U.S. patent application number 11/839292 was filed with the patent office on 2008-03-27 for methods of modulating mitochondrial nad-dependent deacetylase.
Invention is credited to Brian J. North, Bjoern Schwer, Eric M. Verdin.
Application Number | 20080076835 11/839292 |
Document ID | / |
Family ID | 29584501 |
Filed Date | 2008-03-27 |
United States Patent
Application |
20080076835 |
Kind Code |
A1 |
Verdin; Eric M. ; et
al. |
March 27, 2008 |
METHODS OF MODULATING MITOCHONDRIAL NAD-DEPENDENT DEACETYLASE
Abstract
The present invention provides methods for identifying agents
that modulate a level or an activity of a mitochondrial
NAD-dependent deacetylase polypeptide, as well as agents identified
by the methods. The invention further provides methods of
modulating mitochondrial NAD-dependent deacetylase activity in a
cell. The invention further provides methods of modulating
mitochondrial function by modulating the activity of mitochondrial
NAD-dependent deacetylase.
Inventors: |
Verdin; Eric M.; (San
Francisco, CA) ; North; Brian J.; (San Francisco,
CA) ; Schwer; Bjoern; (San Francisco, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
29584501 |
Appl. No.: |
11/839292 |
Filed: |
August 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10444633 |
May 22, 2003 |
7273713 |
|
|
11839292 |
Aug 15, 2007 |
|
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60383069 |
May 23, 2002 |
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Current U.S.
Class: |
514/789 ;
435/7.1; 435/7.5; 435/7.9 |
Current CPC
Class: |
C12Q 1/48 20130101; G01N
2500/00 20130101; C12Q 1/34 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/789 ;
435/007.1; 435/007.5; 435/007.9 |
International
Class: |
G01N 33/53 20060101
G01N033/53; A61K 45/06 20060101 A61K045/06; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. An in vitro method of identifying an agent that modulates an
enzymatic activity of a human mitochondrial NAD-dependent
deacetylase, the method comprising: contacting a mitochondrial
NAD-dependent deacetylase polypeptide with a test agent in an assay
mixture that comprises NAD and an acetylated histone peptide; and
determining the effect, if any, of the test agent on the enzymatic
activity of mitochondrial NAD-dependent deacetylase.
2. The method of claim 1, wherein the human mitochondrial
NAD-dependent deacetylase polypeptide comprises an amino acid
sequence as set forth in SEQ ID NO:01.
3. The method of claim 1, wherein the acetylated histone peptide
comprises amino acids 1-22 of histone 4.
4. The method of claim 1, wherein the acetylated histone peptide
contains a .sup.14C-labeled acetyl group, and determining the
effect of the agent on the enzymatic activity of the deacetylase is
performed by measuring release of the radioactive acetyl group.
5. The method of claim 1, wherein determining the effect of the
agent on the enzymatic activity of the deacetylase is performed by
detecting binding of an antibody specific for acetylated
histone.
6. An in vitro method for identifying an agent that modulates a
level of mitochondrial NAD-dependent deacetylase in a cell, the
method comprising contacting a cell that produces mitochondrial
NAD-dependent deacetylase with a test agent; and determining the
effect, if any, of the test agent on the level of mitochondrial
NAD-dependent deacetylase.
7. The method of claim 6, wherein determining the effect of the
agent on the level of the deacetylase is performed by determining a
level of mitochondrial NAD-dependent deacetylase mRNA in the
cell.
8. The method of claim 6, wherein determining the effect of the
agent on the level of the deacetylase is performed by determining a
level of mitochondrial NAD-dependent deacetylase polypeptide in the
cell.
9. A biologically active agent identified by a screening method
according to claim 1 or claim 6.
10. A pharmaceutical composition comprising a biologically active
agent that reduces a level or an activity of a mitochondrial
NAD-dependent deacetylase protein; and a pharmaceutically
acceptable excipient.
11. A method of treating a disorder caused by mitochondrial
malfunction or dysfunction in an individual, the method comprising
administering to the individual an effective amount of an agent
that modulates a level or activity of a mitochondrial NAD-dependent
deacetylase protein.
12. The method of claim 11, wherein the level or activity of the
mitochondrial NAD-dependent deacetylase protein is increased.
13. The method of claim 11, wherein the level or activity of the
mitochondrial NAD-dependent deacetylase protein is decreased.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of deacetylase
enzymes.
BACKGROUND OF THE INVENTION
[0002] Silent Information Regulator 2 (Sir2) protein is involved in
transcriptional silencing and DNA damage repair in yeast. It also
increases life span in yeast and in Caenorhabditis elegans. Yeast
Sir2 protein has an NAD-dependent histone deacetylase activity that
links Sir2 functions to cellular metabolism. This NAD-dependent
deacetylase activity is conserved from bacteria to humans and
mammalian Sir2 homologues also have NAD-dependent histone
deacetylase activity. The NAD-dependency of Sir2-like enzymes
distinguishes them from the class I and II HDAC histone
deacetylases that use a zinc-catalyzed mechanism. Seven Sir2
homologues have been identified in humans and are designated
hSIRT1-7. Among the human homologues, hSIRT1, hSIRT2 and hSIRT3 the
most homologous to yeast Sir2 and have NAD-dependent deacetylase
activity. At present, very little is known about the in vivo
functions of mammalian SIR2 homologues. hSIRT1 deacetylates the
transcription factor p53 thereby inhibiting p53 activation and
apoptosis in response to DNA damage and oxidative stress.
LITERATURE
[0003] Yang et al. (2000) Genomics 69:355-369; Frye (2000) Biochem.
Biophys. Res. Comm. 273:793-798; GenBank Accession Nos.
NM.sub.--012239 and AF083109.
SUMMARY OF THE INVENTION
[0004] The present invention provides methods for identifying
agents that modulate a level or an activity of a mitochondrial
NAD-dependent deacetylase polypeptide, as well as agents identified
by the methods. The invention further provides methods of
modulating mitochondrial NAD-dependent deacetylase activity in a
cell. The invention further provides methods of modulating
mitochondrial function by modulating the activity of mitochondrial
NAD-dependent deacetylase.
FEATURES OF THE INVENTION
[0005] The present invention features an in vitro method of
identifying an agent that modulates an enzymatic activity of a
human mitochondrial NAD-dependent deacetylase. The method generally
involves contacting a mitochondrial NAD-dependent deacetylase
polypeptide with a test agent in an assay mixture that comprises
nicotinamide adenine dinucleotide (NAD) and an acetylated histone
peptide; and determining the effect, if any, of the test agent on
the enzymatic activity of mitochondrial NAD-dependent deacetylase.
In some embodiments, the human mitochondrial NAD-dependent
deacetylase polypeptide comprises an amino acid sequence as set
forth in SEQ ID NO:01. In some embodiments, the acetylated histone
peptide comprises amino acids 1-22 of histone 4. In some
embodiments, the acetylated histone peptide contains a
.sup.14C-labeled acetyl group, and determining the effect of the
agent on the enzymatic activity of the deacetylase is performed by
measuring release of the radioactive acetyl group. In other
embodiments, determining the effect of the agent on the enzymatic
activity of the deacetylase is performed by detecting binding of an
antibody specific for acetylated histone.
[0006] The present invention further features an in vitro method
for identifying an agent that modulates a level of mitochondrial
NAD-dependent deacetylase in a cell. The method generally involves
contacting a cell that produces mitochondrial NAD-dependent
deacetylase with a test agent; and determining the effect, if any,
of the test agent on the level of mitochondrial NAD-dependent
deacetylase. In some embodiments, determining the effect of the
agent on the level of the deacetylase is performed by determining a
level of mitochondrial NAD-dependent deacetylase mRNA in the cell.
In other embodiments, determining the effect of the agent on the
level of the deacetylase is performed by determining a level of
mitochondrial NAD-dependent deacetylase polypeptide in the
cell.
[0007] The present invention further features a biologically active
agent identified by a screening method according to the invention.
The invention further features a pharmaceutical composition
comprising a biologically active agent that reduces a level or an
activity of a mitochondrial NAD-dependent deacetylase protein; and
a pharmaceutically acceptable excipient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A-F depict deacetylase activity in mitochondria.
[0009] FIGS. 2A-C depict localization of deacetylase activity in
mitochondria.
[0010] FIG. 3 depicts depict requirement of the N-terminal region
of hSIRT3 for mitochondrial targeting.
[0011] FIGS. 4A-C depict mitochondrial import of hSIRT3.
[0012] FIGS. 5A and 5B depict intramitochondrial localization of
hSIRT3.
[0013] FIGS. 6A-C depict proteolytic processing of hSIRT3 by
MPP.
[0014] FIG. 7 depicts the amino acid sequence of human SIRT3 (SEQ
ID NO:01).
[0015] FIG. 8 depicts NAD-dependent HDAC activity of truncated
recombinant SIRT3.
DEFINITIONS
[0016] The terms "polypeptide" and "protein", used interchangeably
herein, refer to a polymeric form of amino acids of any length,
which can include coded and non-coded amino acids, chemically or
biochemically modified or derivatized amino acids, and polypeptides
having modified peptide backbones. The term includes fusion
proteins, including, but not limited to, fusion proteins with a
heterologous amino acid sequence, fusions with heterologous and
homologous leader sequences, with or without N-terminal methionine
residues; immunologically tagged proteins; and the like.
[0017] A "substantially isolated" or "isolated" polypeptide is one
that is substantially free of the macromolecules with which it is
associated in nature. By substantially free is meant at least 50%,
preferably at least 70%, more preferably at least 80%, and even
more preferably at least 90% free of the materials with which it is
associated in nature.
[0018] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as polynucleotides. The term "biological sample"
encompasses a clinical sample, and also includes cells in culture,
cell supernatants, cell lysates, serum, plasma, biological fluid,
and tissue samples.
[0019] The term "disorder associated with mitochondrial
malfunction," as used herein, refers to any disorder that is
directly or indirectly a result of, or caused by, malfunction of a
mitochondrion in a cell.
[0020] The terms "cancer", "neoplasm", "tumor", and "carcinoma",
are used interchangeably herein to refer to cells which exhibit
relatively autonomous growth, so that they exhibit an aberrant
growth phenotype characterized by a significant loss of control of
cell proliferation. Cancerous cells can be benign or malignant.
[0021] As used herein, the terms "treatment", "treating", and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment", as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease.
[0022] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0023] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0024] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0025] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a mitochondrial NAD-dependent deacetylase"
includes a plurality of such deacetylases and reference to "the
agent" includes reference to one or more agents and equivalents
thereof known to those skilled in the art, and so forth.
[0026] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The instant invention provides methods for identifying
agents that modulate an enzymatic activity of a mitochondrial
NAD-dependent deacetylase. The invention further provides agents
identified by the instant methods, as well as methods of modulating
the activity of the mitochondrial NAD-dependent deacetylase. Agents
that modulate the activity of an NAD-dependent mitochondrial
deacetylase are useful to ameliorate disorders associated with
mitochondrial malfunction.
[0028] The instant invention is based on the observation that human
SIRT3 (hSIRT3) is a nuclear-encoded NAD-dependent deacetylase
residing within the mitochondria. hSIRT3 is proteolytically cleaved
by a mitochondrial enzyme to a catalytically active form of having
a molecular weight of about 28 kDa. The identification of the
activity of hSIRT3 allowed development of assays to identify agents
that modulate the activity of this enzyme. Such agents are useful
in treating disorders arising from mitochondrial malfunction.
Screening Methods
[0029] The invention provides in vitro methods of identifying an
agent that modulates a level or an activity of a mitochondrial
NAD-dependent deacetylase. The methods generally involve contacting
a mitochondrial NAD-dependent deacetylase protein, or a cell that
produces a mitochondrial NAD-dependent deacetylase protein, with a
test agent, and determining the effect, if any, on a level or an
activity of the mitochondrial NAD-dependent deacetylase
protein.
[0030] In some embodiments, the methods are cell-free methods.
Cell-free methods generally involve contacting a mitochondrial
NAD-dependent deacetylase with a test agent and determining the
effect, if any, of the test agent on the enzymatic activity of the
mitochondrial NAD-dependent deacetylase.
[0031] In other embodiments, the methods are cell-based methods.
Cell-based methods generally involve contacting a cell that
produces mitochondrial NAD-dependent deacetylase with a test agent
and determining the effect, if any, of the test agent on the level
of mitochondrial NAD-dependent deacetylase mRNA or mitochondrial
NAD-dependent deacetylase protein in the cell.
[0032] As used herein, the term "determining" refers to both
quantitative and qualitative determinations and as such, the term
"determining" is used interchangeably herein with "assaying,"
"measuring," and the like.
[0033] The term "mitochondrial NAD-dependent deacetylase
polypeptide" encompasses human mitochondrial NAD-dependent
deacetylase proteins (e.g., human SIRT3 proteins) having the amino
acid sequences set forth in any of GenBank Accession Nos.
NM.sub.--012239; and AF0831087, where the polypeptide is a
nuclear-encoded, mitochondrial protein and exhibits NAD-dependent
mitochondrial NAD-dependent deacetylase activity. The term
comprises a mitochondrial NAD-dependent deacetylase polypeptide
comprises the amino acid sequence as set forth in SEQ ID NO:01 and
depicted in FIG. 7; catalytically active fragments thereof; and
catalytically active variants thereof. Catalytically active
fragments include fragments lacking from about 1 to about 120
N-terminal amino acids of the sequence set forth in SEQ ID NO:01.
For example, catalytically active fragments lacking from about 1 to
about 10, from about 10 to about 20, from about 20 to about 30,
from about 30 to about 40, from about 40 to about 50, from about 50
to about 60, from about 60 to about 70, from about 70 to about 80,
from about 80 to about 90, from about 90 to about 100, or from
about 100 to about 120 N-terminal amino acids of the sequence set
forth in SEQ ID NO:01 can be used in a subject method. The term
encompasses an enzyme that is proteolytically processed in the
mitochondria by a mitochondrial enzyme referred to as MPP, which
cleaves the 44 kDa form of the enzyme to a catalytically active 28
kDa form. In many embodiments, the 28 kDa form is used in the
instant methods. The term encompasses variants that have
insertions, deletions, and/or conservative amino acid substitutions
that do not affect the ability of the protein to deacetylate an
appropriate substrate (e.g., acetylated histone 4, or an acetylated
fragment thereof) in an NAD-dependent fashion. In some embodiments,
the mitochondrial NAD-dependent deacetylase is recombinant, e.g.,
produced in a cell transfected with an expression construct
comprising a nucleotide sequence that encodes the mitochondrial
NAD-dependent deacetylase.
[0034] The term "mitochondrial NAD-dependent deacetylase
polypeptide" further encompasses fusion proteins comprising a
mitochondrial NAD-dependent deacetylase and a heterologous
polypeptide ("fusion partners"), where suitable fusion partners
include immunological tags such as epitope tags, including, but not
limited to, hemagglutinin, FLAG, and the like; proteins that
provide for a detectable signal, including, but not limited to,
fluorescent proteins (e.g., a green fluorescent protein, a
fluorescent protein from an Anthozoan species, and the like),
enzymes (e.g., .beta.-galactosidase, luciferase, horse radish
peroxidase, etc.), and the like; polypeptides that facilitate
purification or isolation of the fusion protein, e.g., metal ion
binding polypeptides such as 6His tags (e.g., mitochondrial
NAD-dependent deacetylase/6His), GST, and the like. The term
"mitochondrial NAD-dependent deacetylase polypeptide" further
includes a mitochondrial NAD-dependent deacetylase polypeptide
modified to include one or more specific protease cleavage
sites.
[0035] Where the assay is an in vitro cell-free assay, the methods
generally involve contacting a mitochondrial NAD-dependent
deacetylase polypeptide with a test agent. The mitochondrial
NAD-dependent deacetylase polypeptide may be, but need not be,
purified. For example, the mitochondrial NAD-dependent deacetylase
polypeptide can be in a cell lysate, or may be isolated, or
partially purified. Thus, the assay can be conducted in the
presence of additional components, as long as the additional
components do not adversely affect the reaction to an unacceptable
degree.
[0036] Where the assay is an in vitro cell-based assay, any of a
variety of cells can be used. The cells used in the assay are
usually eukaryotic cells, including, but not limited to, rodent
cells, human cells, and yeast cells. The cells may be primary cell
cultures or may be immortalized cell lines. The cells may be
"recombinant," e.g., the cell may have transiently or stably
introduced therein a construct (e.g., a plasmid, a recombinant
viral vector, or any other suitable vector) that comprises a
nucleotide sequence encoding a mitochondrial NAD-dependent
deacetylase polypeptide, or that comprises a nucleotide sequence
that comprises a mitochondrial NAD-dependent deacetylase promoter
operably linked to a reporter gene.
[0037] The terms "candidate agent," "test agent," "agent",
"substance" and "compound" are used interchangeably herein.
Candidate agents encompass numerous chemical classes, typically
synthetic, semi-synthetic, or naturally occurring inorganic or
organic molecules. Candidate agents include those found in large
libraries of synthetic or natural compounds. For example, synthetic
compound libraries are commercially available from Maybridge
Chemical Co. (Trevillet, Cornwall, UK), ComGenex (South San
Francisco, Calif.), and MicroSource (New Milford, Conn.). A rare
chemical library is available from Aldrich (Milwaukee, Wis.) and
can also be used. Alternatively, libraries of natural compounds in
the form of bacterial, fungal, plant and animal extracts are
available from Pan Labs (Bothell, Wash.) or are readily
producible.
[0038] Candidate agents may be small organic or inorganic compounds
having a molecular weight of more than 50 and less than about 2,500
daltons. Candidate agents may comprise functional groups necessary
for structural interaction with proteins, particularly hydrogen
bonding, and may include at least an amine, carbonyl, hydroxyl or
carboxyl group, and may contain at least two of the functional
chemical groups. The candidate agents may comprise cyclical carbon
or heterocyclic structures and/or aromatic or polyaromatic
structures substituted with one or more of the above functional
groups. Candidate agents are also found among biomolecules
including peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof.
[0039] Assays of the invention include controls, where suitable
controls include a sample (e.g., a sample comprising mitochondrial
NAD-dependent deacetylase protein, or a cell that synthesizes
mitochondrial NAD-dependent deacetylase) in the absence of the test
agent. Generally a plurality of assay mixtures is run in parallel
with different agent concentrations to obtain a differential
response to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
[0040] Where the screening assay is a binding assay, one or more of
the molecules may be joined to a label, where the label can
directly or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescers, chemiluminescers, enzymes,
specific binding molecules, particles, e.g. magnetic particles, and
the like. Specific binding molecules include pairs, such as biotin
and streptavidin, digoxin and antidigoxin etc. For the specific
binding members, the complementary member would normally be labeled
with a molecule that provides for detection, in accordance with
known procedures.
[0041] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc. may be used. The components of the assay mixture are
added in any order that provides for the requisite binding or other
activity. Incubations are performed at any suitable temperature,
typically between 4.degree. C. and 40.degree. C. Incubation periods
are selected for optimum activity, but may also be optimized to
facilitate rapid high-throughput screening. Typically between 0.1
and 1 hour will be sufficient.
[0042] The screening methods may be designed a number of different
ways, where a variety of assay configurations and protocols may be
employed, as are known in the art. For example, one of the
components may be bound to a solid support, and the remaining
components contacted with the support bound component. The above
components of the method may be combined at substantially the same
time or at different times.
[0043] Where the assay is a binding assay, following the contact
and incubation steps, the subject methods will generally, though
not necessarily, further include a washing step to remove unbound
components, where such a washing step is generally employed when
required to remove label that would give rise to a background
signal during detection, such as radioactive or fluorescently
labeled non-specifically bound components. Following the optional
washing step, the presence of bound complexes will then be
detected.
[0044] A test agent of interest is one that reduces a level of
mitochondrial NAD-dependent deacetylase protein or inhibits a
mitochondrial NAD-dependent deacetylase activity by at least about
10%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 80%, at least about 90%, or
more, when compared to a control in the absence of the test
agent.
Methods of Detecting Agents that Modulate a Level of Mitochondrial
NAD-Dependent Deacetylase mRNA and/or Mitochondrial NAD-Dependent
Deacetylase Polypeptide
[0045] The subject screening methods include methods of detecting
an agent that modulates a level of a mitochondrial NAD-dependent
deacetylase mRNA and/or mitochondrial NAD-dependent deacetylase
polypeptide in a cell. In some embodiments, the methods involve
contacting a cell that produces mitochondrial NAD-dependent
deacetylase with a test agent, and determining the effect, if any,
of the test agent on the level of mitochondrial NAD-dependent
deacetylase mRNA in the cell.
[0046] A candidate agent is assessed for any cytotoxic activity it
may exhibit toward the cell used in the assay, using well-known
assays, such as trypan blue dye exclusion, an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)
assay, and the like. Agents that do not exhibit cytotoxic activity
are considered candidate agents.
[0047] A wide variety of cell-based assays may be used for
identifying agents which reduce a level of mitochondrial
NAD-dependent deacetylase mRNA in a eukaryotic cell, using, for
example, a cell that normally produces mitochondrial NAD-dependent
deacetylase mRNA, a mammalian cell transformed with a construct
comprising a mitochondrial NAD-dependent deacetylase-encoding cDNA
such that the cDNA is overexpressed, or, alternatively, a construct
comprising a mitochondrial NAD-dependent deacetylase promoter
operably linked to a reporter gene.
[0048] Accordingly, the present invention provides a method for
identifying an agent, particularly a biologically active agent,
that reduces a level of mitochondrial NAD-dependent deacetylase
expression in a cell, the method comprising: combining a candidate
agent to be tested with a cell comprising a nucleic acid which
encodes a mitochondrial NAD-dependent deacetylase polypeptide, or a
construct comprising a mitochondrial NAD-dependent deacetylase
promoter operably linked to a reporter gene; and determining the
effect of said agent on mitochondrial NAD-dependent deacetylase
expression. A decrease of at least about 10%, at least about 20%,
at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 80%, at least about 90%, or more, in the level
(i.e., an amount) of mitochondrial NAD-dependent deacetylase mRNA
and/or polypeptide following contacting the cell with a candidate
agent being tested, compared to a control to which no agent is
added, is an indication that the agent modulates mitochondrial
NAD-dependent deacetylase expression.
[0049] Mitochondrial NAD-dependent deacetylase mRNA and/or
polypeptide whose levels are being measured can be encoded by an
endogenous mitochondrial NAD-dependent deacetylase polynucleotide,
or the mitochondrial NAD-dependent deacetylase polynucleotide can
be one that is comprised within a recombinant vector and introduced
into the cell, i.e., the mitochondrial NAD-dependent deacetylase
mRNA and/or polypeptide can be encoded by an exogenous
mitochondrial NAD-dependent deacetylase polynucleotide. For
example, a recombinant vector may comprise an isolated
mitochondrial NAD-dependent deacetylase transcriptional regulatory
sequence, such as a promoter sequence, operably linked to a
reporter gene (e.g., .beta.-galactosidase, chloramphenicol acetyl
transferase, a fluorescent protein, luciferase, or other gene that
can be easily assayed for expression).
[0050] In these embodiments, the method for identifying an agent
that modulates a level of mitochondrial NAD-dependent deacetylase
expression in a cell, comprises: combining a candidate agent to be
tested with a cell comprising a nucleic acid which comprises a
mitochondrial NAD-dependent deacetylase gene transcriptional
regulatory element operably linked to a reporter gene; and
determining the effect of said agent on reporter gene expression. A
recombinant vector may comprise an isolated mitochondrial
NAD-dependent deacetylase transcriptional regulatory sequence, such
as a promoter sequence, operably linked to sequences coding for a
mitochondrial NAD-dependent deacetylase polypeptide; or the
transcriptional control sequences can be operably linked to coding
sequences for a mitochondrial NAD-dependent deacetylase fusion
protein comprising mitochondrial NAD-dependent deacetylase
polypeptide fused to a polypeptide which facilitates detection. In
these embodiments, the method comprises combining a candidate agent
to be tested with a cell comprising a nucleic acid which comprises
a mitochondrial NAD-dependent deacetylase gene transcriptional
regulatory element operably linked to a mitochondrial NAD-dependent
deacetylase polypeptide-coding sequence; and determining the effect
of said agent on mitochondrial NAD-dependent deacetylase
expression, which determination can be carried out by measuring an
amount of mitochondrial NAD-dependent deacetylase mRNA,
mitochondrial NAD-dependent deacetylase polypeptide, or
mitochondrial NAD-dependent deacetylase fusion polypeptide produced
by the cell.
[0051] Cell-based assays generally comprise the steps of contacting
the cell with an agent to be tested, forming a test sample, and,
after a suitable time, assessing the effect of the agent on
mitochondrial NAD-dependent deacetylase expression. A control
sample comprises the same cell without the candidate agent added.
Mitochondrial NAD-dependent deacetylase expression levels are
measured in both the test sample and the control sample. A
comparison is made between mitochondrial NAD-dependent deacetylase
expression level in the test sample and the control sample.
Mitochondrial NAD-dependent deacetylase expression can be assessed
using conventional assays. For example, when a mammalian cell line
is transformed with a construct that results in expression of
mitochondrial NAD-dependent deacetylase, mitochondrial
NAD-dependent deacetylase mRNA levels can be detected and measured,
or mitochondrial NAD-dependent deacetylase polypeptide levels can
be detected and measured. A suitable period of time for contacting
the agent with the cell can be determined empirically, and is
generally a time sufficient to allow entry of the agent into the
cell and to allow the agent to have a measurable effect on
mitochondrial NAD-dependent deacetylase mRNA and/or polypeptide
levels. Generally, a suitable time is between 10 minutes and 24
hours, or from about 1 hour to about 8 hours.
[0052] Methods of measuring mitochondrial NAD-dependent deacetylase
mRNA levels are known in the art, several of which have been
described above, and any of these methods can be used in the
methods of the present invention to identify an agent which
modulates mitochondrial NAD-dependent deacetylase mRNA level in a
cell, including, but not limited to, a PCR, such as a PCR employing
detectably labeled oligonucleotide primers, and any of a variety of
hybridization assays.
[0053] Similarly, mitochondrial NAD-dependent deacetylase
polypeptide levels can be measured using any standard method,
several of which have been described herein, including, but not
limited to, an immunoassay such as enzyme-linked immunosorbent
assay (ELISA), for example an ELISA employing a detectably labeled
antibody specific for a mitochondrial NAD-dependent deacetylase
polypeptide.
[0054] Mitochondrial NAD-dependent deacetylase polypeptide levels
can also be measured in cells harboring a recombinant construct
comprising a nucleotide sequence that encodes a mitochondrial
NAD-dependent deacetylase fusion protein, where the fusion partner
provides for a detectable signal or can otherwise be detected. For
example, where the fusion partner provides an immunologically
recognizable epitope (an "epitope tag"), an antibody specific for
an epitope of the fusion partner can be used to detect and
quantitate the level of mitochondrial NAD-dependent deacetylase. In
some embodiments, the fusion partner provides for a detectable
signal, and in these embodiments, the detection method is chosen
based on the type of signal generated by the fusion partner. For
example, where the fusion partner is a fluorescent protein,
fluorescence is measured.
[0055] Fluorescent proteins suitable for use include, but are not
limited to, a green fluorescent protein (GFP), including, but not
limited to, a "humanized" version of a GFP, e.g., wherein codons of
the naturally-occurring nucleotide sequence are changed to more
closely match human codon bias; a GFP derived from Aequoria
victoria or a derivative thereof, e.g., a "humanized" derivative
such as Enhanced GFP, which are available commercially, e.g., from
Clontech, Inc.; a GFP from another species such as Renilla
reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described
in, e.g., WO 99/49019 and Peelle et al. (2001) J. Protein Chem.
20:507-519; "humanized" recombinant GFP (hrGFP) (Stratagene); any
of a variety of fluorescent and colored proteins from Anthozoan
species, as described in, e.g., Matz et al. (1999) Nature
Biotechnol. 17:969-973; and the like. Where the fusion partner is
an enzyme that yields a detectable product, the product can be
detected using an appropriate means, e.g., .beta.-galactosidase
can, depending on the substrate, yield colored product, which is
detected spectrophotometrically, or a fluorescent product;
luciferase can yield a luminescent product detectable with a
luminometer; etc.
[0056] Agents that reduce a level of mitochondrial NAD-dependent
deacetylase protein include agents that reduce a level of
enzymatically active mitochondrial NAD-dependent deacetylase. In
some embodiments, an agent that reduces a level of enzymatically
active mitochondrial NAD-dependent deacetylase is an agent that
inhibits activity of a mitochondrial processing peptidase (MPP).
Whether MPP activity is inhibited can be determined using any known
assay, e.g., detecting formation of the 28 kD active form of
mitochondrial NAD-dependent deacetylase.
[0057] A number of methods are available for analyzing nucleic
acids for the presence and/or level of a specific mRNA in a cell.
The mRNA may be assayed directly or reverse transcribed into cDNA
for analysis. The nucleic acid may be amplified by conventional
techniques, such as the polymerase chain reaction (PCR), to provide
sufficient amounts for analysis. The use of the polymerase chain
reaction is described in Saiki, et al. (1985), Science 239:487, and
a review of techniques may be found in Sambrook, et al. Molecular
Cloning: A Laboratory Manual, CSH Press 1989, pp. 14.2-14.33.
Alternatively, various methods are known in the art that utilize
oligonucleotide ligation as a means of detecting polymorphisms, for
examples see Riley et al. (1990), Nucl. Acids Res. 18:2887-2890;
and Delahunty et al. (1996), Am. J. Hum. Genet. 58:1239-1246.
[0058] A detectable label may be included in an amplification
reaction. Suitable labels include fluorochromes, e.g. fluorescein
isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,
allophycocyanin, 6-carboxyfluorescein (6-FAM),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE),
6-carboxy-X-rhodamine (ROX),
6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX),
5-carboxyfluorescein (5-FAM) or
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive
labels, e.g. .sup.32P, .sup.35S, .sup.3H; etc. The label may be a
two stage system, where the amplified DNA is conjugated to biotin,
haptens, etc. having a high affinity binding partner, e.g. avidin,
specific antibodies, etc., where the binding partner is conjugated
to a detectable label. The label may be conjugated to one or both
of the primers. Alternatively, the pool of nucleotides used in the
amplification is labeled, so as to incorporate the label into the
amplification product.
[0059] A variety of different methods for determining the nucleic
acid abundance in a sample are known to those of skill in the art,
where particular methods of interest include those described in:
Pietu et al., Genome Res. (June 1996) .delta.: 492-503; Zhao et
al., Gene (Apr. 24, 1995) 156: 207-213; Soares, Curr. Opin.
Biotechnol. (October 1997) 8: 542-546; Raval, J. Pharmacol Toxicol
Methods (November 1994) 32: 125-127; Chalifour et al., Anal.
Biochem (Feb. 1, 1994) 216: 299-304; Stolz & Tuan, Mol.
Biotechnol. (December 19960 6: 225-230; Hong et al., Bioscience
Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143: 298.
Also of interest are the methods disclosed in WO 97/27317, the
disclosure of which is herein incorporated by reference.
[0060] A number of methods are available for determining the
expression level of a gene or protein in a particular sample. For
example, detection may utilize staining of cells or histological
sections with labeled antibodies, performed in accordance with
conventional methods. Cells are permeabilized to stain cytoplasmic
molecules. The antibodies of interest are added to the cell sample,
and incubated for a period of time sufficient to allow binding to
the epitope, usually at least about 10 minutes. The antibody may be
labeled with radioisotopes, enzymes, fluorescers, chemiluminescers,
or other labels for direct detection. Alternatively, a second stage
antibody or reagent is used to amplify the signal. Such reagents
are well known in the art. For example, the primary antibody may be
conjugated to biotin, with horseradish peroxidase-conjugated avidin
added as a second stage reagent. Final detection uses a substrate
that undergoes a color change in the presence of the peroxidase.
Alternatively, the secondary antibody conjugated to a fluorescent
compound, e.g. fluorescein, rhodamine, Texas red, etc. The absence
or presence of antibody binding may be determined by various
methods, including flow cytometry of dissociated cells, microscopy,
radiography, scintillation counting, etc.
Methods of Detecting Agents that Modulate an Activity of a
Mitochondrial NAD-Dependent Deacetylase Polypeptide
[0061] Methods of detecting an agent that modulates an activity of
a mitochondrial NAD-dependent deacetylase polypeptide include
cell-free and cell-based methods. The methods generally involve
contacting a mitochondrial NAD-dependent deacetylase polypeptide
with a test agent and determining the effect, if any, on the
mitochondrial NAD-dependent deacetylase enzyme activity.
[0062] The deacetylase activity of a mitochondrial NAD-dependent
deacetylase can be determined by incubating the enzyme in the
presence of NAD and an acetylated substrate. Suitable acetylated
substrates include acetylated histone 4, or a fragment thereof,
e.g., amino acids 1-22 of histone 4. The amino acid sequence of
amino acids 1-22 of histone 4 is:
NH.sub.2-MSGRGKGGKGLGKGGAKRHRKV-COOH (SEQ ID NO:02). Additional
exemplary suitable substrates include the following:
NH.sub.2-MSGRGKGGKGLGKGGAKRHRKVLRDNIQGI-COOH (from histone-4; SEQ
ID NO:03); and NH.sub.2-MARTKQTARKSTGGKAPRKQLATKAARKSA-COOH (from
histone-3; SEQ ID NO:04). In the foregoing peptides, the acetylated
lysine residues are in italics.
[0063] The acetylated histone peptide is present in the assay
mixture at a concentration of from about 20 .mu.M to about 1 mM,
from about 30 .mu.M to about 900 .mu.M, from about 40 .mu.M to
about 700 .mu.M, from about 50 .mu.M to about 500 .mu.M, from about
50 .mu.M to about 300 .mu.M, or from about 60 .mu.M to about 100
.mu.M. NAD is present in the assay mixture at a concentration of
about 1 mM. The acetyl group on the histone peptide is
radiolabeled, e.g., [.sup.14C]-acetyl is used. The assay then
involves determining the amount of [.sup.14C]-acetyl that is
released, typically by scintillation counting. Other components,
such as salts, reducing agents, and buffers, may be included.
[0064] In one exemplary embodiment, the enzymatic reaction mixture
comprises 4 mM MgCl.sub.2, 0.2 mM DTT, 50 mM Tris-HCl, pH 9.0,
amino acids 1-22 of histone 4, which peptide is aceylated with a
radiolabel acetyl group, and 1 mM NAD.
[0065] Another method of detecting mitochondrial NAD-dependent
deacetylase activity is to monitor the acetylation status of a
histone substrate using an antibody specific for acetylated histone
substrate. Lack of reactivity of the anti-acetylated histone
antibody with the histone substrate indicates that the histone has
been deacetylated. Thus, in some embodiments, the methods involve
determining binding of an anti-acetylated histone antibody with the
histone substrate. Anti-acetylated antibody/histone binding can be
determined using any type of immunological assay, including
immunoblotting assays, ELISA assays, and the like.
[0066] In some embodiments, the assay is a cell-free assay, wherein
the mitochondrial NAD-dependent deacetylase is contacted with the
test agent, the substrate (i.e., acetylated histone 4 peptide), and
other reaction components (e.g., NAD, buffers, and the like), and
the activity of the mitochondrial NAD-dependent deacetylase
determined. In these embodiments, the mitochondrial NAD-dependent
deacetylase may be purified, but need not be. The mitochondrial
NAD-dependent deacetylase may be present in a cell extract; in an
immunoprecipitate of a cell extract; or may be partially purified,
e.g., at least about 50%, at least about 60%, at least about 70%,
at least about 80%, at least about 90%, or more, purified, e.g.,
free of other macromolecules present in the source of the
mitochondrial NAD-dependent deacetylase. The mitochondrial
NAD-dependent deacetylase may be recombinant, or may be isolated
from a natural source, e.g., a mammalian cell or tissue that
normally produced the enzyme.
Agents
[0067] The present invention further provides biologically active
agents identified using a method of the instant invention. A
biologically active agent of the invention modulates a level or an
activity of a mitochondrial NAD-dependent deacetylase. Agents are
useful to treat various disorders, including cancer,
neurodegenerative disorders, metabolic disorders, and disorders
associated with apoptosis.
[0068] In many embodiments, the agent is a small molecule, e.g., a
small organic or inorganic compound having a molecular weight of
more than 50 and less than about 2,500 daltons. Agents may comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and may include at least
an amine, carbonyl, hydroxyl or carboxyl group, and may contain at
least two of the functional chemical groups. The agents may
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Agents are also found among biomolecules
including peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof.
[0069] In some embodiments, an active agent is a peptide. Suitable
peptides include peptides of from about 3 amino acids to about 50,
from about 5 to about 30, or from about 10 to about 25 amino acids
in length. A peptide of interest inhibits an enzymatic activity of
mitochondrial NAD-dependent deacetylase.
[0070] Peptides can include naturally-occurring and non-naturally
occurring amino acids. Peptides may comprise D-amino acids, a
combination of D- and L-amino acids, and various "designer" amino
acids (e.g., .beta.-methyl amino acids, C.alpha.-methyl amino
acids, and N.alpha.-methyl amino acids, etc.) to convey special
properties to peptides. Additionally, peptide may be a cyclic
peptide. Peptides may include non-classical amino acids in order to
introduce particular conformational motifs. Any known non-classical
amino acid can be used. Non-classical amino acids include, but are
not limited to, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate;
(2S,3S)-methylphenylalanine, (2S,3R)-methyl-phenylalanine,
(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine;
2-aminotetrahydronaphthalene-2-carboxylic acid;
hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate;
.beta.-carboline (D and L); HIC (histidine isoquinoline carboxylic
acid); and HIC (histidine cyclic urea). Amino acid analogs and
peptidomimetics may be incorporated into a peptide to induce or
favor specific secondary structures, including, but not limited to,
LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a .beta.-turn
inducing dipeptide analog; .beta.-sheet inducing analogs;
.beta.-turn inducing analogs; .alpha.-helix inducing analogs;
.gamma.-turn inducing analogs; Gly-Ala turn analog; amide bond
isostere; tretrazol; and the like.
[0071] A peptide may be a depsipeptide, which may be a linear or a
cyclic depsipeptide. Kuisle et al. (1999) Tet. Letters
40:1203-1206. "Depsipeptides" are compounds containing a sequence
of at least two alpha-amino acids and at least one alpha-hydroxy
carboxylic acid, which are bound through at least one normal
peptide link and ester links, derived from the hydroxy carboxylic
acids, where "linear depsipeptides" may comprise rings formed
through S--S bridges, or through an hydroxy or a mercapto group of
an hydroxy-, or mercapto-amino acid and the carboxyl group of
another amino- or hydroxy-acid but do not comprise rings formed
only through peptide or ester links derived from hydroxy carboxylic
acids. "Cyclic depsipeptides" are peptides containing at least one
ring formed only through peptide or ester links, derived from
hydroxy carboxylic acids.
[0072] Peptides may be cyclic or bicyclic. For example, the
C-terminal carboxyl group or a C-terminal ester can be induced to
cyclize by internal displacement of the --OH or the ester (--OR) of
the carboxyl group or ester respectively with the N-terminal amino
group to form a cyclic peptide. For example, after synthesis and
cleavage to give the peptide acid, the free acid is converted to an
activated ester by an appropriate carboxyl group activator such as
dicyclohexylcarbodiimide (DCC) in solution, for example, in
methylene chloride (CH.sub.2Cl.sub.2), dimethyl formamide (DMF)
mixtures. The cyclic peptide is then formed by internal
displacement of the activated ester with the N-terminal amine.
Internal cyclization as opposed to polymerization can be enhanced
by use of very dilute solutions. Methods for making cyclic peptides
are well known in the art
[0073] The term "bicyclic" refers to a peptide in which there
exists two ring closures. The ring closures are formed by covalent
linkages between amino acids in the peptide. A covalent linkage
between two nonadjacent amino acids constitutes a ring closure, as
does a second covalent linkage between a pair of adjacent amino
acids which are already linked by a covalent peptide linkage. The
covalent linkages forming the ring closures may be amide linkages,
i.e., the linkage formed between a free amino on one amino acid and
a free carboxyl of a second amino acid, or linkages formed between
the side chains or "R" groups of amino acids in the peptides. Thus,
bicyclic peptides may be "true" bicyclic peptides, i.e., peptides
cyclized by the formation of a peptide bond between the N-terminus
and the C-terminus of the peptide, or they may be "depsi-bicyclic"
peptides, i.e., peptides in which the terminal amino acids are
covalently linked through their side chain moieties.
[0074] A desamino or descarboxy residue can be incorporated at the
terminii of the peptide, so that there is no terminal amino or
carboxyl group, to decrease susceptibility to proteases or to
restrict the conformation of the peptide. C-terminal functional
groups include amide, amide lower alkyl, amide di(lower alkyl),
lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives
thereof, and the pharmaceutically acceptable salts thereof.
[0075] In addition to the foregoing N-terminal and C-terminal
modifications, a peptide or peptidomimetic can be modified with or
covalently coupled to one or more of a variety of hydrophilic
polymers to increase solubility and circulation half-life of the
peptide. Suitable nonproteinaceous hydrophilic polymers for
coupling to a peptide include, but are not limited to,
polyalkylethers as exemplified by polyethylene glycol and
polypropylene glycol, polylactic acid, polyglycolic acid,
polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose
and cellulose derivatives, dextran and dextran derivatives, etc.
Generally, such hydrophilic polymers have an average molecular
weight ranging from about 500 to about 100,000 daltons, from about
2,000 to about 40,000 daltons, or from about 5,000 to about 20,000
daltons. The peptide can be derivatized with or coupled to such
polymers using any of the methods set forth in Zallipsky, S.,
Bioconjugate Chem., 6:150-165 (1995); Monfardini, C, et al.,
Bioconjugate Chem., 6:62-69 (1995); U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337 or WO
95/34326.
[0076] Another suitable agent for reducing an activity of a
mitochondrial NAD-dependent deacetylase is a peptide aptamer.
Peptide aptamers are peptides or small polypeptides that act as
dominant inhibitors of protein function. Peptide aptamers
specifically bind to target proteins, blocking their function
ability. Kolonin and Finley, PNAS (1998) 95:14266-14271. Due to the
highly selective nature of peptide aptamers, they may be used not
only to target a specific protein, but also to target specific
functions of a given protein (e.g. a signaling function). Further,
peptide aptamers may be expressed in a controlled fashion by use of
promoters which regulate expression in a temporal, spatial or
inducible manner. Peptide aptamers act dominantly; therefore, they
can be used to analyze proteins for which loss-of-function mutants
are not available.
[0077] Peptide aptamers that bind with high affinity and
specificity to a target protein may be isolated by a variety of
techniques known in the art. Peptide aptamers can be isolated from
random peptide libraries by yeast two-hybrid screens (Xu et al.,
PNAS (1997) 94:12473-12478). They can also be isolated from phage
libraries (Hoogenboom et al., Immunotechnology (1998) 4:1-20) or
chemically generated peptides/libraries.
[0078] Intracellularly expressed antibodies, or intrabodies, are
single-chain antibody molecules designed to specifically bind and
inactivate target molecules inside cells. Intrabodies have been
used in cell assays and in whole organisms. Chen et al., Hum. Gen.
Ther. (1994) 5:595-601; Hassanzadeh et al., Febs Lett. (1998) 16(1,
2):75-80 and 81-86. Inducible expression vectors can be constructed
with intrabodies that react specifically with mitochondrial
NAD-dependent deacetylase protein. These vectors can be introduced
into model organisms and studied in the same manner as described
above for aptamers.
[0079] In some of the invention, the active agent is an agent that
modulates, and generally decreases or down regulates, the
expression of the gene encoding mitochondrial NAD-dependent
deacetylase in the host. Such agents include, but are not limited
to, antisense RNA, interfering RNA, ribozymes, and the like.
[0080] In some embodiments, the active agent is an interfering RNA
(RNAi). RNAi includes double-stranded RNA interference (dsRNAi).
Use of RNAi to reduce a level of a particular mRNA and/or protein
is based on the interfering properties of double-stranded RNA
derived from the coding regions of gene. In one example of this
method, complementary sense and antisense RNAs derived from a
substantial portion of the mitochondrial NAD-dependent deacetylase
gene are synthesized in vitro. The resulting sense and antisense
RNAs are annealed in an injection buffer, and the double-stranded
RNA injected or otherwise introduced into the subject (such as in
their food or by soaking in the buffer containing the RNA). See,
e.g., WO99/32619. In another embodiment, dsRNA derived from a
mitochondrial NAD-dependent deacetylase gene is generated in vivo
by simultaneous expression of both sense and antisense RNA from
appropriately positioned promoters operably linked to mitochondrial
NAD-dependent deacetylase coding sequences in both sense and
antisense orientations.
[0081] Antisense molecules can be used to down-regulate expression
of the gene encoding mitochondrial NAD-dependent deacetylase in
cells. Antisense compounds include ribozymes, external guide
sequence (EGS) oligonucleotides (oligozymes), and other short
catalytic RNAs or catalytic oligonucleotides which hybridize to the
target nucleic acid and modulate its expression.
[0082] The anti-sense reagent may be antisense oligonucleotides
(ODN), particularly synthetic ODN having chemical modifications
from native nucleic acids, or nucleic acid constructs that express
such anti-sense molecules as RNA. The antisense sequence is
complementary to the mRNA of the targeted gene, and inhibits
expression of the targeted gene products. Antisense molecules
inhibit gene expression through various mechanisms, e.g. by
reducing the amount of mRNA available for translation, through
activation of RNAse H, or steric hindrance. One or a combination of
antisense molecules may be administered, where a combination may
comprise multiple different sequences.
[0083] Antisense molecules may be produced by expression of all or
a part of the target gene sequence in an appropriate vector, where
the transcriptional initiation is oriented such that an antisense
strand is produced as an RNA molecule. Alternatively, the antisense
molecule is a synthetic oligonucleotide. Antisense oligonucleotides
will generally be at least about 7, usually at least about 12, more
usually at least about 20 nucleotides in length, and not more than
about 500, usually not more than about 50, more usually not more
than about 35 nucleotides in length, where the length is governed
by efficiency of inhibition, specificity, including absence of
cross-reactivity, and the like. It has been found that short
oligonucleotides, of from 7 to 8 bases in length, can be strong and
selective inhibitors of gene expression (see Wagner et al. (1996),
Nature Biotechnol. 14:840-844).
[0084] A specific region or regions of the endogenous sense strand
mRNA sequence is chosen to be complemented by the antisense
sequence. Selection of a specific sequence for the oligonucleotide
may use an empirical method, where several candidate sequences are
assayed for inhibition of expression of the target gene in an in
vitro or animal model. A combination of sequences may also be used,
where several regions of the mRNA sequence are selected for
antisense complementation.
[0085] Antisense oligonucleotides may be chemically synthesized by
methods known in the art (see Wagner et al. (1993), supra, and
Milligan et al., supra.) Preferred oligonucleotides are chemically
modified from the native phosphodiester structure, in order to
increase their intracellular stability and binding affinity. A
number of such modifications have been described in the literature,
which modifications alter the chemistry of the backbone, sugars or
heterocyclic bases.
[0086] Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire ribose phosphodiester backbone with a peptide linkage. Sugar
modifications are also used to enhance stability and affinity. The
.beta.-anomer of deoxyribose may be used, where the base is
inverted with respect to the natural .alpha.-anomer. The 2'-OH of
the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl
sugars, which provides resistance to degradation without comprising
affinity. Modification of the heterocyclic bases must maintain
proper base pairing. Some useful substitutions include deoxyuridine
for deoxythymidine; 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
[0087] Exemplary modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
[0088] Oligonucleotides having a morpholino backbone structure
(Summerton, J. E. and Weller D. D., U.S. Pat. No. 5,034,506) or a
peptide nucleic acid (PNA) backbone (P. E. Nielson, M. Egholm, R.
H. Berg, O. Buchardt, Science 1991, 254: 1497) can also be used.
Morpholino antisense oligonucleotides are amply described in the
literature. See, e.g., Partridge et al. (1996) Antisense Nucl. Acid
Drug Dev. 6:169-175; and Summerton (1999) Biochem. Biophys. Acta
1489:141-158.
[0089] As an alternative to anti-sense inhibitors, catalytic
nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc.
may be used to inhibit gene expression. Ribozymes may be
synthesized in vitro and administered to the patient, or may be
encoded on an expression vector, from which the ribozyme is
synthesized in the targeted cell (for example, see International
patent application WO 9523225, and Beigelman et al. (1995), Nucl.
Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic
activity are described in WO 9506764. Conjugates of anti-sense ODN
with a metal complex, e.g. terpyridylCu(II), capable of mediating
mRNA hydrolysis are described in Bashkin et al. (1995), Appl.
Biochem. Biotechnol. 54:43-56.
Formulations, Dosages, and Routes of Administration
[0090] The invention provides formulations, including
pharmaceutical formulations, comprising an agent that reduces a
level and/or an activity of mitochondrial NAD-dependent
deacetylase. In general, a formulation comprises an effective
amount of an agent that reduces a level and/or an activity of
mitochondrial NAD-dependent deacetylase. An "effective amount"
means a dosage sufficient to produce a desired result, e.g., a
reduction in a level and/or an activity of mitochondrial
NAD-dependent deacetylase, a reduction in histone deacetylation;
and the like. Generally, the desired result is at least a reduction
a level and/or an activity of mitochondrial NAD-dependent
deacetylase as compared to a control.
Formulations
[0091] In the subject methods, the active agent(s) may be
administered to the host using any convenient means capable of
resulting in the desired reduction in a level and/or an activity of
mitochondrial NAD-dependent deacetylase. Thus, the agent can be
incorporated into a variety of formulations for therapeutic
administration. More particularly, the agents of the present
invention can be formulated into pharmaceutical compositions by
combination with appropriate, pharmaceutically acceptable carriers
or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, injections,
inhalants and aerosols.
[0092] In pharmaceutical dosage forms, the agents may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0093] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0094] The agents can be formulated into preparations for injection
by dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0095] The agents can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0096] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0097] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0098] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0099] Other modes of administration will also find use with the
subject invention. For instance, an agent of the invention can be
formulated in suppositories and, in some cases, aerosol and
intranasal compositions. For suppositories, the vehicle composition
will include traditional binders and carriers such as, polyalkylene
glycols, or triglycerides. Such suppositories may be formed from
mixtures containing the active ingredient in the range of about
0.5% to about 10% (w/w), preferably about 1% to about 2%.
[0100] Intranasal formulations will usually include vehicles that
neither cause irritation to the nasal mucosa nor significantly
disturb ciliary function. Diluents such as water, aqueous saline or
other known substances can be employed with the subject invention.
The nasal formulations may also contain preservatives such as, but
not limited to, chlorobutanol and benzalkonium chloride. A
surfactant may be present to enhance absorption of the subject
proteins by the nasal mucosa.
[0101] An agent of the invention can be administered as
injectables. Typically, injectable compositions are prepared as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared. The preparation may also be emulsified or the active
ingredient encapsulated in liposome vehicles.
[0102] Suitable excipient vehicles are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vehicle may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents or pH
buffering agents. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in the art. See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 17th edition, 1985. The composition or formulation to
be administered will, in any event, contain a quantity of the agent
adequate to achieve the desired state in the subject being
treated.
[0103] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
Dosages
[0104] Although the dosage used will vary depending on the clinical
goals to be achieved, a suitable dosage range is one which provides
up to about 1 .mu.g to about 1,000 .mu.g or about 10,000 .mu.g of
an agent that reduces a level and/or an activity of mitochondrial
NAD-dependent deacetylase can be administered in a single dose.
Alternatively, a target dosage of an agent that reduces a level
and/or an activity of mitochondrial NAD-dependent deacetylase can
be considered to be about in the range of about 0.1-1000 .mu.M,
about 0.5-500 .mu.M, about 1-100 .mu.M, or about 5-50 .mu.M in a
sample of host blood drawn within the first 24-48 hours after
administration of the agent.
[0105] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means.
Routes of Administration
[0106] An agent that reduces a level and/or an activity of
mitochondrial NAD-dependent deacetylase is administered to an
individual using any available method and route suitable for drug
delivery, including in vivo and ex vivo methods, as well as
systemic and localized routes of administration.
[0107] Conventional and pharmaceutically acceptable routes of
administration include intranasal, intramuscular, intratracheal,
intratumoral, subcutaneous, intradermal, topical application,
intravenous, rectal, nasal, oral and other parenteral routes of
administration. Routes of administration may be combined, if
desired, or adjusted depending upon the agent and/or the desired
effect. The composition can be administered in a single dose or in
multiple doses.
[0108] The agent can be administered to a host using any available
conventional methods and routes suitable for delivery of
conventional drugs, including systemic or localized routes. In
general, routes of administration contemplated by the invention
include, but are not necessarily limited to, enteral, parenteral,
or inhalational routes.
[0109] Parenteral routes of administration other than inhalation
administration include, but are not necessarily limited to,
topical, transdermal, subcutaneous, intramuscular, intraorbital,
intracapsular, intraspinal, intrasternal, and intravenous routes,
i.e., any route of administration other than through the alimentary
canal. Parenteral administration can be carried to effect systemic
or local delivery of the agent. Where systemic delivery is desired,
administration typically involves invasive or systemically absorbed
topical or mucosal administration of pharmaceutical
preparations.
[0110] The agent can also be delivered to the subject by enteral
administration. Enteral routes of administration include, but are
not necessarily limited to, oral and rectal (e.g., using a
suppository) delivery.
[0111] Methods of administration of the agent through the skin or
mucosa include, but are not necessarily limited to, topical
application of a suitable pharmaceutical preparation, transdermal
transmission, injection and epidermal administration. For
transdermal transmission, absorption promoters or iontophoresis are
suitable methods. Iontophoretic transmission may be accomplished
using commercially available "patches" which deliver their product
continuously via electric pulses through unbroken skin for periods
of several days or more.
[0112] By treatment is meant at least an amelioration of the
symptoms associated with the pathological condition afflicting the
host, where amelioration is used in a broad sense to refer to at
least a reduction in the magnitude of a parameter, e.g. symptom,
associated with the pathological condition being treated, such as
an allergic hypersensitivity. As such, treatment also includes
situations where the pathological condition, or at least symptoms
associated therewith, are completely inhibited, e.g. prevented from
happening, or stopped, e.g. terminated, such that the host no
longer suffers from the pathological condition, or at least the
symptoms that characterize the pathological condition.
[0113] A variety of hosts (wherein the term "host" is used
interchangeably herein with the terms "subject" and "patient") are
treatable according to the subject methods. Generally such hosts
are "mammals" or "mammalian," where these terms are used broadly to
describe organisms which are within the class mammalia, including
the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice,
guinea pigs, and rats), and primates (e.g., humans, chimpanzees,
and monkeys). In many embodiments, the hosts will be humans.
[0114] Kits with unit doses of the active agent, e.g. in oral or
injectable doses, are provided. In such kits, in addition to the
containers containing the unit doses will be an informational
package insert describing the use and attendant benefits of the
drugs in treating pathological condition of interest. Preferred
compounds and unit doses are those described herein above.
Therapeutic Methods
[0115] The invention further provides methods of treating various
disorders, by modulating a level or an activity of a mitochondrial
NAD-dependent deacetylase. The methods generally involve
administering to an individual in need thereof an effective amount
of an agent that modulates a level or an activity of a
mitochondrial NAD-dependent deacetylase. In some embodiments, the
methods involve decreasing a level or activity of a mitochondrial
NAD-dependent deacetylase. In other embodiments, the methods
involve increasing a level or activity of a mitochondrial
NAD-dependent deacetylase.
[0116] Increasing a level or activity of a mitochondrial
NAD-dependent deacetylase provides a protective effect against
apoptosis. In some embodiments, an effective amount of an agent
that increases a level of activity of a mitochondrial NAD-dependent
deactylase is an amount that is effective to decrease apoptosis by
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, or at least about 90% or more, when
compared to the level of apoptosis in an individual not treated
with the agent.
[0117] Decreasing a level or activity of a mitochondrial
NAD-dependent deacetylase increases apoptosis. Increasing apoptosis
is desirable in the context of reducing unwanted cellular
proliferation. In some embodiments, an effective amount of an agent
that decreases a level of activity of a mitochondrial NAD-dependent
deactylase is an amount that is effective to increase apoptosis by
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, or at least about 90% or more, when
compared to the level of apoptosis in an individual not treated
with the agent.
[0118] Disorders amenable to treatment using a method according to
the invention are disorders related to, associated with, or caused
(directly or indirectly) by mitochondrial malfunction or
dysfunction. Disorders amenable to treatment using a method
according to the invention include cancer; neurodegenerative
disorders; metabolic disorders; ischemia-reperfusion injury; and
disorders associated with apoptosis or cell death.
[0119] Indications which can be treated using the methods of the
invention for reducing apoptosis or cell death in a eukaryotic
cell, include, but are not limited to, cell death or apoptosis
associated with Alzheimer's disease, Parkinson's disease,
rheumatoid arthritis, septic shock, sepsis, stroke, central nervous
system inflammation, osteoporosis, ischemia (e.g., resulting from
stroke or myocardial infarction), reperfusion injury, cell death
associated with cardiovascular disease, polycystic kidney disease,
cell death of endothelial cells in cardiovascular disease,
degenerative liver disease, multiple sclerosis, amyotropic lateral
sclerosis, cerebellar degeneration, ischemic injury, cerebral
infarction, myocardial infarction, myelodysplastic syndromes,
aplastic anemia, male pattern baldness, and head injury damage.
Also included are any hypoxic or anoxic conditions, e.g.,
conditions relating to or resulting from ischemia, myocardial
infarction, cerebral infarction, stroke, bypass heart surgery,
organ transplantation, neuronal damage, and the like.
[0120] Cell death-related indications which can be treated using
methods of the invention for activating apoptosis or cell death
include, but are not limited to, undesired, excessive, or
uncontrolled cellular proliferation, including, for example,
neoplastic cells; as well as any undesired cell or cell type in
which induction of cell death is desired, e.g., virus-infected
cells and self-reactive immune cells. The methods may be used to
treat follicular lymphomas, carcinomas associated with p53
mutations; autoimmune disorders, such as, for example, systemic
lupus erythematosus (SLE), immune-mediated glomerulonephritis;
hormone-dependent tumors, such as, for example, breast cancer,
prostate cancer and ovary cancer; and viral infections, such as,
for example, herpesviruses, poxviruses and adenoviruses.
[0121] Whether a therapeutic method of the invention is effective
in modulating cell death/apoptosis can be determined using any
known assay. Cell death can be measured using any known method, and
is generally measured using any of a variety of known methods for
measuring cell viability. Such assays are generally based on entry
into the cell of a detectable compound (or a compound that becomes
detectable upon interacting with, or being acted on by, an
intracellular component) that would normally be excluded from a
normal, living cell by its intact cell membrane. Such compounds
include substrates for intracellular enzymes, including, but not
limited to, a fluorescent substrate for esterase; dyes that are
excluded from living cell, including, but not limited to, trypan
blue; and DNA-binding compounds, including, but not limited to, an
ethidium compound such as ethidium bromide and ethidium homodimer,
and propidium iodide.
[0122] Apoptosis can be assayed using any known method. Assays can
be conducted on cell populations or an individual cell, and include
morphological assays and biochemical assays. A non-limiting example
of a method of determining the level of apoptosis in a cell
population is TUNEL (TdT-mediated dUTP nick-end labeling) labeling
of the 3'-OH free end of DNA fragments produced during apoptosis
(Gavrieli et al. (1992) J. Cell Biol. 119:493). The TUNEL method
consists of catalytically adding a nucleotide, which has been
conjugated to a chromogen system or a to a fluorescent tag, to the
3'-OH end of the 180-bp (base pair) oligomer DNA fragments in order
to detect the fragments. The presence of a DNA ladder of 180-bp
oligomers is indicative of apoptosis. Procedures to detect cell
death based on the TUNEL method are available commercially, e.g.,
from Boehringer Mannheim (Cell Death Kit) and Oncor (Apoptag Plus).
Another marker that is currently available is annexin, sold under
the trademark APOPTEST.TM.. This marker is used in the "Apoptosis
Detection Kit," which is also commercially available, e.g., from
R&D Systems. During apoptosis, a cell membrane's phospholipid
asymmetry changes such that the phospholipids are exposed on the
outer membrane. Annexins are a homologous group of proteins that
bind phospholipids in the presence of calcium. A second reagent,
propidium iodide (PI), is a DNA binding fluorochrome. When a cell
population is exposed to both reagents, apoptotic cells stain
positive for annexin and negative for PI, necrotic cells stain
positive for both, live cells stain negative for both. Other
methods of testing for apoptosis are known in the art and can be
used, including, e.g., the method disclosed in U.S. Pat. No.
6,048,703.
EXAMPLES
[0123] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric. Standard abbreviations may be
used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s,
second(s); min, minute(s); hr, hour(s); and the like.
Example 1
Characterization of a Mitochondrial NAD-Dependent Histone
Deacetylase
Experimental Procedures
Plasmid Construction
[0124] Plasmids expressing hSIRT3 were constructed by polymerase
chain reaction (PCR) amplification of the hSIRT3 coding sequence
using primers containing EcoRI sites and pCR2.1-SIRT3 as a
template. Amplified sequences were digested with EcoRI and cloned
into a modified pcDNA3.1+ vector (Invitrogen, Carlsbad, Calif.) to
yield a C-terminally FLAG-tagged hSIRT3. hSIRT3.DELTA.1-25-FLAG was
constructed by using modified N-terminal PCR primers introducing
EcorI sites and a methionine start codon before amino acid 26 of
the wild-type protein. Site-directed mutagenesis (QuikChange.TM.
Mutagenesis Kit, Stratagene, La Jolla, Calif.) was used for
construction of hSIRT3N229A-FLAG, hSIRT3H248Y-FLAG,
hSIRT3R7/13G-FLAG, hSIRT3R17/21G-FLAG, hSIRT3R7/13/17/21G-FLAG,
hSIRT3R7/13Q-FLAG, hSIRT3R17/21Q-FLAG, hSIRT3R7/13/17/21Q-FLAG,
hSIRT3L12P/R13P-FLAG and hSIRT3R99/100G-FLAG. All constructs were
verified by DNA sequencing. pSu9-DHFR was provided by J. Brix and
N. Pfanner (Institut fuer Biochemie und Molekularbiologie,
Freiburg, Germany).
GFP Fusion Constructs
[0125] To generate fusion proteins of GFP with wild-type hSIRT3 or
with amino acid 26-399 of hSIRT3, corresponding coding sequences
were PCR amplified and cloned into pEGFP-N1 (Clontech, Palo Alto,
Calif.).
Cell Culture and Transfection
[0126] HEK293T and HeLa cells were cultured in DMEM supplemented
with 10% FCS, 2 mM L-glutamine, 100 U Penicillin and 100 .mu.g
Streptomycin per ml and grown in 5% CO.sub.2 at 37.degree. C.
Calcium phosphate transfection was used to transfect HEK293T cells
(19). HeLa cells were transfected with Lipofectamine (Life
Technologies, Rockville, Md.).
Immunoblot Analysis
[0127] Antibodies used for immunoblotting included anti-mtHsp70
(Clone JG1, Affinity Bioreagents, Golden, Colo.), anti-Hsp60 (Clone
4B9/89, Affinity Bioreagents, Golden, Colo.), anti-Hsp90.alpha.
(StressGen, Victoria, Canada), anti-cytochrome c oxidase subunit IV
(Clone 20E8-C12, Molecular Probes, Eugene, Oreg.), anti-FLAG M2
(Sigma, St. Louis, Mo.), anti-cytochrome c (Clone 7H, 8.2; C12,
Pharmingen, San Diego, Calif.). hSIRT3 antisera were raised in
rabbits against a C-terminal peptide
(H.sub.2N-DLVQRETGKLDGPDK-COOH; SEQ ID NO:05). Western blots were
revealed with enhanced chemiluminescence (Amersham Pharmacia,
Piscataway, N.J.). Membranes were either nitrocellulose (Hybond
ECL, Amersham Pharmacia, Piscataway, N.J.) or PVDF (Immun-Blot.TM.,
Bio-Rad, Hercules, Calif.).
Immunofluorescence and Confocal Microscopy
[0128] HeLa cells grown on coverslips were incubated for 45 min
with 30 nM MitoTracker (CMXRos, Molecular Probes, Inc., Eugene,
Oreg.) in DMEM min at 37.degree. C., transferred to fresh DMEM and
further incubated for 60 min. Cells on coverslips were rinsed in
phosphate-buffered saline (PBS), fixed in 3.7% formaldehyde/PBS for
30 min, washed again in PBS and mounted. Images were acquired on a
BioRad Radiance 2000 laser scanning microscope equipped with an
Olympus BX60 microscope using an Olympus PlanApo 60.times./1.40 oil
objective. Excitation laser line was 488 nm for enhanced green
fluorescent protein (eGFP) and 578 nm for MitoTracker.
Preparation of Subcellular Fractions
[0129] Subcellular fractionation was performed according to
published procedures with minor modifications (20,21). All steps
were performed at 4.degree. C. In brief, cells were homogenized in
ice-cold buffer A (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl.sub.2, 1
mM EDTA, 1 mM EGTA, 1 mM dithiotreithol, 0.1 mM
phenylmethylsulfonyl fluoride, 20 mM HEPES-KOH, pH 7.5) and
homogenized in a Dounce homogenizer (Wheaton, Millville, N.J.).
Homogenization was checked by phase-contrast microscopy. The
homogenate was centrifuged twice at 800.times.g to remove nuclei
and unbroken cells. Mitochondria were sedimented by centrifugation
at 7,000.times.g for 15 min at 4.degree. C., washed twice with
buffer A and resuspended in TXIP-1 buffer (1% Triton X-100 (v/v),
150 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl, pH 7.4) supplemented with
protease inhibitors. Postmitochondrial supernatants were
fractionated by ultracentrifugation at 100,000.times.g for 30 min
at 4.degree. C. The supernatant constituting the cytosolic S-100
fraction was removed and the pellet was resuspended in TXIP-1
buffer. Protein concentrations of the fractions were determined (DC
Protein Assay, Bio-Rad, Hercules, Calif.) and equal amounts of each
fraction were separated by SDS-PAGE and blotted to
nitrocellulose.
Isolation of Mitochondria from Mammalian Cells
[0130] Mitochondria were isolated by differential centrifugation
according to published procedures (21). After several washes in SEM
buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH, pH 7.2),
mitochondria were resuspended in SEM buffer. To further purify
mitochondria, a crude mitochondrial fraction was layered on a
discontinuous sucrose gradient (1-1.5 M) in T.sub.10E.sub.1 buffer
(1 mM EDTA, 10 mM Tris-HCl, pH 7.5). After centrifugation for 20
min at 60,000.times.g at 4.degree. C., mitochondria were recovered
from the 1.0 M/1.5 M interface, carefully adjusted to 250 mM
sucrose and washed twice in SEM buffer.
Immunoprecipitation
[0131] Cells or isolated mitochondria were lysed in ice-cold TXIP-1
buffer containing either PMSF or protease inhibitor cocktail
(Roche, Indianapolis, Ind.). Lysates were centrifuged at
16,000.times.g for 5 min at 4.degree. C. and Anti-FLAG monoclonal
M2 antibody (Sigma, St. Louis, Mo.) covalently coupled to agarose
was added. Samples were incubated at 4.degree. C. for 12 hrs,
centrifuged and washed 4 times in TXIP-1 buffer. For the
deacetylation assays, the fourth wash was carried out in SIRT
deacetylase buffer (4 mM MgCl2, 0.2 mM dithiothreitol, 50 mM
Tris-HCl, pH 9.0)
Import of Radiolabeled Proteins into Isolated Mitochondria
[0132] Import into isolated mitochondria was carried out as
previously reported (22). Proteins were synthesized in the presence
of [.sup.35S]-methionine by coupled transcription-translation in
reticulocyte lysate (Promega, Madison, Wis.) (23). In vitro
translation reactions were centrifuged at 108,000.times.g,
2.degree. C. for 15 min and adjusted to 250 mM sucrose. Import
reactions contained 5% (v/v) reticulocyte lysate in import buffer
(3% (w/v) fatty-acid free bovine serum albumin (BSA), 250 mM
sucrose, 80 mM KCl, 5 mM MgCl.sub.2, 2 mM KH.sub.2PO.sub.4, 5 mM
L-methionine, 10 mM 3-[N-morpholino]propanesulfonic acid-KOH, pH
7.2). In each import reaction, 50 .mu.g of freshly isolated
mammalian mitochondria were mixed with radiolabeled proteins and
incubated at 30.degree. C. ATP (2 mM) and sodium succinate (10 mM)
were added to maintain coupling of isolated mitochondria. Import
was stopped by addition of valinomycin (1 .mu.M) and transfer to
0.degree. C.
[0133] Where indicated, samples were treated with proteinase K (50
.mu.g/ml) for 10 min on ice. Protease treatment was stopped by
addition of 2 mM phenylmethylsulfonyl fluoride (PMSF). Mitochondria
were reisolated by centrifugation at 10,000.times.g for 5 min at
4.degree. C., washed in SEM buffer and recentrifuged as above.
Mitochondrial pellets were resuspended in SDS sample buffer
containing dithiothreitol (DTT) and heated to 95.degree. C. for 5
min. Samples were subjected to SDS-PAGE. Dried gels were exposed to
Biomax MR film (Kodak, Rochester, N.Y.) at -70.degree. C. and
analyzed on a Fuji FUJIX BAS 1000 phosphorimager. Where indicated,
mitochondrial transmembrane potential was disrupted by blocking of
complex III of the respiratory chain (Antimycin, 8 .mu.M), blocking
of the F.sub.0/F.sub.1-ATPase (Oligomycin, 20 .mu.M) and potassium
flux (Valinomycin, 1 .mu.M).
[0134] Swelling experiments were performed according to published
protocols (24). Mitochondria were isolated from hSIRT3-FLAG
transfected HEK293T cells, washed and treated with proteinase K
(150 .mu.g/ml) to remove nonimported protein. Mitochondria were
reisolated at 10 000.times.g for 5 min, washed with SEM buffer and
recentrifuged. Mitochondrial pellets were resuspended in SM buffer
(250 mM sucrose, 10 mM MOPS-KOH, pH 7.2) and swollen by diluting
them tenfold dilution into M buffer (10 mM MOPS-KOH, pH 7.2) and
incubation on ice for 15 min. Mitoplasts and non-swollen
mitochondria were treated with proteinase K (150 .mu.g/ml) for 10
min at 0.degree. C. Protease digestion was stopped by addition of 2
mM PMSF and mitoplasts and mitochondria were reisolated by
centrifugation, washed and lysed in sample buffer. Samples were
separated by SDS-PAGE and blotted onto PVDF membrane. Radiolabeled
proteins were detected by autoradiography.
Fractionation of Mitochondrial Proteins by Alkaline Treatment
[0135] These experiments were performed using published protocols
(25,26). In brief, washed mitochondrial pellets were resuspended in
freshly prepared 0.1 M sodium carbonate, pH 11.5, and incubated at
0.degree. C. for 30 min. Mitochondrial membranes were sedimented by
ultracentrifugation at 100,000.times.g for 30 min at 4.degree. C.
The pellet was resuspended in SDS sample buffer and proteins in the
supernatant were concentrated by trichloracetate precipitation and
finally resuspended in sample buffer.
In Vitro Deacetylase Assay
[0136] Deacetylase assays were performed in a total volume of 100
.mu.l SIRT deacetylase buffer (4 mM MgCl.sub.2, 0.2 mM
dithiothreitol, 50 mM Tris-HCl, pH 9.0) containing
immunoprecipitated proteins or mitochondrial lysates and a peptide
corresponding to the first the first 22 amino acids of histone 4
chemically acetylated in vitro (27). Where indicated, 1 mM NAD, 5
mM nicotinamide (both from Sigma, St. Louis, Mo.) or 400 nM TSA
(WAKO, Richmond, Va.) were added. Deacetylation reactions were
stopped after 2 hours of incubation at room temperature by adding
25 .mu.l stop solution (0.1 M HCl, 0.16 M acetic acid). Released
acetate was extracted into 500 .mu.l ethyl acetate and samples were
vigorously shaken for 15 minutes. After centrifugation for 5
minutes, 400 .mu.l of the ethyl acetate fraction was mixed with 5
ml scintillation fluid (Packard, Meriden, Conn.) and the released
radioactivity was measured using a liquid scintillation
counter.
Mitochondrial Processing Peptidase Cleavage Assay
[0137] Purified recombinant yeast MPP (28) was obtained from G.
Isaya (Mayo Clinic and Foundation, Rochester, Minn.). Cleavage of
radiolabeled in vitro translated proteins was carried out in
reaction buffer (1 mM dithiothreitol, 1 mM MnCl.sub.2, 10 mM
Hepes-KOH, pH 7.4). Purified MPP or reaction buffer was added to
each sample followed by incubation at 27.degree. C. for 45 min.
Reactions were stopped by addition of SDS sample buffer and boiling
at 95.degree. C. for 5 min. Samples were separated by SDS-PAGE and
analyzed by phosphorimaging.
Results
Mitochondria Contain Sir2-Like Deacetylase Activity.
[0138] A systematic survey of subcellular fractions for the
presence of histone deacetylase activities led to the detection of
a deacetylase activity in human mitochondrial fractions prepared
from HEK293 T cells (FIG. 1A). This activity was strictly dependent
on the presence of NAD and was suppressed by nicotinamide (Vitamin
B3), a product of NAD hydrolysis (29-31) reported to inhibit
Sir2-like proteins (32) (FIG. 1A). In contrast, trichostatin A
(TSA), a specific inhibitor of class I and class II deacetylases,
had no effect on the deacetylase activity present in mitochondria
(FIG. 1A). Under the same conditions, TSA treatment led to a
significant inhibition of the activity of a prototypic class II
HDAC, HDAC6. The observed NAD-dependent deacetylase activity
sensitive to inhibition by nicotinamide but not by TSA indicated
the presence of Sir2-like class III protein deacetylases in
mitochondria.
hSIRT3 Mediates NAD-Dependent Deacetylase Activity in the
Mitochondria.
[0139] Transfection of each hSIRT cDNA in mammalian cells followed
by immunoprecipitation and incubation with a histone H4 peptide
substrate showed that hSIRT 1, 2 and 3 exhibited bona fide
NAD-dependent deacetylase activity while hSIRT4, 5, 6 and 7 showed
no detectable activity. To determine which hSIRT protein
contributed to the mitochondrial activity, expression vectors for
hSIRT1, hSIRT2 and hSIRT3 (epitope-tagged with FLAG at the
C-terminus), or a control vector, were transfected into HEK293T
cells. Cells were harvested and half of the preparation was used to
prepare a whole cell lysate while the other half was used to
isolate and purify mitochondria (mitochondrial lysate). hSIRT
proteins were immunoprecipitated with anti-FLAG antibodies from
whole cell or from mitochondria lysates and tested by western
blotting for the presence of the protein. All three proteins were
expressed and detected in whole cell lysates (FIG. 1B).
Interestingly, two forms of hSIRT3 were detected, a 44 KDa product
of the expected size given the cDNA sequence (predicted molecular
weight=43.6 KDa) and a smaller, 28 KDa product (FIG. 1B).). In
contrast, anti-FLAG immunoprecipitates prepared from mitochondria,
showed only the presence of hSIRT3 (28 KDa product) but not of
hSIRT1 and 2 (FIG. 1B). Testing of the same immunoprecipitates for
enzymatic activity yielded the same results. While all three hSIRTs
showed robust NAD-dependent enzymatic activity after
immunoprecipitation from whole cell lysates (FIG. 1C), only
anti-FLAG immunoprecipitates from cells transfected with hSIRT3
showed mitochondrial deacetylase activity (FIG. 1D). These results
are consistent with the model that hSIRT3 can target mitochondria
and mediate NAD-dependent deacetylase activity within that
subcellular compartment.
[0140] When total mitochondrial lysates prepared from cells
transfected with hSIRT3 were analyzed in the same in vitro
deacetylase assay, an increase in NAD-dependent deacetylase
activity was observed in comparison to cells transfected with a
control plasmid (FIG. 1E). The mitochondrial lysates overexpressing
hSIRT3 exhibited the same properties as untransfected mitochondrial
lysates in terms of sensitivity to nicotinamide and TSA. In
contrast, transfection of two catalytically inactive mutants,
hSIRT3-N229A and hSIRT3-H248Y, had no effect on the activity of the
lysates (FIG. 1E). These two mutants were designed by homology to
similar mutations reported to abrogate the activity of Sir2-like
proteins. Both mutants were shown in separate experiments to be
catalytically inactive in whole cell lysates. Importantly, both
mutants were efficient targeted to mitochondria, were equally well
expressed after transfection and were processed to the smaller 28
KDa product as wild type hSIRT3 (FIG. 1F). These observations are
consistent with the selective targeting of exogenous hSIRT3 to
mitochondria.
[0141] FIG. 1 A: Mitochondria contain Sir2-like deacetylase
activity. Mitochondrial lysates were prepared from HEK293T cells
and protein content was determined. Equal amounts of lysate were
assayed for deacetylase activity on a histone H4 peptide in either
the presence or absence of NAD (1 mM) or in combination with
nicotinamide (5 mM) or TSA (400 nM). Samples were incubated for 2
hrs at 25.degree. C. Released acetate was quantitated as described
in Materials and Methods. Representative results are shown.
[0142] FIG. 1B: In vitro deacetylase activity assay. hSIRT proteins
were immunoprecipitated from whole cell lysate from transfected
HEK293T cells using anti-FLAG antibodies. Immunoprecipitated
proteins were assayed in the presence or absence of NAD (1 mM).
[0143] FIG. 1C: Purified mitochondria from HEK293T cells
transfected with hSIRT proteins were lysed and FLAG-tagged proteins
were immunoprecipitated and analyzed for in vitro deacetylase
activity.
[0144] FIG. 1D: Western blot analysis of the immunoprecipitates
obtained from whole cell lysate (upper panel) or purified
mitochondria (lower panel). 50% of the immunoprecipitate used in
the deacetylase assay was detected using anti-FLAG M2
antibodies.
[0145] FIG. 1E: Transfection of hSIRT3 increases the NAD-dependent
deacetylase activity of mitochondria. Mitochondria were isolated
from HEK293T cells transfected with hSIRT3-FLAG, hSIRT3N229A-FLAG,
hSIRT3H248Y-FLAG or control vector (pFLAG) and lysed in TXIP-1
buffer. In vitro deacetylase activities of equal amounts of
mitochondrial lysate are shown.
[0146] FIG. 1F: Mitochondria were analyzed for the presence of
hSIRT3 wild-type and mutants by western blotting.
Endogenous and Exogenous hSIRT3 are Mitochondrial Proteins.
[0147] To further determine the subcellular localization of hSIRT3
in cells, a fusion protein with green fluorescent protein was
generated (hSIRT3-GFP). Confocal laser scanning microscopy of HeLa
cells transfected with hSIRT3-GFP revealed that it localized
exclusively to cytoplasmic substructures consistent with
mitochondria. This prediction was verified by costaining with a
mitochondria-specific dye, MitoTracker red, which showed total
overlapping of the two signals. This experiment indicated that
hSIRT3 exclusively localizes to mitochondria.
[0148] This observation was further verified using cell
fractionation experiments. Cells transfected with hSIRT3-FLAG were
used to prepare subcellular fractions according to established
protocols (21). Equal amounts of protein from each subcellular
fraction were subjected to SDS-PAGE and immunoblotting using an
antibody specific for the C-terminal FLAG-epitope. hSIRT3-FLAG and
cytochrome c were only be detected within the heavy membrane
fraction representing mitochondria (HM, FIG. 2A). Two FLAG-reactive
bands were detected within the mitochondrial fraction as discussed
above (see FIG. 2A). Immunoblotting of subfractions prepared from
untransfected cell confirmed that both bands were specific for
hSIRT3-FLAG.
Endogenous Mitochondrial hSIRT3 Protein has NAD-Dependent
Deacetylase Activity.
[0149] To examine the subcellular localization of endogenous
hSIRT3, a specific antiserum was raised against the a peptide
corresponding the last 15 amino acids of hSIRT3
(N-DLVQRETGKLDGPDK-C; SEQ ID NO:06). This antiserum recognized two
peptides of .about.44 and .about.28 KDa in mitochondria fraction
while the preimmune antiserum obtained from the same rabbit was
unreactive to these proteins (FIG. 2B). These two bands
corresponded in size to the .about.44 and 28 kDa fragment detected
after transfection of the FLAG-tagged hSIRT3. Immunoprecipitation
of mitochondria fraction with this antiserum showed the presence of
a specific NAD-dependent deacetylase activity which was not present
with the preimmune serum or with Protein G sepharose alone (FIG.
2C). These experiments demonstrate that endogenous hSIRT3 is
located in the mitochondria and is associated with NAD-dependent
deacetylase activity in that compartment.
[0150] FIG. 2A: Subcellular fractionation of HEK293T cells
transfected with hSIRT3-FLAG were homogenized and fractionated by
differential centrifugation. Equal amounts (30 .mu.g) of HM (heavy
membranes), LM (light membranes) and S-100 (cytosolic proteins)
fraction were analyzed by immunoblotting. hSIRT3-FLAG was revealed
by detection with monoclonal M2 anti-FLAG antibodies. Two
hSIRT3-FLAG specific forms (asterisks) were detected.
Nitrocellulose membranes were stripped and reprobed with antibodies
against cytochrome c (cyt c) and Hsp90.alpha..
[0151] FIG. 2 B: Detection of endogenous hSIRT3 protein in
mitochondrial lysates. Mitochondria were prepared from HEK293T
cells. Lysates were analyzed by western blotting using a polyclonal
rabbit hSIRT3 antiserum (35 .mu.g/ml) or a preimmune serum (35
.mu.g/ml) obtained from the same rabbit.
[0152] FIG. 2C: Endogenous hSIRT3 protein has NAD-dependent
deacetylase activity in vitro. hSIRT3 was immunoprecipitated from
HEK293T cells lysed in TXIP-1 buffer using hSIRT3 antiserum (0.35
mg/ml), preimmune serum (0.35 mg/ml) or protein G sepharose. Equal
amounts of immunoprecipitate were analyzed for in vitro deacetylase
activity.
The N-Terminus of hSIRT3 is Required for Mitochondrial Import.
[0153] Mitochondrial targeting signals frequently contain an
amphipatic .alpha.-helix and tend to contain positively charged,
hydrophobic and hydroxylated amino acid residues (15-18). Secondary
structure prediction of hSIRT3 revealed that an N-terminal peptide
corresponding to residues 1-25 has a high probability to contain an
amphipatic alpha-helix (34,35) (FIG. 3, middle panel). When plotted
as a helical wheel (FIG. 3, right panel) residues 4 to 21, showed a
cluster of positively charged arginine residues on one side of the
helix opposed by hydrophobic residues on the other side, a typical
feature of mitochondrial presequences (reviewed in (36)). To test
the importance of this putative alpha-helix in hSIRT3 mitochondrial
import, amino acid residues 1 to 25 were deleted from hSIRT3 and
fused it to GFP (hSIRT3.DELTA.1-25-GFP). Expression of this
construct in HeLa cells showed pancellular distribution. No
significant colocalization between the fusion protein and
MitoTracker-stained mitochondria could be observed. This
localization was in sharp contrast to the subcellular localization
observed after expression of full-length hSIRT3 protein fused to
GFP and indicated that the N-terminal 25 amino acids of hSIRT3 are
necessary for mitochondrial targeting.
[0154] FIG. 3. The N-terminal region of hSIRT3 is required for
mitochondrial targeting. Schematic diagram of hSIRT3. The hatched
box illustrates the region involved in mitochondrial targeting
(left panel). Parts of the N-terminal region show a high
probability to form an amphiphatic .alpha. helix (middle panel).
Illustration of residues 4 to 21 as a helical wheel plot reveals a
cluster of basic amino acids (black) on one side of the putative
helix (right panel).
[0155] To further define the requirement for mitochondrial import
of hSIRT3, cell-free mitochondrial in vitro import assays were
used. Similar assays have been used to elucidate the import
requirements of a variety of mitochondrial proteins.
[.sup.35S]-labeled hSIRT3 or hSIRT3.DELTA.1-25 proteins were
synthesized in rabbit reticulocyte lysates and incubated with
isolated mammalian mitochondria at 30.degree. C. for 2, 5 or 15
minutes in the presence of succinate and ATP. Mitochondria were
reisolated from the mixture by centrifugation and cosedimenting
proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis followed by autoradiography. A
time-dependent accumulation of hSIRT3, but not of
hSIRT3.DELTA.1-25, into mitochondria was observed (FIGS. 4A and B).
The import of SIRT across the mitochondrial membrane was dependent
on the mitochondrial transmembrane potential (.DELTA..PSI.m) since
import was inhibited in the presence of antimycin (8 .mu.M),
oligomycin, (20 .mu.M) and valinomycin (1 .mu.M). (FIG. 4A, lane
4).
[0156] When the proteinase K digestion performed at the end of the
import reaction was omitted, it was noted that both hSIRT3 and
hSIRT3.DELTA.1-25 could bind to the outer surface of mitochondria
in vitro, indicating that adhesion to mitochondria was not
dependent on the N-terminal 25 amino acids of hSIRT3. To exclude
the possibility that proteins had aggregated and cosedimented
nonspecifically, similar experiments were carried out in the
absence of mitochondria, but no unspecific sedimentation
occurred.
[0157] To further define the sequence and structural requirements
necessary for import of hSIRT3 into mitochondria, a series of point
mutations in the first 25 amino acids was generated. We used two
different strategies. First, we disrupted the .alpha. helix by
introducing 2 prolines at position 12 and 13. Second, we modified
the charge of the amphipathic helix by replacing arginine residues
with glycine or glutamine. The polar but uncharged glutamine
residues were predicted to preserve the .alpha. helical
conformation while changing the amphipathic character of the
.alpha. helix. To study the import efficiency, mutants and wild
type hSIRT3 were synthesized in rabbit reticulocyte lysates in the
presence of [.sup.35S]-methionine and assayed using the in vitro
import assay described above. Mutation of R7 and R13 in either
glycine or glutamine resulted in a loss of mitochondrial import. In
contrast, mutation of R17 and R21 reduced import by .about.50%
(FIG. 4C). When all arginine residues were mutated into glutamine
or glycine import efficiency was even further reduced. Disruption
of the putative helical structure by two prolines led to a loss in
mitochondrial import similar to the R7/13G mutant. These results
demonstrate the importance of the positively charged residues and
of the .alpha. helical structure of region 1-25 in hSIRT3 for its
import into mitochondria.
[0158] FIG. 4. Mitochondrial import of hSIRT3. FIG. 4A,
[.sup.35S]-labeled hSIRT3-FLAG or hSIRT3.DELTA.1-25-FLAG
synthesized in rabbit reticulocyte lysate was imported into
isolated mammalian mitochondria at 30.degree. C. To assay import in
the absence of .DELTA..PSI.m (lane 4), valinomycin (1 .mu.M),
antimycin (8 .mu.M) and oligomycin (20 .mu.M) were added to
mitochondria 5 min prior to the addition of proteins. At indicated
timepoints, further import was stopped by dissipating .DELTA..PSI.m
(addition of 1 .mu.M valinomycin) and incubation at 0.degree. C.
Samples from each timepoint were treated with proteinase K (50
.mu.g/ml) for 10 min at 0.degree. C. to remove nonimported
proteins. After reisolation of mitochondria and SDS-PAGE, the
amounts of imported proteins were quantified by phosphorimaging.
FIG. 4B, Quantitation of imported protein by phosphorimaging. FIG.
4C, Schematic illustration of mutants used to address the effects
of charged residues and conformation on hSIRT3 import. FIG. 4D,
[.sup.35S]-labeled hSIRT3 wild-type or mutants were imported into
isolated mitochondria for 20 min at 30.degree. C. Import was
stopped as described above and nonimported proteins were removed by
proteinase K treatment. Reisolated and washed mitochondria were
lysed in SDS sample buffer and analyzed by SDS-PAGE. Standards
representing 50% of the input used in the individual import
reactions were loaded adjacent to each import sample. FIG. 4E,
Import efficiency of individual hSIRT3 mutants was quantitated in
relation to their standards by phosphorimaging. The import
efficiency of hSIRT3 was set to 100%.
hSIRT3 is a Mitochondrial Matrix Protein
[0159] As discussed above, the observation that the mitochondrial
transmembrane potential (.DELTA..PSI.m) was required for hSIRT3
import into mitochondria suggested that hSIRT3 is likely to be
imported across the inner mitochondrial membrane. To further define
the exact localization of hSIRT3 in the mitochondria, we took
advantage of established methods addressing the submitochondrial
localization of proteins. First, mitochondria were isolated from
HEK293T cells expressing hSIRT3-FLAG. Mitoplasts were prepared by
incubation in hypotonic MOPS-buffer. This treatment leads to the
rupture of the outer mitochondrial membrane and to the release of
soluble proteins located in the intermembrane space. Mitoplasts and
mitochondria were reisolated by centrifugation and analyzed by
western blotting (FIG. 5A). The .about.28 kDa form of hSIRT3 was
not affected by the breakage of the outer mitochondrial membrane
and subsequent proteinase K digestion (FIG. 5A).
[0160] To exclude the possibility that hSIRT3-FLAG had formed a
protease-stable aggregate, mitochondria from cells transfected with
hSIRT3-FLAG were lysed in 0.5% Triton X-100 followed by proteinase
K digestion. Under these conditions, hSIRT3 was completely
degraded. In this respect, hSIRT3 behaved in a manner similar to
the matrix protein Hsp60 (FIG. 5A). Confirmation of the rupture of
the outer membrane by the hypotonic treatment was obtained by
blotting against the intermembrane space protein cytochrome c. In
contrast to hSIRT3, cytochrome c was lost after protease treatment
of mitoplasts (FIG. 5A). The results were consistent with three
different locations for hSIRT3: 1. mitochondrial matrix; 2.
peripherally attached to the inner side of the inner mitochondrial
membrane; 3. integral inner mitochondrial membrane protein.
[0161] To differentiate between these possibilities, we performed
alkaline extraction experiments of mitochondria with sodium
carbonate at pH 11.5. This treatment releases soluble and
peripheral membrane proteins to the supernatant, while integral
membrane proteins sediment with the membranes in the pellet (25).
Following this treatment, the .about.28 KDa form of hSIRT3 was
found in the supernatant, indicating that this form was either a
soluble matrix protein or was peripherally attached to the inner
face of the inner membrane (FIG. 5A). Interestingly, the .about.44
KDa form of hSIRT3 was detected mostly in the pellet, suggesting
that this form of SIRT3 is associated with the inner mitochondrial
membrane. As expected, the soluble matrix chaperonin mtHsp70 was
detected in the supernatant after alkaline extraction, whereas the
inner-membrane protein COXIV was associated with the membrane
fraction (FIG. 5B). These experiments indicate that the 28 KDa form
or hSIRT3 is a soluble matrix protein.
[0162] FIG. 5. A, Intramitochondrial localization of hSIRT3.
Mitochondria were isolated from hSIRT3-FLAG transfected HEK293T
cells and treated with proteinase K (150 .mu.g/ml) for 10 min at
0.degree. C. to remove proteins bound to the outer mitochondrial
surface. Proteinase K treatment was stopped by incubation with 2 mM
PMSF for 10 min at 0.degree. C. Mitochondrial preparations were
divided and one half was diluted with hypotonic EM buffer to create
mitoplasts. The other half was mock-treated with isotonic SEM
buffer. After incubation for 20 min at 0.degree. C., proteinase K
(150 .mu.g/ml) was added for 10 min at 0.degree. C. Protease
treatment was stopped as described above and mitochondria (M, left
lane) and mitoplasts (MP, right lane) were reisolated and analyzed
by western blotting. Opening of the outer mitochondrial membrane
was confirmed by detection of endogenous intermembrane space
protein cytochrome c (cyt c). Integrity of the inner mitochondrial
membrane was determined using the matrix protein Hsp60 as a marker.
hSIRT3-FLAG was detected using anti-FLAG M2 antibodies.
[0163] FIG. 5B, Alkaline extraction of mitochondria from
hSIRT3-FLAG transfected HEK293T cells. Mitochondria were isolated
and treated with proteinase K (150 .mu.g/ml) for 10 min at
0.degree. C. PMSF (2 mM) was added to stop proteinase K digestion.
Mitochondria were reisolated and washed in SEM buffer. The
preparation was devided and one half was resuspended in SDS sample
buffer (Total, left lane). The other half of the preparation was
resuspended in 100 mM sodium carbonate (Na.sub.2CO.sub.3), pH 11.5,
and incubated for 30 min at 0.degree. C. The extract was
centrifuged at 100,000.times.g at 4.degree. C. and the
mitochondrial membranes (Pellet, middle lane) were resuspended in
SDS sample buffer. The supernatant containing the soluble and
peripheral membrane proteins was TCA precipitated (Soluble, right
lane). Samples were analyzed by western blotting. hSIRT3 was
detected using anti-FLAG antibodies. Alkaline extraction was
controlled by detection of the marker proteins COXIV and
mtHsp70.
Proteolytic Processing of hSIRT3
[0164] As discussed above, the majority of hSIRT3 is present in
mitochondria as a truncated .about.28 KDa protein. Since this form
is reactive to the anti-FLAG antibody after transfection of a
C-terminal FLAG fusion protein, we concluded that hSIRT3 is
proteolytically cleaved at its N-terminus. The majority of
mitochondrial proteins carrying N-terminal targeting signals is
processed by matrix processing peptidase (MPP) after import into
the mitochondrial matrix (38). Incubation of radiolabelled hSIRT3
with recombinant yeast MPP led to its cleavage to a product of
.about.28 KDa, undistinguishable in size from the product detect in
vivo in mitochondria (FIG. 6A). Cleavage of a fusion protein
between subunit 9 of F0/F1-ATPase and DHFR (Su9-DHFR) by MPP in
vitro resulted in the appearance of digestion products similar to
what has been previously reported (28). Based on the size of the
processed hSIRT3 protein, we scanned the primary sequence of hSIRT3
for putative MPP recognition motifs. MPP specifically processes
many mitochondrial precursor proteins but no consensus processing
site has emerged. However, an arginine at -2 relative to the
cleavage site and additional aromatic or hydrophobic residues in
position 1 relative to the cleavage site appear necessary for
cleavage (39-41).
[0165] Several hSIRT3 mutants targeting arginine residues at
positions 99, 100, 133, 135, 139 and 158 were constructed by
site-directed mutagenesis and synthesized in rabbit reticulocyte in
the presence of [.sup.35S]-methionine. A mutant carrying two
glycines substituted for arginines at position 99 and 100 showed
abrogation of cleavage by MPP in vitro (FIG. 6B), while other
mutants were unaffected. These results indicate that residues
R99/100 are critical for the processing of hSIRT3 by MPP.
Transfection of this construct into mammalian cells led to a
partial inhibition of the processing of hSIRT3 into the 28 KDa
fragment and a new fragment of higher molecular weight was
detected.
Catalytic Activation of a Latent hSIRT3 by MPP--Mediated
Proteolytic Processing
[0166] It was noted that the in vitro translated hSIRT3 protein was
catalytically inactive in our in vitro deacetylase assay.
Similarly, hSIRT3 expressed in E. coli was not processed and was
poorly active enzymatically. The hypothesis that proteolytic
processing of hSIRT3 might lead to its catalytic activation was
tested. Unlabeled hSIRT3 was synthesized in vitro using rabbit
reticulocyte lysate. Samples were split in half and subjected to
cleavage by recombinant MPP in vitro. Reactions were diluted and
hSIRT3 was immunoprecipitated and assayed for deacetylase activity
in the presence or absence of NAD. Remarkably, the hSIRT3 processed
by MPP showed NAD-dependent deacetylase activity, whereas the
full-length uncleaved hSIRT3 remained inactive (FIG. 6C). These
results linked processing of hSIRT3 to the activation of its
NAD-dependent deacetylase activity. To control that no unspecific
factors or MPP itself had caused the observed NAD-dependent
deacetylase activity, we used the catalytic inactive hSIRT3-H248Y
mutant. When this mutant was assayed in the same way as hSIRT3, no
NAD-dependent deacetylase activity after incubation and cleavage
with MPP (FIG. 6C, left and right panels). These results
demonstrate that proteolytic processing of hSIRT3 by MPP leads to
the activation of its latent enzymatic activity.
[0167] FIG. 6. Proteolytic processing of hSIRT3 by MPP. FIG. 6A,
Cleavage of radiolabeled hSIRT3-FLAG (left panel) or pSu9-DHFR
(right panel) was assayed in HDM buffer in the presence or absence
of purified recombinant yeast MPP (1 .mu.l) for 45 min at
27.degree. C. in a total volume of 20 .mu.l. Samples were analyzed
by SDS-PAGE and autoradiography. p, precursor form; m, mature form
of pSu9-DHFR. FIG. 6B, Schematic illustration of the mutant showing
abrogated MPP cleavage (upper panel). Radiolabeled wild-type
hSIRT3-FLAG or hSIRT3R99/100G-FLAG were analyzed for MPP
processing. Assay conditions were as described (see A). Efficiency
of proteolytic processing by recombinant yeast MPP was quantitated
using phosphorimaging (left panel). Autoradiography of the same
experiment (right panel). FIG. 6C, MPP processing activates
NAD-dependent deacetylase activity of hSIRT3. Unlabeled hSIRT3-FLAG
or hSIRT3H248Y-FLAG synthesized in rabbit reticulocyte lysate was
incubated with recombinant yeast MPP or an equal amount of water
for 45 min at 27.degree. C. Samples were diluted with TXIP-1
buffer. FLAG-tagged proteins were immunoprecipitated with anti-FLAG
M2 antibodies covalently bound to agarose for 2 hrs at 4.degree. C.
Immunoprecipitates were washed and analyzed for in vitro
deacetylase activity in the presence or absence of NAD (1 mM). FIG.
6D, Western blot analysis of immunoprecipitates used in the
deacetylase assay.
Example 2
Enzymatically Active Recombinant SIRT 3 Protein
[0168] hSIRT3 is an NAD dependent, class III HDAC. SIRT3 localizes
to the mitochondrial matrix via an amphipathic .alpha.-helix rich
NH.sub.2-terminal. Once in the mitochondrial matrix, hSIRT3 is
proteolytically cleaved by mitochondrial matrix processing
peptidase (MPP) between residues Ser101 and Ile102. Full length
hSIRT3 is enzymatically inactive, but exhibits HDAC activity in
vitro after MPP cleavage. Based on these observations, it was
predicted that a recombinant form of SIRT3 lacking the first 100
amino acids to mimic the cleavage that occurs in the mitochondria
would be active as an HDAC.
Cloning Strategy
[0169] PCR primers were designed to amplify hSIRT3 from Ser101 to
Lys399 using a pcDNA3.1-SIRT3-Flag plasmid (pEV821) as a template.
Primer sequence is as follows:
forward--GTGAATTCATATCTTTTTCTGTGGGTGC (SEQ ID NO:07),
reverse--GTGAATTCGCCCTTGAATCATC (SEQ ID NO:08). Both primers
included an EcoR1 site so the PCR amplicon could be digested with
EcoR1 for subcloning into other vectors. The following PCR
parameters were used: 94.degree. C. --5', (94.degree. C. --30'',
55.degree. C. --30'', 72.degree. C. --60'').times.30 cycles,
72.degree. C. --7'. The EcoR1 digested amplicon was then subcloned
into the expression vector pTrcHis. Frame form B of pTrcHis was
used for subcloning to express an in frame, amino terminal
6.times.His tagged SIRT3 (101-399 aa) recombinant protein.
Expression and Purification
[0170] DH5.alpha. bacteria were transformed with the pTrcHis-SIRT3
(101-399) plasmid (pEV1453). Transformed bacteria were induced with
1.0 mM IPTG at 37 C for 2 h. The resulting 6.times.His-tagged
protein was purified under native conditions at 4.degree. C. by
Ni-NTA affinity chromotography (Qiagen).
[0171] First, bacteria were pelleted and cleared lysate was
prepared under native conditions. The pellet was resuspended (50 mM
NaH.sub.2PO.sub.4, pH8.0; 300 mM NaCl; 10 mM imidazole) and
incubated on ice for 30 minutes in the presence of 1 mg/ml
lysozyme. This mixture was then sonicated on ice (four 10-15 second
bursts at 40-60% power) and centrifuged at 4.degree. C. at 14,000
rpm for 25 minutes. Supernatant (cleared lysate) was bound to
Ni-NTA resin (batch method, Qiagen) on a rotary mixer at 4.degree.
C. for 60 minutes. Batch mixture was loaded into poly prep column
(BioRad) and flow-through collected. The resin bed was washed twice
with 4 ml of wash buffer (50 mM NaH.sub.2PO.sub.4, pH8.0; 300 mM
NaCl; 20 mM imidazole), and the tagged proteins eluted 4 times with
0.5 ml elution buffer (50 mM NaH.sub.2PO.sub.4, pH8.0; 300 mM NaCl;
250 mM imidazole). SDS-PAGE analysis revealed the second elution
fraction contained a majority of recombinant protein along with
contaminating proteins. A concentrating spin column (Vivaspin 6)
was used to concentrate the recombinant protein and remove excess
imidazole.
Activity Assay
[0172] Recombinant SIRT3 (1.5 .mu.g) was resuspended in 100 .mu.L
deacetylase buffer (50 mM Tris-HCl [pH 9.0], 4 mM MgCl.sub.2, and
0.2 mM DTT) with different concentrations of NAD (Sigma) and 20,000
cpm of the acetylated peptide substrate (in vitro acetylated
histone H4 peptide).
[0173] The results are shown in FIG. 8. SIRT3 showed a
dose-dependent HDAC activity similar to the activity demonstrated
for SIRT2. This experiment demonstrate that recombinant SIRT3 can
function as a deacetylase in vitro and offers a new tool for the
screening of SIRT3 inhibitors and the study of its enzymatic
activity.
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[0237] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *