic effect,
generation of mitochondrial membrane potential, and maintenance of
mitochondrial morphology. (3) The method according to (1) or (2),
in which the PHB2 protein function is the capability of the PHB2
protein to interact with a protein that is encoded by a gene
selected from the group consisting of a VDAC2 gene, a Hax-1 gene, a
PHB1 gene, an ANT2 gene, and an OPA1 gene. (4) The method according
to (3), in which the capability to interact is the capability to
form a complex. (5) The method according to (1) or (2), in which
the PHB2 protein function is the nucleus-mitochondria translocation
function of the PHB2 protein. (6) The method according to (5), in
which the cells expressing the PHB2 gene further express a nuclear
receptor selected from the group consisting of ER.alpha.,
PPAR.alpha., and PFAR.gamma.2. (7) The method according to (6), in
which the cells expressing the PHB2 gene are cultured in the
presence of estradiol. (8) A PHB2 gene regulator, which is obtained
by the method according to any one of (1) to (7). (9) A PHB2 gene
regulator, containing the following siRNA (a) or (b): (a) siRNA
consisting of the nucleotide sequence represented by any one of SEQ
ID NOS: 1 to 20; and (b) siRNA consisting of a nucleotide sequence
derived from that of the siRNA according to (a) by deletion,
substitution, or addition of one or several nucleotides and having
activity to inhibit PHB2 gene expression. (10) A PHB2 gene
regulator, containing an antagonist or an agonist of a nuclear
receptor selected from the group consisting of ER.alpha.,
PPAR.alpha., and PPAR.alpha..gamma.2. (11) The PHB2 gene regulator
according to any one of (8) to (10), in which the PHB2 gene
regulator is selected from the group consisting of an
anti-apoptotic agent, an agent for regulating mitochondrial
membrane potential, and an agent for regulating mitochondrial
membrane morphology. (12) A therapeutic drug for
mitochondrial-function-related disease, containing the PHB2 gene
regulator according to any one of (8) to (11). (13) A method for
detecting a mitochondrial-function-related disease, comprising the
steps of: measuring PHB2 gene expression or a PHB2 protein function
in a biological sample derived from a subject; and determining that
the subject has or is suspected of having a
mitochondrial-function-related disease using as an indicator the
presence of an abnormality in PHB2 gene expression or the PHB2
protein function. (14) The method according to (13), in which the
PHB2 protein function is selected from the group consisting of an
anti-apoptotic effect, generation of mitochondrial membrane
potential, and maintenance of mitochondrial morphology. (15) The
method according to (13) or (14), in which the PHB2 protein
function is the capability of the PHB2 protein to interact with a
protein that is encoded by a gene selected from the group
consisting of a VDAC2 gene, a Hax-1 gene, a PHB I gene, an ANT2
gene, and an OPA1 gene. (16) The method according to (15), in which
the capability to interact is the capability to form a complex.
(17) The method according to (13) or (14), in which the PHB2
protein function is the nucleus-mitochondria translocation function
of the PHB2 protein. (18) A method for screening for an agent for
regulating mitochondrial functions, comprising the steps of:
causing cells capable of expressing a PHB2 gene but having
mitochondrial dysfunction to come into contact with a candidate
substance under culture; and determining that the candidate
substance is an agent for regulating mitochondrial functions using
as an indicator the recovery or normalization of mitochondrial
functions as a result of regulation of PHB2 gene expression or the
PHB2 protein function in the mitochondrially dysfunctional cells
caused to come into contact with the candidate substance, when the
cells are compared with mitochondrially dysfunctional cells in the
absence of the candidate substance. (19) The method according to
(18), in which the PHB2 protein function is selected from the group
consisting of an anti-apoptotic effect, generation of mitochondrial
membrane potential, and maintenance of mitochondrial morphology.
(20) The method according to (18) or (19), in which the PHB2
protein function is the capability of the PHB2 protein to interact
with a protein that is encoded by a gene selected from the group
consisting of a VDAC2 gene, a Hax-1 gene, a PHB1 gene, an ANT2
gene, and an OPA1 gene. (21) The method according to (20), in which
the capability to interact is the capability to form a complex.
(22) The method according to (18) or (19), in which the PHB2
protein function is the nucleus-mitochondria translocation function
of the PHB2 protein. (23) The method according to (22), in which
the mitochondrially dysfunctional cells further express a nuclear
receptor selected from the group consisting of ER.alpha.,
PPAR.alpha., and PPAR.gamma.2. (24) The method according to (23),
in which the mitochondrially dysfunctional cells are cultured in
the presence of estradiol.
[0016] The present invention is explained in detail as follows.
[0017] Interaction factors of human PHB2 protein in mitochondria
were searched by immunoprecipitation analysis and mass spectrometry
using purified mitochondrial fractions derived from HeLa cells. As
a result, a mitochondrial protein Hax-1 having an anti-apoptotic
effect (NCBI accession No: NP.sub.--006109) (Cilenti L. et al., J
Biol. Chem., 279: 50295-50301, 2004), a VDAC2 protein, which is a
configuration factor of PTP (permeability transition pore)
(Swiss-Prot accession No: P45880), an ANT2 protein (NCBI accession
No: NP.sub.--001143), and a PHB1 protein (NCBI accession No:
NP.sub.--002625) were identified as interaction factors.
Furthermore, it was revealed by an in vitro binding experiment that
the PHB2 protein directly interacts with the Hax-1 protein.
Furthermore, when HeLa cells were subjected to PHB2 knockdown using
an RNA interference method, decreased expression levels of the PHB1
protein and the Hax-1 protein, reduced mitochondrial membrane
potential, induction of caspase-dependent cell death, decreased
levels of the OPA1 protein (NCBI accession No: NP.sub.--056375),
which is a regulatory factor for mitochondrial membrane morphology,
and mitochondrial fragmentation (change in mitochondrial
morphology) were observed.
[0018] Meanwhile, Hax-1 knockdown was found to have no effect on
the expression level of a PHB2 protein in Hax-1 knockdown cells,
but it was found to induce apoptosis to a degree similar to that
induced by PHB2 knockdown. Thus, it was thought that apoptosis
induced by PHB2 knockdown specifically results from a decrease in
the level of the Hax-1 protein. Moreover, regarding mitochondrial
morphology, mitochondrial fragmentation could not be observed in
Hax-1 knockdown cells.
[0019] Based on the above findings, it was demonstrated that the
PHB2 protein is involved in (1) stabilization of the Hax-1 protein,
which is an interaction factor in mitochondria, (2) an
anti-apoptotic effect that is mediated by (1), (3) generation of
mitochondrial membrane potential, which is thought to be mediated
by binding to the VDAC2 protein and the ANT2 protein, which form a
PTP complex, and (4) regulation of mitochondrial morphology
mediated by the OPA1 protein.
[0020] Accordingly, in mitochondria, the PHB2 protein exhibits an
effect of physically or physiologically interacting with the Hax-1
protein, the VDAC2 protein, the ANT2 protein, the PHB1 protein, the
OPA1 protein, and the like. The PHB2 protein also has an
anti-apoptotic effect, an effect of generating mitochondrial
membrane potential, and an effect of maintaining mitochondrial
morphology.
[0021] Meanwhile, the PHB2 protein has been reported to be present
in the nucleus of mammals, to function for transcriptional
repression, and to recruit histone deacetylase (HDAC) (J. Biol.
Chem. 279 (23), 24834-24843 (2004)). Localization of the PHB2
protein in the nucleus in MCF-7 cells derived from human breast
cancer has been reported (J. Biol. Chem. 279 (23), 24834-24843
(2004)). In the present application, a mechanism by which the PHB2
protein is localized in mitochondria and the nucleus has been
revealed. In HeLa cells, unlike MCF-7 cells, the PHB2 protein is
localized in mitochondria alone and is not localized in the
nucleus. However, it was revealed that the PHB2 protein
translocates from mitochondria to the nucleus in the presence of an
estrogen receptor (hereinafter, referred to as "ER") in an
estradiol-dependent manner (hereinafter, referred to as "E2"). A
region required for the translocation of PHB2 to the nucleus is
dependent on the carboxyl terminus of the PHB2 protein.
Furthermore, a mitochondrial targeting signal and a weak
transmembrane domain are present in the amino terminus of the PHB2
protein. This portion alone translocates to mitochondria.
Translocation of PHB2 to the nucleus takes place also in the
presence of PPAR.alpha. or PPAR.gamma.2, which is a non-ER nuclear
receptor.
[0022] As described above, it was revealed in the present
application that the PHB2 protein is a protein that is localized in
both mitochondria and the nucleus and functions for generation of
mitochondrial membrane potential, an anti-apoptotic effect,
maintenance of mitochondrial morphology, and the like through
transcriptional repression in the nucleus and stabilization of
various proteins in mitochondria.
[0023] The present invention is described as follows based on the
above-explained novel functions of the PHB2 protein.
[0024] The method for screening for a PHB2 gene regulator according
to the present invention comprises: causing cells expressing a PHB2
gene to come into contact with a candidate substance under culture;
and determining that the candidate substance is a PHB2 gene
regulator if PHB2 gene expression or PHB2 protein functions are
regulated in the PHB2-gene-expressing cells caused to come into
contact with the candidate substance, when the cells are compared
with PHB2-gene-expressing cells in the absence of the candidate
substance.
[0025] A human PHB2 gene is DNA consisting of the nucleotide
sequence of SEQ ID NO: 21, which has been registered with the NCBI
under NCBI accession No: NM.sub.--007273. In addition, CDS is the
nucleotide sequence between nucleotides 186 and 1085 in the
nucleotide sequence of SEQ ID NO: 21. The human PHB2 protein is
registered with the NCBI under NCBI accession No: NP.sub.--009204
and is a protein consisting of the amino acid sequence of SEQ ID
NO: 22. Examples of PHB2 genes or PHB2 proteins derived from
non-human organisms include PHB2_YEAST (swissprot: accession
P50085) derived from Saccharomyces cerevisiae, PHB2_CAEEL
(swissprot: accession P50093) derived from Caenorhabditis elegans,
ATPHB2 (PROHIBITIN 2) [Arabidopsis thaliana] (NCBI accession No:
NP.sub.--973755) derived from Arabidopsis thaliana, prohibitin 2
[Xenopus tropicalis] (NCBI accession No: NP.sub.--001016551)
derived from Xenopus tropicalis, PHB2--MOUSE (swissprot: accession
035129) derived from a mouse, and PHB2_RAT (swissprot: accession
Q5XIH7) derived from a rat. Examples of such PHB2 gene in the
present invention include not only DNAs consisting of nucleotide
sequences of the above SEQ ID NOS: or accession Nos. Furthermore,
DNA consisting of a nucleotide sequence derived from that of the
above DNA by deletion, substitution, or addition of 1 or a
plurality of (e.g., 1 to 10 and preferably 1 to 5) nucleotides and
encoding a protein having PHB2 protein functions is also included
herein. Moreover, examples of such PHB2 gene also include DNAs each
having 90% or more, preferably 95% or more, 97% or more, and more
preferably 98% or more, and particularly preferably 99% or more
identity with DNA consisting of the nucleotide sequence of the
above SEQ ID NO: or accession No and encoding a protein having PHB2
protein functions. Alternatively, not only DNAs encoding the amino
acid sequences of the above SEQ ID NOS: or accession Nos., but also
DNAs each encoding a protein consisting of an amino acid sequence
derived from the above amino acid sequence by deletion,
substitution, or addition of 1 or a plurality of (e.g., 1 to 10 and
preferably 1 to 5) amino acids and having PHB2 protein functions
are also included in the examples of the PHB2 gene.
[0026] Here, examples of PHB2 protein functions include an
anti-apoptotic effect, generation of mitochondrial membrane
potential, maintenance of mitochondrial morphology, capability
(e.g., the capability to form a complex, the capability for signal
transduction, the capability for protein stabilization, protein
conformation and/or, the capability for functional regulation
(physiological interaction is also included herein)) of the PHB2
protein to interact with a protein that is encoded by a gene
selected from the group consisting of the VDAC2 gene, the Hax-1
gene, the PHB1 gene, the ANT2 gene, and the OPA1 gene (hereinafter,
the proteins are referred to as the VDAC2 protein, the Hax-1
protein, the PHB1 protein, the ANT2 protein, and the OPA1 protein,
respectively), and a nucleus-mitochondria translocation function.
"Capability to form a complex" in the description refers to the
capability to form a complex that is formed via direct or indirect
binding in mitochondria, nucleus, cytoplasm, and the like. Examples
of components of such a complex include proteins or nucleic acids,
or some types of lipid. Furthermore, "nucleus-mitochondria
translocation function" refers to a function to translocate between
mitochondria and the nucleus. Examples of such function include a
function to translocate between mitochondria and cytoplasm and a
function to translocate between cytoplasm and the nucleus.
Furthermore, an example of such mechanism for nucleus-mitochondria
translocation is a transport mechanism by which transporters that
mediate such translocation or ligands that stimulate the same are
included.
[0027] Cells expressing a PHB2 gene may be any cells, as long as
they express the PHB2 gene. Examples of such cells include cells of
human-derived cell lines, such as HeLa cells (derived from human
cervical cancer), MCF-7 cells (derived from human breast cancer),
U-2OS cells (derived from human osteosarcoma), and human
fibroblasts. Examples of such cells further include B6.1 cells
(derived from mouse myeloma), mouse embryonic stem cells, and SWISS
3T3 cells (derived from mouse fibroblasts). Furthermore, examples
of PHB2-gene-expressing cells also include cells transfected with
the above PHB2 gene (or in which the PHB2 gene has been
overexpressed). For example, a PHB2 gene contained in a PCR product
or a vector can be introduced into cells by an electroporation
method, a calcium phosphate method, a lipofection method, or the
like. An example of a method for confirming the expression of a
PHB2 gene in PHB2-gene-expressing cells at the mRNA level is a
method that involves confirming by RT-PCR, quantitative PCR, or
Northern blotting using primers or a probe specific to the PHB2
gene. Moreover, at the protein level, PHB2 gene expression can be
confirmed using an immunological method such as ELISA, flow
cytometry, or Western blotting using an antibody specific to the
PHB2 protein, for example.
[0028] In the meantime, in the present invention, examples of a
candidate substance include nucleic acids, peptides, proteins,
synthetic compounds, culture supernatants of microorganisms,
natural ingredients derived from plants or marine organisms, plant
extracts, and animal tissue extracts.
[0029] The method for screening for a PHB2 gene regulator according
to the present invention comprises causing cells expressing a PHB2
gene to come into contact with a candidate substance under culture.
Such PHB2-gene-expressing cells can be cultured by adequately
selecting medium and culture conditions (e.g., temperature and pH)
depending on each cell. Here, contact means the status that the
candidate substance has an effect on cells expressing the PHB2
gene. For example, a candidate substance may be simply added to
medium of PHB2-gene-expressing cells. Alternatively, a candidate
substance may be embedded in or bound to a liposome and then added
to medium. Furthermore, a candidate substance may be added together
with a type of carrier substance (protein, lipid, or the like) to
medium. Furthermore, a candidate substance may be directly
introduced into PHB2-gene-expressing cells via microinjection or
the like. The time for culturing may also be any time length as
long as it is sufficient for a candidate substance to have an
effect on PHB2-gene-expressing cells. Such time for culturing may
range from 1 to 72 hours and preferably may range from 12 to 24
hours, for example.
[0030] Subsequently, the method for screening for a PHB2 gene
regulator according to the present invention comprises determining
whether or not PHB2 gene expression or PHB2 protein functions are
regulated in the PHB2-gene-expressing cells caused to come into
contact with the candidate substance, when the cells are compared
with PHB2-gene-expressing cells in the absence of the candidate
substance. Here, regulation of PHB2 gene expression means a
decrease or an increase in PHB2 gene expression at the mRNA level
or the protein level. Moreover, regulation of PHB2 protein
functions means enhanced PHB2 protein functions described
above.
[0031] In a method for evaluating the regulation of PHB2 gene
expression, mRNA or a protein is extracted from cells expressing
the PHB2 gene after culturing. Subsequently, a PHB2 gene expression
level in the thus obtained mRNA or protein is compared with a PHB2
gene expression level in cells expressing the PHB2 gene cultured in
the absence of the candidate substance. In addition, a PHB2 gene
expression level at the mRNA level or protein level can be measured
according to the above-described method.
[0032] It can be determined that a candidate substance is a PHB2
gene regulator: if PHB2 gene expression levels are significantly
increased (e.g., 1.5- to 100-fold and preferably 2- to 5-fold) at
the mRNA level or protein level in PHB2-gene-expressing cells
caused to come into contact with the candidate substance; or if
PHB2 gene expression levels are significantly decreased (e.g.,
decreased to 1/2 to 1/1000 and preferably 1/4 to 1/10), when the
cells are compared with PHB2-gene-expressing cells cultured in the
absence of a candidate substance.
[0033] Meanwhile, regulation of PHB2 protein functions can be
evaluated separately according to each function.
[0034] The anti-apoptotic effect can be evaluated by detecting DNA
fragmentation, cell membrane structural changes due to binding of
Annexin V, disappearance of mitochondrial membrane potential, the
activity of an apoptosis-associated enzyme, such as caspase, and/or
transfer of cytochrome c from mitochondria to cytoplasm, for
example.
[0035] Generation of mitochondrial membrane potential can be
evaluated by subjecting PHB2-gene-expressing cells to staining
using a mitochondria staining marker Rh123 depending on the
mitochondrial membrane potential and then observing the degrees of
staining under a microscope, for example.
[0036] Maintenance of mitochondrial morphology can be evaluated by
subjecting PHB2-gene-expressing cells to staining of mitochondria
using MitoTracker Red and then observing the mitochondrial
morphology via microscopic observation, for example.
[0037] Moreover, the capability of the PHB2 protein to interact
with the VDAC2 protein, the Hax-1 protein, the PHB1 protein, the
ANT2 protein, or the OPA1 protein (e.g., capability to form a
complex) can be evaluated by subjecting a protein derived from
PHB2-gene-expressing cells to immunological analysis using an
antibody specific to each protein and then observing association
between the proteins, for example.
[0038] Furthermore, regarding the nucleus-mitochondria
translocation function, translocation of a PHB2 protein from
mitochondria to the nucleus can be evaluated by culturing
PHB2-gene-expressing cells that express ER.alpha., PPAR.alpha., or
PPAR.gamma.2 (in the presence of E2 in the case of ER.alpha.) and
then staining using an antibody specific to the PHB2 protein, for
example. The amount of E2 to be added to medium ranges from
1.times.10.sup.-12 to 1.times.10.sup.-4M and preferably ranges from
1.times.10.sup.-7 to 1.times.10.sup.-6M, for example.
[0039] It can be determined that a candidate substance is a PHB2
gene regulator when the PHB2 protein functions were observed to be
significantly enhanced (e.g., enhanced 1.5- to 100-fold and
preferably 2- to 10-fold) in PHB2-gene-expressing cells caused to
come into contact with the candidate substance, when the cells are
compared with PHB2-gene-expressing cells cultured in the absence of
the candidate substance.
[0040] Meanwhile, according to the above method for screening for a
PHB2 gene regulator according to the present invention, an agent
for regulating mitochondrial functions can be screened for. The
method for screening for an agent for regulating mitochondrial
functions according to the present invention comprises causing
cells capable of expressing the PHB2 gene but having mitochondrial
dysfunction to come into contact with a candidate substance under
culture; and determining that the candidate substance is an agent
for regulating mitochondrial functions using as an indicator the
recovery or normalization of mitochondrial functions as a result of
regulation of PHB2 gene expression or PHB2 protein functions in the
mitochondrially dysfunctional cells caused to come into contact
with the candidate substance, when the cells are compared with
mitochondrially dysfunctional cells in the absence of the candidate
substance.
[0041] Here, cells capable of expressing the PHB2 gene but having
mitochondrial dysfunction (hereinafter, referred to as simply
"mitochondrially dysfunctional cells") are cells expressing the
PHB2 gene and having abnormalities in mitochondrial functions as
described above. Examples of such cells include cells subjected to
PHB2 knockdown and caused to express another mutant PHB2.
[0042] In the method for screening for an agent for regulating
mitochondrial functions according to the present invention,
according to the above method for screening for a PHB2 gene
regulator according to the present invention, whether or not
mitochondrial functions are recovered or normalized (as a result of
regulation of PHB2 gene expression or PHB2 protein functions in
mitochondrially dysfunctional cells caused to come into contact
with candidate substances) is determined via comparison of the
cells with mitochondrially dysfunctional cells in the absence of
the candidate substances. Based on such indicator, an agent for
regulating mitochondrial functions can be selected from among the
above candidate substances.
[0043] Examples of the PHB2 gene regulator according to the present
invention include a PHB2 gene regulator that is obtained by the
above method for screening for a PHB2 gene regulator, a PHB2 gene
regulator containing the following siRNA (a) or (b): (a) siRNA
consisting of the nucleotide sequence represented by any one of SEQ
ID NOS: 1 to 20; or (b) siRNA consisting of a nucleotide sequence
derived from that of the siRNA of (a) above by deletion,
substitution, or addition of one or several (e.g., 1 to 5 and
preferably 1 to 3) nucleotides and having activity of inhibiting
PHB2 gene expression, and a PHB2 gene regulator containing an
antagonist or an agonist for an ER.alpha., PPAR.alpha., or
PPAR.gamma.2 nuclear receptor. Furthermore, siRNA having 90% or
more, preferably 95% or more, 97% or more, more preferably 98% or
more, and particularly preferably 99% or more identity with the
siRNA of (a) above and having activity of inhibiting PHB2 gene
expression can also be an example of the PHB2 gene regulator
according to the present invention.
[0044] The siRNA (a) or (b) above can be chemically synthesized by
a known method for nucleic acid synthesis, for example.
[0045] Examples of an antagonist or an agonist for an ER.alpha.,
PPAR.alpha., or PPAR.gamma.2 nuclear receptor include antagonists
of ER, such as ICI182,780, DDE, and Tamoxifen, agonists of ER, such
as DES (Diethylstilbestrol), genistein, nonyl phenol, and bisphenol
A, agonists of PPAR.alpha., such as WY-14643, ETYA, Benzafibrate,
and LY171883, antagonists of PPAR.alpha., such as GW6471, agonists
of PPAR.gamma., such as GW9662 and Troglitazone, and antagonists of
PPAR.gamma., such as thiazolidinedione.
[0046] Further examples of the PHB2 gene regulator include agonists
or antagonists of other nuclear receptors of TR, PR, RXR, and the
like.
[0047] Furthermore, as a PHB2 gene regulator, a chimeric protein
comprising a mitochondrial targeting signal and a transmembrane
domain (in the human PHB1 protein, amino acid residues 1 to 50 from
the N-terminus) of a PHB1 protein and the C-terminus of the PHB2
protein (in the human PHB2 protein, amino acid residues 51 to 299
from the N-terminus) can be used, for example.
[0048] In addition, based on PHB2 protein functions, the PHB2 gene
regulator can be used as an anti-apoptotic agent, an agent for
regulating mitochondrial membrane potential, or an agent for
regulating the morphology of mitochondrial membrane.
[0049] The therapeutic drug for mitochondrial-function-related
disease according to the present invention is characterized by
containing the PHB2 gene regulator according to the present
invention. Here, the mitochondrial-function-related disease means
disease associated with mitochondrial dysfunction. Examples of such
mitochondrial-function-related disease include obesity, diabetes,
Parkinson's disease, Alzheimer's disease, and cancer (e.g., breast
cancer), but are not limited thereto.
[0050] The therapeutic drug for mitochondrial-function-related
disease according to the present invention may directly be the PHB2
gene regulator according to the present invention or may be a drug
that is formulated into a dosage form (e.g., a tablet, a powder, an
emulsion, or a capsule) using a generally employed solid or liquid
carrier, emulsifying and dispersing agent, or the like. Examples of
the above carrier include water, gelatin, starch, magnesium
stearate, lactose, and plant oil. The content of the PHB2 gene
regulator in the therapeutic drug for
mitochondrial-function-related disease according to the present
invention and the dosage of the therapeutic drug can be adequately
varied depending on purposes of administration, routes of
administration, dosage forms, and the like.
[0051] Pharmacological evaluation of the therapeutic drug for
mitochondrial-function-related disease according to the present
invention can be performed, for example, at the in vitro level
using the above-described mitochondrially dysfunctional cells or at
the in vivo level using animal models for
mitochondrial-function-related disease such as Alzheimer's disease.
For example, pharmacological evaluation can be performed based on
whether or not mitochondrial dysfunction is restored or normalized
in mitochondrially dysfunctional cells cultured in the presence of
the therapeutic drug for mitochondrial-function-related disease
according to the present invention, when the cells are compared
with cells in the absence of the therapeutic drug. Alternatively,
pharmacological evaluation can also be performed based on whether
or not a mitochondrial-function-related disease can be treated or
ameliorated in a mitochondrial-function-related disease animal
model to which the therapeutic drug for
mitochondrial-function-related disease according to the present
invention has been administered, when the animal model is compared
with an animal model to which no such therapeutic drug has been
administered.
[0052] The method for detecting a mitochondrial-function-related
disease according to the present invention comprises: measuring
PHB2 gene expression or PHB2 protein functions in a biological
sample derived from a subject; and determining that the subject has
or is suspected of having a mitochondrial-function-related disease
using as an indicator the presence of an abnormality in PHB2 gene
expression or PHB2 protein functions.
[0053] Examples of a biological sample include subject-derived
cells such as fibroblasts, myoblasts, white blood cells,
spermatids, and egg cells, tissues and organs containing these
cells, body fluids such as blood and saliva, and excretory
substances.
[0054] The method for detecting a mitochondrial-function-related
disease according to the present invention can be performed
according to the above method for screening for a PHB2 gene
regulator according to the present invention. Specifically, first,
PHB2 gene expression or PHB2 protein functions in cells in a
biological sample are measured. Subsequently, whether or not PHB2
gene expression or PHB2 protein functions are abnormal is
determined via comparison with cells derived from a normal subject.
Here, the abnormality in PHB2 gene expression or PHB2 protein
functions means an increase or a decrease in PHB2 gene expression
or a decrease in PHB2 protein functions, for example. When an
abnormality is found in PHB2 gene expression or PHB2 protein
functions, it can be determined that the subject from which the
biological sample is derived has or is suspected of having a
mitochondrial-function-related disease.
[0055] As explained above, based on the novel functions of the PHB2
protein, a PHB2 gene regulator can be identified and an agent for
regulating mitochondrial functions or a therapeutic drug for
mitochondrial-function-related disease can be provided.
[0056] This specification includes the contents as disclosed in the
specification and/or drawings of Japanese Patent Application No.
2005-339354, which is a priority document of the present
application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 shows confocal laser scanning micrographs showing
HeLa cells expressing PHB1-FLAG or PHB2-FLAG in Example 1.
[0058] FIG. 2 shows photographs showing HeLa cells expressing each
gene, which were stained with Rh123, in Example 2.
[0059] FIG. 3 shows the results of IP analysis and MASS analysis in
Example 3.
[0060] FIG. 4 shows the results of an in vitro binding experiment
concerning the interaction between a PHB2 protein and an
Hax-1protein in Example 4.
[0061] FIG. 5 shows the results of PHB2 knockdown by an RNAi method
in Example 5.
[0062] FIG. 6 shows the results of examining mitochondrial membrane
potential and caspase-dependent cell death (apoptosis) in HeLa
cells that were subjected to PHB1 or PHB2 knockdown in Example
6.
[0063] FIG. 7 shows the results of Hax-1 knockdown by an RNAi
method in Example 7.
[0064] FIG. 8 shows photographs showing PHB1, PHB2, or Hax-1
knockdown cells in which mitochondria were stained with MitoTracker
Red in Example 8.
[0065] FIG. 9 shows the results of Western blot analysis using an
antibody against an OPA1 protein in Example 8.
[0066] FIG. 10 shows micrographs showing MCF-7 cells and HeLa cells
expressing PHB1-GFP or PHB2-GFP in Example 9.
[0067] FIG. 11 shows confocal laser scanning micrographs showing
HeLa cells that were caused to coexpress PHB2-GFP and ER.alpha. in
Example 10.
[0068] FIG. 12 shows the results of structural analysis of PHB2
proteins in Example 11.
[0069] FIG. 13 shows micrographs showing HeLa cells expressing
PHB1-GFP or PHB2-GFP and PPAR.alpha. or PPAR.gamma.2 in Example
12.
[0070] FIG. 14 shows relative luciferase activity in HeLa cells
transfected with each gene in Example 13.
BEST MODES FOR CARRYING OUT THE INVENTION
Examples
[0071] Hereafter, the present invention is described in greater
detail with reference to the following examples, although the
technical scope of the present invention is not limited
thereto.
[0072] In addition, proteins mentioned in these Examples were all
derived from human, except for proteins (e.g., FLAG tag, GST, GFP,
and luciferase) used as labels or reporters.
Example 1
Intracellular Localization of PHB 1 Protein or PHB2 Protein in HeLa
Cell
[0073] A gene encoding a PHB1 protein (hereinafter, referred to as
"PHB1-FLAG") or a PHB2 protein (hereinafter, referred to as
"PHB2-FLAG"), in which a FLAG tag had been fused to the carboxyl
terminus, was expressed transiently in HeLa cells (derived from
human cervical cancer).
[0074] The method employed herein is as follows. cDNA encoding
PHB1-FLAG or PHB2-FLAG was inserted into a mammalian cell
expression vector pCMV-SPORT and then HeLa cells were transfected
with the vector. After approximately 12 hours, these cells were
fixed and then subjected to immunostaining. Antibodies used herein
are as follows: an anti-FLAG rabbit polyclonal antibody and an
anti-cytochrome C (cyt.c) mouse monoclonal antibody as a control
antibody for staining mitochondria were used as primary antibodies;
and as secondary antibodies, an Alexa488-labeled anti-rabbit
antibody and a Cy3-labeled anti-mouse antibody were used. These
cells were observed under a confocal laser scanning microscope.
[0075] FIG. 1 shows the results. FIG. 1 shows confocal laser
scanning micrographs showing HeLa cells expressing PHB1-FLAG or
PHB2-FLAG. In FIG. 1, "a-FLAG" denotes images (green) of staining
with the anti-FLAG antibody and ".alpha.-cyt.c" denotes images
(red) of staining with the anti-cytochrome C antibody. Moreover,
"Merge" shows merged images of the two.
[0076] As shown in FIG. 1, when PHB1-FLAG and PHB2-FLAG were each
transiently expressed by HeLa cells, PHB1-FLAG and PHB2-FLAG were
found to be localized in mitochondria. PHB1-FLAG and PHB2-FLAG in
HeLa cells were found to be co-localized with cyt.c, which is a
mitochondria marker.
Example 2
Disappearance of Mitochondrial Membrane Potential by PHB1 Protein
or PHB2 Protein Expression
[0077] As described in Example 1, when PHB1-FLAG and PHB2-FLAG were
separately transiently expressed by HeLa cells, PHB1-FLAG and
PHB2-FLAG were found to be localized in mitochondria.
[0078] HeLa cells expressing PHB1-FLAG or PHB2-FLAG were then
stained with Rh123, which is a mitochondrial staining marker,
depending on mitochondrial membrane potential. Similarly, a gene
encoding a fusion protein (hereinafter, referred to as "SLP2-FLAG")
in which a FLAG tag had been fused to the carboxyl terminus of an
SLP2 protein (NCBI accession No: NP.sub.--038470) (J Biol. Chem.
2000 Mar. 17; 275(11): 8062-71) was transiently expressed by HeLa
cells, followed by staining with Rh123. Furthermore, as a control,
HeLa cells that had not been transfected with any genes were
stained with Rh123.
[0079] FIG. 2 shows the results. FIG. 2 shows photographs showing
HeLa cells expressing each gene, such cells having been stained
with Rh123. In FIG. 2, "TI" denotes photographs of transmission
images.
[0080] As shown in FIG. 2, it was revealed that HeLa cells
overexpressing PHB1-FLAG or PHB2-FLAG were not stained with Rh123.
This indicates disappearance of mitochondrial membrane potential
due to forced expression of PHB1-FLAG or PHB2-FLAG, suggesting
possible involvement of the PHB protein in regulation of membrane
potential in mitochondria.
Example 3
Molecular Functional Analysis of PHB2 Protein
[0081] To elucidate the molecular functions of the PHB2 protein, an
interaction factor of the PHB2 protein in mitochondria was
searched.
[0082] First, immunoprecipitation (IP) analysis and mass
spectrometry (MASS analysis) were conducted using purified
mitochondrial fractions derived from HeLa cells expressing
PHB2-FLAG, which had been prepared in Example 1.
[0083] In IP analysis, an immunoprecipitation method was performed
using an anti-FLAG antibody, the precipitates were subjected to 12%
SDS-PAGE, protein staining was performed, and then proteins that
had been co-precipitated with the PHB2-FLAG protein were
detected.
[0084] Meanwhile, in MASS analysis, the bands of proteins obtained
by IP analysis were excised and then subjected to digestion using
trypsin within gel, the thus digested peptides were extracted, and
then peptide fragments were identified using nanoscale high
performance liquid chromatography and tandem mass spectrometer
(nano LC-MS/MS). Subsequently, proteins were identified by database
analysis.
[0085] FIG. 3 shows the results of IP analysis and MASS
analysis.
[0086] As is understood from FIG. 3, mitochondrial protein Hax-1
(NCBI accession No: NP.sub.--006109) having an anti-apoptotic
effect, the VDAC2 protein (Swiss-Prot accession No: P45880), which
is a configuration factor of PTP (permeability transition pore), an
ANT2 protein (NCBI accession No: NP.sub.--001143), and the PHB1
protein (NCBI accession No: NP.sub.--002625) known to form a
complex with the PHB2 protein (Curr. Biol. 7 (8), 607-610 (1997))
were obtained as factors interacting with the PHB2 protein.
Example 4
Interaction Between PHB2 Protein and Hax-1 Protein
[0087] Interaction of the PHB2 protein with the Hax-1 protein, the
VDAC2 protein, and the PHB1 protein was confirmed via IP analysis
described in Example 3 and Western blot analysis using specific
antibodies against the PHB2 protein, the Hax-1 protein, the VDAC2
protein, and the PHB1 protein.
[0088] Accordingly, interaction between the PHB2 protein and the
Hax-1 protein was examined by an in vitro binding experiment.
[0089] Methods employed herein are as follows. A glutathione
S-transferase (GST)-PHB2 fusion protein (the protein prepared by
fusing a GST protein to the N-terminus of the PHB2 protein) that
had been synthesized using Escherichia coli was mixed in vitro with
a PHB1-FLAG protein and a Hax-1-FLAG protein (the protein prepared
by fusing a FLAG tag to the C-terminus of the Hax-1 protein), which
had been translated in vitro using a reticulocyte lysate solution.
GST fusion proteins were then pulled down using
glutathione-sepharose beads, so as to obtain precipitates.
Subsequently, the precipitates were subjected to SDS-PAGE and then
the Western blot method was performed using an anti-FLAG antibody.
Thus, whether or not each FLAG fusion protein had bound to the PHB2
protein was examined.
[0090] FIG. 4 shows the results. In FIG. 4, "Input" denotes each
FLAG fusion protein mixed therein. Furthermore, "GST-pulldown"
denotes a precipitate obtained via precipitation using
glutathione-sepharose beads. Furthermore, "-" denotes that the
protein was not mixed in the reaction system, "+" denotes that the
protein was mixed in the reaction system.
[0091] As shown in FIG. 4, it was revealed that the PHB2 protein
directly interacts with the Hax-1 protein.
Example 5
PHB2 Knockdown by RNAi Method
[0092] PHB2 knockdown was performed in HeLa cells by an RNAi
method. siRNA consisting of the nucleotide sequence of SEQ ID NO: 1
was used herein. Furthermore, PHB 1 knockdown was also performed
using siRNA (SEQ ID NO: 23) for PHB 1.
[0093] The method employed herein is as follows. A plasmid was
constructed by inserting nucleotide sequences for expression of
short hairpin RNAs partially matching the cDNA of PHB2 and cDNA of
PHB1 into a pScilencer 3.1-H1 puro vector having a puromycin
resistance gene. Cultured cells were transfected with the thus
prepared plasmids. Subsequently, puromycin was added to medium, so
that cells in which target genes had been knocked down could be
obtained.
[0094] FIG. 5A to C show the results. FIG. 5A shows the results of
Western blot analysis using an antibody against each protein when
PHB1 knockdown or PHB2 knockdown was performed. FIG. 5B shows the
results of Western blot analysis using an antibody against the
Hax-1 protein when PHB1 knockdown or PHB2 knockdown was performed.
FIG. 5C shows the results of RT-PCR by which the Hax-1 mRNA level
was measured when PHB1 knockdown or PHB2 knockdown was performed.
In FIGS. 5A to C, "siPHB1" or "siPHB2" indicates the result of PHB1
knockdown or PHB2 knockdown performed by the RNAi method. "Control"
denotes cells not transfected with any genes and "puro-vec."
denotes cells transfected with a puromycin resistance gene and then
subjected to drug selection.
[0095] As shown in FIGS. 5A to C, when HeLa cells were subjected to
PHB2 knockdown using the RNAi method, the expression levels of the
Hax-1 protein and the PHB1 protein, which are interaction factors,
were found to be decreased. However, the mRNA of PHB1 and the mRNA
of PHB2 were not found to be decreased. It has been reported that
yeast PHB interacts with a novel synthesized mitochondrial protein
so as to stabilize the protein (Mol. Cell. Biol. 19, 3435-3442
(1999) and EMBO J. 19, 2444-2451 (2000)). Accordingly, it is
thought that human PHB2 protein may also stabilize the Hax-1
protein, which is an interaction factor.
Example 6
Decrease in Mitochondrial Membrane Potential and Induction of
Caspase-Dependent Cell Death (Apoptosis) in PHB2 Knockdown Hela
Cell
[0096] In this example, mitochondrial membrane potential and
caspase-dependent cell death (apoptosis) in HeLa cells subjected in
Example 5 to PHB1 knockdown or PHB2 knockdown were examined.
[0097] A method similar to the method employed for knockdown
performed in Example 5 was employed.
[0098] FIGS. 6A to C show the results. FIGS. 6A and B show relative
cell counts of HeLa cells subjected to PHB1 knockdown and HeLa
cells subjected to PHB2 knockdown. In FIG. 6B, "Z-VAD-FMK" is an
inhibitor for the caspase which is activated in apoptosis, and "+"
or "-" with reference to Z-VAD-FMK denotes addition or no addition
to the medium. FIG. 6C shows the results of Rh123 staining in HeLa
cells subjected to PHB1 knockdown or PHB2 knockdown. In FIGS. 6A to
C, "siPHB1" or "siPHB2" indicates the result of PHB1 knockdown or
PHB2 knockdown by the RNAi method; and "puro-vec." indicates the
result of transfection with a plasmid vector having a puromycin
resistance gene.
[0099] As shown in FIGS. 6A and B, it was revealed that
caspase-dependent cell death (apoptosis) had been induced in HeLa
cells subjected to PHB1 knockdown or PHB2 knockdown. Moreover, as
shown in FIG. 6C, it was revealed that mitochondrial membrane
potential had been decreased in some of HeLa cells subjected to
PHB1 knockdown or PHB2 knockdown.
Example 7
Hax-1 Knockdown by RNAi Method
[0100] In this Example, HeLa cells were subjected to the knockdown
of Hax-1, which is an interaction factor of PHB2. siRNA consisting
of the nucleotide sequence of SEQ ID NO: 24 was used as siRNA for
Hax-1.
[0101] The method employed herein is as follows. A plasmid was
constructed by inserting a nucleotide sequence for the expression
of short hairpin RNA partially matching Hax-1 cDNA into a
pScilencer 3.1-H1 puro vector having puromycin resistance gene
according to the method of Example 5.
[0102] FIGS. 7A to C show the results. FIG. 7A shows the result of
Western blot analysis using an antibody against each protein when
Hax-1 knockdown was performed. FIGS. 7B and C show relative cell
counts of HeLa cells subjected to Hax-1 knockdown. In FIG. 7,
"Z-VAD-FMK" and "+" or "-" with reference to Z-VAD-FMK have the
same meaning as they do in FIG. 6. In FIGS. 7A to C, "siHax-1"
indicates the result of Hax-1 knockdown performed by the RNAi
method and "puro-vec." indicates the result of transfection with a
plasmid vector having a puromycin resistance gene.
[0103] As shown in FIGS. 7A to C, Hax-1 knockdown did not affect
the expression levels of the PHB1 protein and the PHB2 protein.
However, it induced apoptosis similar to that induced by PHB2
knockdown. Hence, it was thought that a decrease in Hax-1 protein
expression level is a cause of apoptosis induced by PHB2 knockdown.
Therefore, it was demonstrated that the PHB2 protein exerts its
anti-apoptotic effect via maintenance of the expression level of
the Hax-1 protein.
Example 8
Change in Mitochondrial Morphology Due to PHB2 Knockdown
[0104] In this Example, changes in mitochondrial morphology in HeLa
cells subjected to PHB1 knockdown, PHB2 knockdown, or Hax-1
knockdown described in Examples 5 and 7 were examined.
[0105] The method employed herein is as follows. Knockdown cells
prepared in Examples 5 and 7 were stained with MitoTracker Red, and
then the morphology of mitochondria was observed under a confocal
laser scanning microscope.
[0106] FIG. 8 shows the results. In FIG. 8, "puro-vec." indicates
the results of transfection with a plasmid vector having a
puromycin resistance gene. Furthermore, "siPHB1," "siPHB2," and
"siHax-1" denote the results of subjecting HeLa cells to PHB1
knockdown, PHB2 knockdown, and Hax-1 knockdown, respectively.
[0107] As shown in FIG. 8, mitochondrial fragmentation (a change in
mitochondrial morphology) was observed in PHB1 or PHB2 knockdown
cells in which mitochondria had been stained with MitoTracker Red.
Such mitochondrial fragmentation was not observed in Hax-1
knockdown cells. Hence, this mitochondrial fragmentation was
thought to take place in a manner specific to PHB protein knockdown
and to be the result of a morphological change not mediated by
Hax-1.
[0108] Furthermore, the above PHB1 knockdown, PHB2 knockdown, or
Hax-1 knockdown cells were subjected to Western blot analysis using
an antibody against an OPA1 protein, which is a mitochondrial
fusion factor.
[0109] The method employed herein is as follows. A cell extract was
prepared from each type of knockdown cell and then subjected to the
Western blot method. An anti-OPA1 polyclonal rabbit antibody was
used as a primary antibody.
[0110] FIG. 9 shows the results. In FIG. 9, "control" denotes
untransfected cells. Moreover, "puro-vec.," "siPHB1," "siPHB2," and
"siHax-1" indicate the results of cells similar to FIG. 8.
[0111] As shown in FIG. 9, a significantly decreased expression
level of the OPA1 protein was observed in PHB2 knockdown cells.
Hence, it was concluded that mitochondrial fragmentation due to a
decrease in OPA1 protein level had taken place. On the other hand,
the OPA1 protein level was also found to be decreased in Hax-1
knockdown cells. Moreover, a decrease in OPA1 protein expression
level due to PHB1 knockdown was not significant compared with the
same due to PHB2 knockdown, but mitochondrial fragmentation was
induced to a degree equivalent to that induced by PHB2 knockdown
(FIG. 8).
[0112] It is known that the OPA1 protein maintains mitochondrial
morphology through a balance with other factors (e.g., Drp1) for
regulating mitochondrial morphology (Cell. 2004 Dec. 17; 119 (6):
873-87). Mitochondrial fragmentation in PHB1 knockdown cells is
thought to be associated with some factors other than the OPA1
expression level.
Example 9
Intracellular Localization of PHB1 Protein or PHB2 Protein in MCF-7
Cell
[0113] As shown in Example 1, the PHB1 protein and the PHB2 protein
were localized in mitochondria of HeLa cells.
[0114] In this Example, intracellular localization of the PHB1
protein and the PHB2 protein in MCF-7 cells (derived from human
breast cancer) was examined, in addition to that in HeLa cells
(derived from human cervical cancer).
[0115] The method employed herein is as follows. A mammalian cell
expression vector was constructed by ligating cDNA encoding the
PHB2 or PHB1 protein (fusion proteins are referred to as "PHB2-GFP"
and "PHB1-GFP," respectively) in which a green fluorescent protein
(GFP) had been ligated to the C-terminus. Cultured cells were then
transfected with the vector, following which the intracellular
localization of the GFP fusion proteins was observed under a
confocal laser scanning microscope.
[0116] FIG. 10 shows the results. FIG. 10 shows micrographs showing
MCF-7 cells expressing PHB1-GFP or PHB2-GFP and HeLa cells
expressing PHB1-GFP or PHB2-GFP. As shown in FIG. 10, when MCF-7
cells and HeLa cells were transfected with a gene encoding
PHB1-GFP, PHB1-GFP was found to be localized in mitochondria in
both types of cells. On the other hand, PHB2-GFP was found to be
localized in the mitochondria of HeLa cells, but in the case of
MCF-7 cells, some PHB2-GFPs were also found to be localized in the
nucleus.
Example 10
Interaction Between PHB2 Protein and Nuclear Receptor ER.alpha.
[0117] In this Example, interaction between the PHB2 protein and a
nuclear receptor ER.alpha. was examined.
[0118] The method employed herein is as follows. HeLa cells were
transfected with an ER.alpha. gene and a PHB2-GFP gene incorporated
in an expression vector. Subsequently, the cells were fixed and
then cell immunostaining was performed using an anti-ER.alpha.
rabbit polyclonal antibody as a primary antibody and a Cy3-labeled
anti-rabbit antibody as a secondary antibody.
[0119] FIG. 11 shows the results. FIG. 11 shows confocal laser
scanning micrographs obtained when PHB2-GFP and ER.alpha. had been
co-expressed by HeLa cells. In FIG. 11, "TI" denotes transmission
images and ".alpha.-ER.alpha." denotes anti-ER.alpha.
antibody-stained images (red). Furthermore, "PHB2-GFP" denotes GFP
images (green), and "merge" denotes merged images. "E2(-)" denotes
cells cultured in the absence of E2 and "E2(+)" denotes cells
cultured in the presence of E2.
[0120] As shown in FIG. 11, co-expression of PHB2-GFP with
ER.alpha. in HeLa cells caused PHB2-GFP to be localized in the
nucleus in the presence of E2. It is known based on a previous
report that the PHB2 protein binds to the nuclear receptor
ER.alpha. (J. Biol. Chem. Vol. 275, No. 46, pp. 35848-35856, 2000).
Therefore, it was considered that PHB2 binds to ER.alpha. in an
E2-dependent manner, so as to translocate to the nucleus.
Example 11
Structural Analysis of PHB2 Protein
[0121] As shown in the above Example, it was revealed that the PHB2
protein is localized in mitochondria and the nucleus. To elucidate
the mechanism, deletion mutants of the PHB2 protein, a chimeric
protein having the C-terminus of the PHB2 protein, and the like
were prepared. Similarly, a deletion mutant of the PHB1 protein was
prepared.
[0122] FIG. 12A shows the prepared deletion mutants of the PHB2
protein, the chimeric protein, and the deletion mutant of the PHB1
protein. "PHB2N" is a deletion mutant consisting of the amino acid
residues 1 to 50 from the N-terminus of the PHB2 protein. "PHB2C"
is a deletion mutant consisting of the amino acid residues 51 to
299 from the N-terminus of the PHB2 protein. Furthermore, "PHB1C"
is a deletion mutant consisting of the amino acid residues 45 to
272 from the N-terminus of the PHB1 protein. Furthermore, a
chimeric protein "PHB1N-2C" is a mutant prepared by ligating the
amino acid residues 1 to 50 from the N-terminus of the PHB1 protein
(having a signal for localization within the inner membrane) to the
amino acid residues 51 to 299 from the N-terminus of the PHB2
protein. In FIG. 12A, "cc" denotes a coiled-coil domain.
[0123] The method employed herein for preparing a gene encoding
such a deletion mutant is as follows. Primers complementary to
arbitrary nucleotide portions were prepared, an arbitrary region
was amplified by PCR, and then resultant was incorporated into an
expression vector, so as to construct an expression vector
containing a gene encoding each mutant.
[0124] FIGS. 12B to D show the results.
[0125] FIG. 12B shows the results of cell immunostaining using HeLa
cells expressing a PHB2N-FLAG (the protein prepared by fusing a
FLAG tag to the C-terminus of the PHB2N protein). In FIG. 12B,
".alpha.-FLAG" denotes an image of staining with an anti-FLAG
antibody and ".alpha.-cyt.c" denotes an image of staining with an
anti-cytochrome C antibody. Furthermore, "merge" denotes a merged
image.
[0126] FIG. 12C shows intracellular localization of PHB1C-GFP (the
protein prepared by fusing a GFP protein to the C-terminus of the
PHB1C protein) or PHB2C-GFP (the protein prepared by fusing a GFP
protein to the C-terminus of the PHB2C protein) in HeLa cells.
"E2(-)" denotes HeLa cells cultured in the absence of E2 and
"+ER.alpha. E2(+)" denotes ER.alpha.-expressing HeLa cells cultured
in the presence of E2.
[0127] Furthermore, FIG. 12D shows the results of examining the
effects of full-length PHB2 (PHB2-FLAG), the PHB2N terminus
(PHB2N-FLAG), and the PHB1N-2C chimeric protein (PHB1N-2C-FLAG) on
mitochondrial membrane potential in HeLa cells. Each expressed
protein is a fusion protein having a FLAG tag on the C-terminus. In
addition, detection of mitochondrial membrane potential was
performed via staining with the use of TMRE for staining of
mitochondria depending on membrane potential. In FIG. 12D, "TI"
denotes transmission images.
[0128] As shown in FIG. 12B, the PHB2N-FLAG was expressed in
mitochondria. Hence, the presence of a mitochondrial targeting
signal in the amino terminus of the PHB2 protein was revealed. In
the PHB2 protein and the PHB2N protein in FIG. 12A, each box shown
with oblique lines denotes the mitochondrial targeting signal, and
a box adjacent thereto denotes a transmembrane domain.
[0129] Furthermore, as shown in FIG. 12C, PHB2C-GFP translocated to
the nucleus in the presence of E2. Hence, the presence of an
E2-dependent nuclear translocation signal in the carboxyl terminus
of the PHB2 protein was revealed. On the other hand, PHB1C-GFP did
not translocate to the nucleus, even in the presence of E2. Hence,
it was revealed that a nuclear translocation signal was absent on
the carboxyl terminus of the PHB1 protein and that the signal (or
nuclear translocation function) was specific to the PHB2
protein.
[0130] Furthermore, as shown in FIG. 12D, it was revealed that the
chimeric protein (PHB1N-2C-FLAG) of the C-terminus of PHB2
regulates mitochondrial membrane potential. When transfection was
performed with a gene encoding a chimeric protein prepared by
fusing the C-terminus of PHB2 to the N-terminus containing PHB1
mitochondrial targeting signal and a transmembrane domain, no
changes were observed in mitochondrial functions in the case of the
PHB2 N-terminus alone (PHB2N-FLAG). However, mitochondrial membrane
potential was revealed to decrease in a manner similar to that in
the case of PHB2 as a result of expression of the above chimeric
protein. In addition, decreased membrane potential was not observed
in the case of the C-terminus alone of PHB1 or the C-terminus alone
of PHB2.
[0131] Furthermore, it was confirmed by the following experiment
that the chimeric protein of the C-terminus of PHB2 causes
disappearance of mitochondrial membrane potential.
[0132] PHB2-GFP as a positive control (the protein prepared by
fusing a GFP protein to the C-terminus of the full-length PHB2
protein), MTS-GFP as a negative control (the protein prepared by
fusing a GFP protein to the C-terminus of the mitochondrial
targeting signal (MTS) of another protein (human cytochrome C
oxidase subunit 8A, 1-29a.a. (NP.sub.--004065)), and PHB1N-2C-GFP
(the protein prepared by fusing a GFP protein to the C-terminus of
the PHB1N-2C chimeric protein) were each expressed by HeLa cells.
The presence or the absence of mitochondrial membrane potential was
observed under a confocal laser scanning microscope after staining
with TMRE (tetramethylrhodamine ethyl ester).
[0133] FIG. 12E shows the results. In FIG. 12E, "GFP" denotes
fluorescence images resulting from the GFP protein, "TMRE" denotes
TMRE staining images, and "merge" denotes merged images.
[0134] As shown in FIG. 12E, membrane potential decreased
significantly in cells expressing the positive control and the
chimeric protein (PHB1N-2C-GFP). However, no decrease in membrane
potential was observed in mitochondria expressing the negative
control. Specifically, the PHB2 chimeric protein was shown to be an
example of an agent for regulating mitochondrial functions.
Example 12
Interaction between PHB2 Protein and Nuclear Receptor PPAR.alpha.
or PPAR.gamma.2
[0135] It is considered in Example 10 that PHB2 binds to ER.alpha.
in an E2-dependent manner so as to translocate to the nucleus.
[0136] In this example, interaction between the PHB2 protein and
PPAR.alpha. or PPAR.gamma.2, which is a nuclear receptor similar to
ER.alpha., was examined.
[0137] The method employed herein is as follows. Cells in which a
gene encoding PPAR.alpha. or PPAR.gamma.2 and a gene encoding
PHB2-GFP or PHB1-GFP incorporated into an expression vector had
been co-expressed were observed under a confocal laser scanning
microscope.
[0138] FIG. 13 shows the results. FIG. 13 shows micrographs of HeLa
cells expressing PHB1-GFP or PHB2-GFP and PPAR.alpha. or
PPAR.gamma.2. As shown in FIG. 13, it was confirmed in HeLa cells
that nuclear translocation of the PHB2 protein takes place in the
presence not only of ER.alpha., but also of a similar nuclear
receptor, PPAR.alpha. or PPAR.gamma.2, and that the PHB2 protein is
specifically localized in the nucleus.
Example 13
Examination of the Transcriptional Repression Activity of the PHB2
Protein
[0139] In this example, to examine the transcriptional repression
activity of the PHB2 protein, dual luciferase assay was performed
using a reporter gene (ERE-Luc) in which a luciferase gene had been
ligated downstream of an estrogen-responsive element.
[0140] The method employed herein is as follows. HeLa cells were
transfected with an ERE-Luc gene and the expression vectors, into
each of which an ER.alpha. gene, a PHB-2 gene, or a PGC-1.alpha.
gene had been inserted. Furthermore, transfection with Renilla
luciferase having a CMV promoter was simultaneously performed as a
control, and then luciferase activity was measured using the
luciferase as the internal control.
[0141] FIG. 14 shows the results. FIG. 14 shows the relative
luciferase activity in HeLa cells transfected with each gene.
"E2(-)" denotes cells cultured in the absence of E2 and "E2(+)"
denotes cells cultured in the presence of E2. In FIG. 14, "+" or
"-" denotes the presence or the absence of transfection with each
gene. In addition, in FIG. 14, each bar indicates 1SD (standard
deviation) and the average value of the values obtained by the
experiment that had been conducted independently 3 times.
[0142] As shown in FIG. 14, transfection of HeLa cells with the
ER.alpha. gene and the PGC-1.alpha. gene, which is a coactivator of
ER.alpha., resulted in enhanced transcriptional activity, and it
further enhanced transcriptional activity particularly in the
presence of E2. When the PHB2 protein was co-expressed in the HeLa
cells with the ER.alpha. gene and the PGC-1.alpha. gene, a
transcriptional-activity-repressing effect could be detected.
Therefore, it was revealed that the PHB2 protein functions for
transcriptional regulation of the PGC-1a protein, which is a
coactivator of ER.alpha., in a repressive manner.
INDUSTRIAL APPLICABILITY
[0143] According to the present invention, a PHB2 gene regulator, a
therapeutic drug for mitochondrial-function-related disease, and an
agent for regulating mitochondrial functions can be provided.
Moreover, according to the present invention, a
mitochondrial-function-related disease can be detected.
[0144] All publications, patents, and patent applications cited
herein are incorporated herein by reference in their entirety.
Sequence CWU 1
1
24119RNAArtificial SequenceDescription of Artificial Sequence siRNA
1gaacccuggc uacaucaaa 19219RNAArtificial SequenceDescription of
Artificial Sequence siRNA 2cccuggcuac aucaaacuu 19319RNAArtificial
SequenceDescription of Artificial Sequence siRNA 3guggaucugc
uucuccagu 19419RNAArtificial SequenceDescription of Artificial
Sequence siRNA 4ccccucuugg auuaaggaa 19519RNAArtificial
SequenceDescription of Artificial Sequence siRNA 5gauucgagca
gcccagaau 19619RNAArtificial SequenceDescription of Artificial
Sequence siRNA 6gacugaagac uagccccuu 19719RNAArtificial
SequenceDescription of Artificial Sequence siRNA 7gaaaugagcc
uagucacca 19819RNAArtificial SequenceDescription of Artificial
Sequence siRNA 8agaccuacag auggugaau 19919RNAArtificial
SequenceDescription of Artificial Sequence siRNA 9ccuacaggau
gaaaguuuc 191019RNAArtificial SequenceDescription of Artificial
Sequence siRNA 10ggguaagaaa ugagccuag 191119RNAArtificial
SequenceDescription of Artificial Sequence siRNA 11ucguaucuau
cucacagcu 191219RNAArtificial SequenceDescription of Artificial
Sequence siRNA 12augagccuag ucaccaaga 191319RNAArtificial
SequenceDescription of Artificial Sequence siRNA 13gaugcuugga
gaagcacug 191419RNAArtificial SequenceDescription of Artificial
Sequence siRNA 14ccuugugcug aaccuacag 191519RNAArtificial
SequenceDescription of Artificial Sequence siRNA 15cagcggcaga
aaauugugc 191619RNAArtificial SequenceDescription of Artificial
Sequence siRNA 16uucuugguag aaaaagcaa 191719RNAArtificial
SequenceDescription of Artificial Sequence siRNA 17acuucgcaag
auucgagca 191819RNAArtificial SequenceDescription of Artificial
Sequence siRNA 18guucaaugcc ucacagcug 191919RNAArtificial
SequenceDescription of Artificial Sequence siRNA 19ggaagacuga
agacuagcc 192019RNAArtificial SequenceDescription of Artificial
Sequence siRNA 20ucuguguuca ccguggaag 19211416DNAHomo
sapiensCDS(186)..(1085) 21aagttcgggt ccgtagtggg ctaaggggga
gggtttcaaa gggagcgcac ttccgctgcc 60ctttctttcg ccagccttac gggcccgaac
cctcgtgtga agggtgcagt acctaagccg 120gagcggggta gaggcgggcc
ggcaccccct tctgacctcc agtgccgccg gcctcaagat 180cagac atg gcc cag
aac ttg aag gac ttg gcg gga cgg ctg ccc gcc ggg 230 Met Ala Gln Asn
Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly 1 5 10 15ccc cgg ggc
atg ggc acg gcc ctg aag ctg ttg ctg ggg gcc ggc gcc 278Pro Arg Gly
Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly Ala 20 25 30gtg gcc
tac ggt gtg cgc gaa tct gtg ttc acc gtg gaa ggc ggg cac 326Val Ala
Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly Gly His 35 40 45aga
gcc atc ttc ttc aat cgg atc ggt gga gtg cag cag gac act atc 374Arg
Ala Ile Phe Phe Asn Arg Ile Gly Gly Val Gln Gln Asp Thr Ile 50 55
60ctg gcc gag ggc ctt cac ttc agg atc cct tgg ttc cag tac ccc att
422Leu Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr Pro Ile
65 70 75atc tat gac att cgg gcc aga cct cga aaa atc tcc tcc cct aca
ggc 470Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr
Gly80 85 90 95tcc aaa gac cta cag atg gtg aat atc tcc ctg cga gtg
ttg tct cga 518Ser Lys Asp Leu Gln Met Val Asn Ile Ser Leu Arg Val
Leu Ser Arg 100 105 110ccc aat gct cag gag ctt cct agc atg tac cag
cgc cta ggg ctg gac 566Pro Asn Ala Gln Glu Leu Pro Ser Met Tyr Gln
Arg Leu Gly Leu Asp 115 120 125tac gag gaa cga gtg ttg ccg tcc att
gtc aac gag gtg ctc aag agt 614Tyr Glu Glu Arg Val Leu Pro Ser Ile
Val Asn Glu Val Leu Lys Ser 130 135 140gtg gtg gcc aag ttc aat gcc
tca cag ctg atc acc cag cgg gcc cag 662Val Val Ala Lys Phe Asn Ala
Ser Gln Leu Ile Thr Gln Arg Ala Gln 145 150 155gta tcc ctg ttg atc
cgc cgg gag ctg aca gag agg gcc aag gac ttc 710Val Ser Leu Leu Ile
Arg Arg Glu Leu Thr Glu Arg Ala Lys Asp Phe160 165 170 175agc ctc
atc ctg gat gat gtg gcc atc aca gag ctg agc ttt agc cga 758Ser Leu
Ile Leu Asp Asp Val Ala Ile Thr Glu Leu Ser Phe Ser Arg 180 185
190gag tac aca gct gct gta gaa gcc aaa caa gtg gcc cag cag gag gcc
806Glu Tyr Thr Ala Ala Val Glu Ala Lys Gln Val Ala Gln Gln Glu Ala
195 200 205cag cgg gcc caa ttc ttg gta gaa aaa gca aag cag gaa cag
cgg cag 854Gln Arg Ala Gln Phe Leu Val Glu Lys Ala Lys Gln Glu Gln
Arg Gln 210 215 220aaa att gtg cag gcc gag ggt gag gcc gag gct gcc
aag atg ctt gga 902Lys Ile Val Gln Ala Glu Gly Glu Ala Glu Ala Ala
Lys Met Leu Gly 225 230 235gaa gca ctg agc aag aac cct ggc tac atc
aaa ctt cgc aag att cga 950Glu Ala Leu Ser Lys Asn Pro Gly Tyr Ile
Lys Leu Arg Lys Ile Arg240 245 250 255gca gcc cag aat atc tcc aag
acg atc gcc aca tca cag aat cgt atc 998Ala Ala Gln Asn Ile Ser Lys
Thr Ile Ala Thr Ser Gln Asn Arg Ile 260 265 270tat ctc aca gct gac
aac ctt gtg ctg aac cta cag gat gaa agt ttc 1046Tyr Leu Thr Ala Asp
Asn Leu Val Leu Asn Leu Gln Asp Glu Ser Phe 275 280 285acc agg gga
agt gac agc ctc atc aag ggt aag aaa tga gcctagtcac 1095Thr Arg Gly
Ser Asp Ser Leu Ile Lys Gly Lys Lys 290 295caagaactcc acccccagag
gaagtggatc tgcttctcca gtttttgagg agccagccag 1155gggtccagca
cagccctacc ccgccccagt atcatgcgat ggtcccccac accggttccc
1215tgaacccctc ttggattaag gaagactgaa gactagcccc ttttctggga
aattactttc 1275ctcctccctg tgttaactgg ggctgttggg gacagtgcgt
gatttctcag tgatttccta 1335cagtgttgtt ccctccctca aggctgggag
gagataaaca ccaacccagg aattctcaat 1395aaatttttat tacttaacct g
141622299PRTHomo sapiens 22Met Ala Gln Asn Leu Lys Asp Leu Ala Gly
Arg Leu Pro Ala Gly Pro1 5 10 15Arg Gly Met Gly Thr Ala Leu Lys Leu
Leu Leu Gly Ala Gly Ala Val 20 25 30Ala Tyr Gly Val Arg Glu Ser Val
Phe Thr Val Glu Gly Gly His Arg 35 40 45Ala Ile Phe Phe Asn Arg Ile
Gly Gly Val Gln Gln Asp Thr Ile Leu 50 55 60Ala Glu Gly Leu His Phe
Arg Ile Pro Trp Phe Gln Tyr Pro Ile Ile65 70 75 80Tyr Asp Ile Arg
Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly Ser 85 90 95Lys Asp Leu
Gln Met Val Asn Ile Ser Leu Arg Val Leu Ser Arg Pro 100 105 110Asn
Ala Gln Glu Leu Pro Ser Met Tyr Gln Arg Leu Gly Leu Asp Tyr 115 120
125Glu Glu Arg Val Leu Pro Ser Ile Val Asn Glu Val Leu Lys Ser Val
130 135 140Val Ala Lys Phe Asn Ala Ser Gln Leu Ile Thr Gln Arg Ala
Gln Val145 150 155 160Ser Leu Leu Ile Arg Arg Glu Leu Thr Glu Arg
Ala Lys Asp Phe Ser 165 170 175Leu Ile Leu Asp Asp Val Ala Ile Thr
Glu Leu Ser Phe Ser Arg Glu 180 185 190Tyr Thr Ala Ala Val Glu Ala
Lys Gln Val Ala Gln Gln Glu Ala Gln 195 200 205Arg Ala Gln Phe Leu
Val Glu Lys Ala Lys Gln Glu Gln Arg Gln Lys 210 215 220Ile Val Gln
Ala Glu Gly Glu Ala Glu Ala Ala Lys Met Leu Gly Glu225 230 235
240Ala Leu Ser Lys Asn Pro Gly Tyr Ile Lys Leu Arg Lys Ile Arg Ala
245 250 255Ala Gln Asn Ile Ser Lys Thr Ile Ala Thr Ser Gln Asn Arg
Ile Tyr 260 265 270Leu Thr Ala Asp Asn Leu Val Leu Asn Leu Gln Asp
Glu Ser Phe Thr 275 280 285Arg Gly Ser Asp Ser Leu Ile Lys Gly Lys
Lys 290 2952321RNAArtificial SequenceDescription of Artificial
Sequence siRNA 23aacacagccu uccuucugcu c 212419RNAArtificial
SequenceDescription of Artificial Sequence siRNA 24ccagagagga
caaugaucu 19
* * * * *