U.S. patent application number 10/383592 was filed with the patent office on 2004-04-15 for mechanism of mitochondrial membrane permeabilization by hiv-1 vpr, mimetics of vpr and methods of screening active molecules having the ability to alter and/or prevent and/or mimic the interaction of vpr with ant.
Invention is credited to Belzacq, Anne-Sophie, Brenner-Jan, Catherine, Edelmann, Lena, Hoebeke, Johan, Jacotot, Etienne Daniel Francois, Kroemer, Guido, Roques, Bernard Pierre.
Application Number | 20040072146 10/383592 |
Document ID | / |
Family ID | 26925199 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072146 |
Kind Code |
A1 |
Jacotot, Etienne Daniel Francois ;
et al. |
April 15, 2004 |
Mechanism of mitochondrial membrane permeabilization by HIV-1 Vpr,
mimetics of Vpr and methods of screening active molecules having
the ability to alter and/or prevent and/or mimic the interaction of
Vpr with ANT
Abstract
The invention is directed to the induction of mitochondrial
membrane permeabilization via the physical and functional
interaction of the HIV-1 Vpr protein with the mitochondrial inner
membrane protein ANT (adenine nucleotide translocator, also called
adenine nucleotide translocase or ADP/ATP carrier). Reagents and
methods for inducing and/or inhibiting the binding of Vpr to ANT,
mitochondrial membrane permeabilization, and apoptosis are
provided.
Inventors: |
Jacotot, Etienne Daniel
Francois; (Paris, FR) ; Kroemer, Guido;
(Paris, FR) ; Roques, Bernard Pierre; (Paris,
FR) ; Edelmann, Lena; (Boulogne, FR) ;
Hoebeke, Johan; (Schiltighcim, FR) ; Brenner-Jan,
Catherine; (LeChesnay, FR) ; Belzacq,
Anne-Sophie; (Paris, FR) |
Correspondence
Address: |
Finnegan, Henderson, Farabow,
Garrett & Dunner, L.L.P.
1300 I Street, N.W.
Washington
DC
20005-3315
US
|
Family ID: |
26925199 |
Appl. No.: |
10/383592 |
Filed: |
March 10, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10383592 |
Mar 10, 2003 |
|
|
|
PCT/EP01/11316 |
Sep 11, 2001 |
|
|
|
60231539 |
Sep 11, 2000 |
|
|
|
60232841 |
Sep 15, 2000 |
|
|
|
Current U.S.
Class: |
435/5 ;
424/144.1 |
Current CPC
Class: |
G01N 2500/02 20130101;
G01N 33/6872 20130101; C12N 2740/16322 20130101; G01N 2500/10
20130101; A61K 38/00 20130101; G01N 2500/20 20130101; C07K 14/47
20130101; G01N 2333/163 20130101; A61P 43/00 20180101; C07K 14/4747
20130101; G01N 33/5432 20130101; C07K 14/005 20130101; G01N 33/5079
20130101 |
Class at
Publication: |
435/005 ;
424/144.1 |
International
Class: |
C12Q 001/70; A61K
039/395 |
Claims
What is claimed is:
1. A method of preventing interaction of Vpr with ANT comprising:
(a) providing a molecule capable of preventing the binding of
full-length Vpr to ANT; and (b) contacting said molecule with an
ANT fragment; wherein said molecule prevents the interaction of
said ANT fragment with Vpr.
2. The method claim 1, wherein said method prevents channel
formation in mitochondrial membranes.
3. The method claim 1, wherein said method prevents
permeabilization of mitochondrial membranes.
4. The method claim 1, wherein said method prevents cell death.
5. The method of claim 4, wherein said method prevents cell death
by apoptosis.
6. The method of claim 1, wherein said molecule is Bcl-2 or a
fragment thereof.
7. A method of screening for molecules that compete with the
binding of the C-terminal moeity of Vpr to ANT comprising: (a)
providing a Vpr fragment capable of binding to ANT; (b) contacting
said Vpr fragment with an ANT fragment capable of binding to Vpr in
the presence and absence of a test molecule; and (c) detecting the
binding of said Vpr fragment to said ANT fragment in the presence
and absence of a test molecule.
8. The method of claim 7, wherein said fragment comprises
full-length Vpr.
9. The method of claim 7, wherein said fragment comprises amino
acids 52-96 of HIV-1 Vpr.
10. A method of screening for molecules that mimic Vpr or Vpr
fragments in its capacity to interact physically of with ANT
comprising: a) providing a Vpr or Vpr fragment capable of
interacting with ANT, b) contacting said Vpr or Vpr fragment with
an ANT fragment capable of interacting with Vpr or Vpr fragment in
the presence of absence of a test molecule; and c) detecting the
binding of said Vpr or Vpr fragment to said ANT fragment in the
presence of absence of a test molecule.
11. A peptidic or non-peptidic molecule that prevents
permeabilization of mitochondrial membranes, wherein said molecule
prevents the binding of Vpr to ANT.
12. A peptidic or non-peptidic molecule that causes
permeabilization of mitochondrial membranes, wherein said molecule
enhances the binding of Vpr to ANT.
13. A pharmaceutical and diagnostic composition comprising a
molecule of claim 11 or 12.
14. A method for causing or preventing permeabilization of
mitochondrial membranes comprising administering a composition of
claim 13 to a patient.
15. A method of screening for genetic or epigenetic alterations in
the expression or structure of the three ANT isoforms in humans
comprising: (a) providing a fragment of Vpr, wherein said fragment
is capable of binding to ANT, with a sample comprising human ANT;
(b) mixing said fragment with a biological sample comprising human
ANT; (c) mixing said fragment with a control sample comprising
human ANT; (d) detecting the binding of Vpr to ANT in said
biological sample and said control sample; (e) correlating a
difference in binding with a genetic or epigenetic alteration of
ANT; and (f) optionally detecting a difference in the ANT capacity
to form channel in liposome or in planar lipids bilayers.
16. A method of quantifying the level of the three human ANT
isoforms in a cell comprising: (a) mixing Vpr with a biological
sample comprising ANT in an amount effective to bind to ANT; and
(b) quantitating the level of binding of Vpr to ANT.
17. A method of screening active molecules of interest that induce
or prevent formation of a lethal pore by ANT comprising: (a)
providing purified ANT in artificial lipid bilayers or liposomes;
(b) contacting molecules of interest to be screened with said ANT;
and (c) detecting lethal pore formation by measuring the release of
labeled substrate.
18. A method of screening active molecules of interest that inhibit
the formation of a lethal pore without preventing antiport function
comprising: (a) providing a composition comprising purified ANT in
artificial lipid bilayers or liposomes with a molecule that induces
the formation of a lethal pore; (b) contacting said composition in
the presence or absence of a test molecule. (c) detecting by
fluorescence the presence of the antiport function; and (d)
detecting by another fluorescence the test molecule that inhibits
the formation of a lethal pore.
19. A method of screening active molecules of interest according to
the claim 18, wherein in step a) the active molecule that induces
the formation of a lethal pore is Vpr, a fragment of Vpr, or a
variant of Vpr.
20. A method of screening active molecules of interest according to
claim 18, wherein in step a) the active molecule that induces the
formation of a lethal pore is selected from the group comprising:
atractyloside, mastoparan, terbutyl or diamide.
21. A method of screening active molecules of interest according to
claim 18, wherein in step a) the active molecule that induces the
formation of a lethal pore is selected from the group of
pro-apoptotic molecules of Bcl-2 family.
22. A method of screening active molecules of interest according to
claim 18, wherein in step a) the active molecule that induces the
formation of lethal pore is a BAX molecule selected from the group
of pro-apoptotic molecules of Bcl-2 family.
23. An isolated or purified peptide having the sequence:
DRHKQFWRYFAGN.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/231,539, filed Sep. 11, 2000, and of U.S.
Provisional Application Ser. No. 60/232,841, filed Sep. 15, 2000,
both of which are hereby incorporated by reference.
DESCRIPTION OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention is directed to discovery that the proapoptotic
HIV-1-encoded protein Vpr induces mitochondrial membrane
permeabilization via its physical and functional Interaction with
the mitochondrial Inner membrane protein ANT (adenine nucleotide
translocator, also called adenine nucleotide translocase or ADP/ATP
carrier). HIV-1 Viral protein R (Vpr) interacts with the
permeability transition pore complex (PTPC) to trigger ANT pore
formation and/or mitochondrial membrane permeabilization (MMP) and
consequent cell death (by apoptosis or any related mechanism of
cell death).
[0004] 2. Background of the Invention
[0005] It is now recognized that mitochondria play an important
role in controlling the life and death (the apoptosis) of cells
(Kroemer and Reed 2000). Thus it seems that a growing number of
molecules are involved in signal transduction, and that many
metabolites (and certain viral effectors) act on the mitochondria
and influence the permeabilization of mitochondrial membranes.
Also, a certain number of experimental anti-cancer drugs kill cells
by acting directly on mitochondrial membranes (Ravagnan et al.,
1999; Larochette et al., 1999; Marchetti et al., 1999; Fulda et
al., 1999; Belzacq et al., 2000). Therefore, the use of specific
pro-apoptotic agents for mitochondria seems to be a concept that is
emerging in anti-cancer chemotherapy (for reference: Costantini, et
al., 2000). A possible outcome could be the use of cytoprotective
molecules to treat illnesses associated with excess apoptosis
(AIDS, neurodegenerative diseases, etc.) owing to their ability to
stabilize mitochondrial membranes. Against this background, the
identification (mode of action) of those molecular components that
control the permeability of the mitochondrial membranes has become
a major topic in biomedicine.
[0006] MMP is a key event of apoptotic cell death associated with
the release of caspase activators and caspase-independent death
effectors from the intermembrane space, dissipaton of the inner
transmembrane potential (.DELTA..PSI.m), as well as a perturbation
of oxidative phosphorylation G. Kroemer, N. Zamzami, S. A. Susin,
Immunol. Today 18, 44-51 (1997). D. R. Green, J. C. Reed, Science
281, 1309-1312 (1998). J. J. Lemasters, et al., Biochim. Biophys.
Acta 1366, 177-196 (1998). D. C. Wallace, Science 283, 1482-1488
(1999). M. G. Vander Heiden, C. B. Thompson, Nat. Cell Biol. 1,
E209-E216 (1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes
Dev. 13, 1988-1911 (1999). G. Kroemer, J. C. Reed, Nat. Med. 6,
513-519 (2000). Pro- and anti-apoptotic members of the Bcl-2 family
regulate inner and outer MMP through interactions with the adenine
nucleotide translocator (ANT; in the inner membrane, IM), the
voltage-dependent anion channel (VDAC; in the outer membrane, OM)
and/or through autonomous channel-forming activities G. Kroemer, N.
Zamzami, S. A. Susin, Immunol. Today 18, 44-51 (1997). D. R. Green,
J. C. Reed, Science 281, 1309-1312 (1998). J. J. Lemasters, et al.,
Biochim. Biophys. Acta 1366, 177-196 (1998). D. C. Wallace, Science
283, 1482-1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat.
Cell Biol. 1, E209-E216 (1999). A. Gross, J. M. McDonnell, S. J.
Korsmeyer, Genes Dev. 13, 1988-1911 (1999). G. Kroemer, J. C. Reed,
Nat. Med. 6, 513-519 (2000). I. Marzo, et al., Science 281,
2027-2031 (1998). S. Shimizu, M. Narita, Y. Tsujimoto, Nature 399,
483-487 (1999). S. Shimizu, A. Konishi, T. Kodama, Y. Tsujimoto,
Proc. Natl. Acad. Sci. USA 97, 3100-3105 (2000). S. Desagher. et
al., J. Cell Biol. 144, 891-901 (1999).
[0007] ANT and VDAC are major components of the permeability
transition pore complex (PTPC), a polyprotein structure organized
at sites at which the two mitochondrial membranes are apposed. G.
Kroemer, N. Zamzami, S. A. Susin, Immunol. Today 18, 44-51 (1997).
D. R. Green, J. C. Reed, Science 281, 1309-1312(1998). J. J.
Lemasters, et al., Biochim. Biophys. Acta 1366, 177-196 (1998). D.
C. Wallace, Science 283, 1482-1488 (1999). M. G. Vander Heiden, C.
B. Thompson, Nat. Cell Biol. 1, E209-E216 (1999). A. Gross, J. M.
McDonnell, S. J. Korsmeyer, Genes Dev. 13, 1988-1911 (1999). G.
Kroemer, J. C. Reed, Nat. Med. 6, 513-519 (2000). M. Crompton,
Biochem. J. 341, 233-249 (1999).
[0008] The adenine nucleotide translocator (ANT) plays an important
role in the process that triggers the permeabilization of
mitchondrial membranes, and subsequent apoptosis (Marzo, et al.,
1998; Brenner, et al., 2000). In the cellular context, ANT Is
inserted into the internal membrane of mitochondria and has two
opposing functions. On the one hand, ANT is a vital antiport for
cellular bioenergetics and is specific to ATP and ADP. On the other
hand, ANT can form a non-specific lethal pore through the action of
certain ligands (natural or xenobiotic) that eliminate the
mitochondrial electrochemical gradient.
[0009] The HIV-1 regulatory protein Vpr has pleiotropic effects on
viral replication and cellular proliferation, differentiation,
cytokine production, and NF-kB-mediated transcription. M. Emerman,
M. H. Malim, Science 280, 1880-1884 (1998). A. D. Frankel, J. A. T.
Young, Annu. Rev. Biochem. 67, 1-25 (1998). M. Bukrinsky, A.
Adzhubei, J. Med. Virol 9, 39-49 (1999). In addition, Vpr can
localize to mitochondria. I. G. Macreadie, et al., Proc. Natl.
Acad. Sci. USA 92, 2770-2774 (1995). I. G. Macreadie, et al., FEBS
Lett. 410, 145-149 (1997). K. Muthami, L. J. Montaner, V. Ayyavoo,
D. B. Weine. DNA and Cell Biology 19, 179-188 (2000). E. Jacotot,
et al., J. Exp. Med. 191, 33-45 (2000). Full length (Vpr1-96) or
truncated synthetic forms of Vpr act on the PTPC to induce all
mitochondrial hallmarks of apoptosis, including .DELTA..PSI..sub.m
loss and the release of cytochrome c and apoptosis inducing factor
(AIF). E. Jacotot, et al., J. Exp. Med. 191, 33-45 (2000). The
MMP-inducing activity of Vpr resides in its C-terminal moiety
(Vpr52-96), within an .alpha.-helical motif of 12 amino acids
(Vpr71-82) containing several critical arginine (R) residues (R73,
R77, R80) which are strongly conserved among different pathogenic
HIV-1 isolates. L. G. Macreadie, et al., Proc. Natl. Acad. Sci. USA
92, 2770-2774 (1995). I. G. Macreadie, et al., FEBS Lett. 410,
145-149 (1997). E. Jacotot, et al., J. Exp. Med. 191, 33-45
(2000).
[0010] Depending on the apoptotic stimulus, permeabilization may
affect the OM and IM in a variable fashion and may or may be not
accompanied by matrix swelling. G. Kroemer, N. Zamzami, S. A.
Susin, Immunol. Today 18, 44-51 (1997). D. R. Green, J. C. Reed.
Science 281, 1309-1312 (1998). J. J. Lemasters, et al., Biochim.
Biophys. Acta 1366, 177-196 (1998). D. C. Wallace, Science 283,
1482-1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat. Cell
Biol. 1, E209-E216 (1999). A. Gross, J. M. McDonnell, S. J.
Korsmeyer, Genes Dev. 13, 1988-1911 (1999). G. Kroemer, J. C. Reed.
Nat. Med. 6, 513-519 (2000). In vitro experiments performed on
purified mitochondria or proteins reconstituted into artificial
membranes suggest at least two competing models of MMP. On the one
hand, pore formation by ANT has been proposed to account for IM
permeabilization, osmotic matrix swelling, and consequent OM
rupture, resulting because the surface area of the IM with its
folded christae exceeds that of the OM. In support of this
hypothesis, pro-apoptotic molecules such as Bax, atractyloside,
Ca.sup.2+, and thiol oxidants cause ANT (which normally is a
strictly specific ADP/ATP antiporter) to form a non-specific pore
(I. Marzo, et al., Science 281, 2027-2031 (1998); N. Brustovetsky,
M. Klingenberg, Biochemistry 35, 8483-8488 (1996); C. Brenner, et
al., Oncogene 19, 329-336 (2000)). On the other hand, VDAC has been
suggested to account for a primary OM permeabilization not
affecting IM (S. Shimizu, M. Narita, Y. Tsujimoto, Nature 399,
483-487 (1999). S. Shimizu, A. Konishi, T. Kodama, Y. Tsujimoto,
Proc. Nat. Acad. Sci. USA 97, 3100-3105 (2000)). In favor of this
hypothesis, the permeabilization of VDAC-containing liposomes to
sucrose or cytochrome c is enhanced by Bax and inhibited by Bcl-2
in vitro. S. Shimizu, M. Narita, Y. Tsujimoto, Nature 399, 483-487
(1999). S. Shimizu, A. Konishi. T. Kodama, Y. Tsujimoto, Proc.
Natl. Acad. Sci. USA 97, 3100-3105 (2000).
[0011] Recent studies have revealed the existence of several viral
apoptosis inhibitors acting on mitochondria. For example,
adenovirus, Epstein Barr virus, Herpes virus saimiri, and Kaposi
sarcorma-associated human herpes virus 8 produce
apoptosis-suppressive Bcl-2 homologs. E. H.-Y. Cheng, et al., Proc.
Natl. Acad. Sci. USA 94, 690-694 (1997). J. H. Han, D. Modha, E.
White, Oncogene 17, 2993-3005 (1998). T. Derfuss, et al., J. Virol.
72, 5897-5904 (1998). W. L. Marshall, et al., J. Virol. 73,
5181-5185 (1999). In addition, several viruses encode
PTPC-interacting proteins without any obvious homology to the
Bcl-2/Bax family. The cytomegalovirus apoptosis inhibitor pUL37x
(V. S. Goldmacher, et al., Proc. Natl. Acad. Sci. USA 96,
12536-12541 (1999).) and Vpr, an HIV-1-encoded apoptosis inducer,
selectively bind to ANT. The proapoptotic p13 (II) protein derived
from the X-II ORF of HTLV-1 is also targeted to mitochondria via a
peptide motif that bears structural similarities to the
mitochondriotoxic domain of Vpr. V. Ciminale, et al., Oncogene 18,
4505-4514 (1999). Moreover, the pro-apoptotic MMP-inducing
hepatitis virus B protein X interacts with VDAC. Z Rahmani, K. W.
Huh, R. Lasher, A. Siddiqui, J. Virol. 74, 2840-2846 (2000). Thus,
both VDAC and ANT emerge as major targets of viral apoptosis
regulation and, perhaps, as targets for pharmacological
intervention on viral pathogenesis and/or other pathologies linked
to apoptosis dysregulations (i.e., cancer, ischemia,
neurodegenerative diseases, etc.). Apoptosis is a process that
develops in several phases: (1) an initiation phase, which is
extremely heterogeneous and during which the biochemical pathways
paticipating in the process depend on the apoptosis-inducing agent;
(2) a decision phase, which is common to different types of
apoptosis, during which the cell "decides" to commit suicide; and
(3) a common degradation phase, which is characterized by the
activation of catabolic hydrolases (caspases and nucleases).
Although the activation of caspases (cysteine proteases cleaving at
aspartic acid [Asp] residues) and nucleases is necessary for the
acquisitions of the full apoptotic morphology, it appears clear
that inhibition of such enzymes does not inhibit cell death induced
by a number of different triggers: Bax, Bak, c-Myo, PML, FADD,
glucocorticoid receptor occupancy, tumor necrosis factor, growth
factor withdrawal, CXCR4 cross-linking, and chemotherapeutic
agents, such as etoposide, camptothecin, or cisplatin. In the
absence of caspase activation, cells manifest a retarded cytolysis
without characteristics of advanced apoptosis, such as total
chromatin condensation, oligonucleosomal DNA fragmentation, and
formation of apoptotic bodies. However, before cells lyse, they do
manifest a permeabilization of both mitochondrial membranes with
dissipation of the inner transmembrane potential
(.DELTA..PSI..sub.m) and/or the release of apoptogenic proteins,
such as cytochrome c and apoptosis-inducing factor (AIF) via the
outer membrane. These results have invalidated the hypothesis that
caspase activation is always required for apoptotic cell death to
occur. Rather, cell death is intimately associated with the
permeabilization of mitochondrial membranes.
[0012] The understanding of apoptosis has recently been facilitated
by the development of cell-free Systems. Instead of considering the
cell as a black box, subcellular fractions (e.g., mitochrondria,
nuclei, and cytosol) are mixed together with the aim to
reconstitute the apoptosis phenomenon by recapitulating the
essential steps of the process in vitro. It appears that
proapoptotic second messengers, whose nature depends on the
apoptosis-inducing agent, accumulate in the cytosol during the
initiation phase. These agents then induce mitochondrial membrane
permeabilization, allowing cells to enter the decision phase. The
apoptotic changes of mitochondria consist in a .DELTA..PSI..sub.m
loss, transient swelling of the mitochondrial matrix, mechanical
rupture of the outer membrane and/or its nonspecific
permeabilization by giant protein-permanent pores, and release of
soluble intermembrane proteins (SIMPs) through the outer membrane.
Once the mitochondrial membrane barrier function is lost, several
factors, e.g., the metabolic consequences at the bioenergetic
level, the loss of redox homeostasis, and the perturbation of ion
homeostais, contribute to cell death. The activation of proteases
(caspases) and nucleases by SIMP's is necessary for the acquisition
of apoptotic morphology. This latter phase corresponds to the
degradation step, beyond the point of no return of the apoptotic
process. Different SIMPs provide a molecular link between
mitochondrial membrane permeabilization and the activation of
catabolic hydrolases: cytochrome c (a heme protein that
participates in caspase activation), certain procaspases (in
particular, procaspases 2 aid 9, which in some cell types, are
selectively enriched in mitochondria), and AIF, AIF is a
nuclear-encoded intermembrane flavoprotein that translocates to the
nucleus where it induces the caspases-independent peripheral
chromatin condensation and the degradation of DNA into 50-kilobase
pair fragments.
[0013] The mechanism of mitochondrial membrane permeabilization is
not completely understood. Some investigators prefer the hypothesis
that proapoptotic members of the Bcl-2 family are inserted in the
outer membrane where they oligomerize and form cytochrome c
permeant pores in an autonomous fashion, not requiring the
interaction with other mitochondrial membrane proteins. However,
Bax-induced membrane permeabilization is inhibited by cyclosporin A
(CsA) and bongkrekic acid (BA), two inhibitors of formation of the
permeability transition pore (or "megachannel"), suggesting that
sessile mitochrondrial proteins (the targets of CsA and BA) are
involved in this process. The permeability transition pore has a
polyprotein structure that is formed at the contact sites between
the inner and outer membranes. One of the key proteins of the
permeability transition pore complex (PTPC) is the adenine
nucleotide translocator (ANT). ANT, the target of BA, is the most
abundant inner membrane protein, ANT normally functions as a
specific carrier protein for the exchange of adenosine triphosphate
(ATP) and adenosine diphosphate (ADP), but it can become a
nonspecific pore.
[0014] An interesting property of the PTPC is that the
permeabilization of the inner and/or outer mitochondrial membranes
compromises the bioenergetic equilibrium of the cell (e.g., it
provokes the oxidation of reduced NADPH and glutathione, the
depletion of ATP, and the dissipation of .DELTA..PSI..sub.mand
effects the homeostasis of intracellular ions (e.g. by releasing
Ca.sup.2+ from the matrix). Intriguingly, all of these changes
themselves increase the probability of PTPCs opening. This has two
important implications. First, the consequences of PTPC opening
themselves favor opening of the PTPC in a self-amplification loop
that coordinates the lethal response among mitochondria within the
same cells. Second, this implies that the final result of PTPC
opening is a stereotyped ensemble of biochemical alterations, which
does not depend on the initiating stimulus, be it a specific
proapoptotic signal transduction cascade or nonspecific damage at
the energy or redox levels.
[0015] Chemotherapy aims at the specific eradication of cancer
cells, mostly through the induction of apoptosis. Gene therapy can
employ Bax-delivering vectors, thereby indirectly targeting
mitochondria to induce apoptosis. In contrast to such proteins,
certain peptides readily penetrate the plasma membrane and thus can
be used as the pharmacologic agents. Mastoparan, a peptide isolated
from wasp venom, is the first peptide known to induce mitochondral
membrane permeabilization via a CsA-inhibitable mechanism and to
induce apoptosis via a mitochondrial effect when added to intact
cells. This peptide has an .alpha.-helical structure and possesses
some positive charges that are distributed on one side of the
helix. A similar peptide (KLAKLAKKLAKLAK or (KLAKLAK).sub.2
(K=lysine, L=amine, and A=leucine) has been found recently to
disrupt mitochondral membranes when it is added to purified
mitochondria, although the mechanisms of this effect have not been
elucidated. (Ellerby, H. M. et al., Anti-cancer activity of
targeted pro-apoptotic peptides, Nature Med. 5, 1032-1038
(1999)).
[0016] The proapoptotic 96 amino acid protein viral protein R (Vpr)
from human immunodeficiency virus-I contains a comparable
structural motif (aa 71-82), i.e., an .alpha.-helix with several
cationic charges that concentrate on the same side of the helix.
Vpr, as well as Vpr derivatives containing this "mitochondriotoxic"
domain cause a rapid CsA and BA-inhibited dissipation of the
.DELTA..PSI..sub.m as well as the mitochondrial release of
apoptogenic proteins, such as cytochrome c or AIF. The same
structural motifs relevant for cell killing appear to be
responsible for the mitochondriotoxic effects of Vpr. Vpr favors
the permeabilization of artificial membranes containing the
purified PTPC or defined PTPC components such as the ANT combined
with Bax, but this effect is prevented by the addition of
recombinant Bcl-2. According to surface plasmon resonance studies,
the Vpr C-terminus binds purified ANT with a high affinity in the
nanomolar range. E. Jacotot et al., J. Exp. Med. 191, 33-45 (2000),
which is specifically incorporated herein by reference. In
addition, a biotinylated Vpr-derived peptide (Vpr52-96) may be
employed as bait to specifically purify a mitochondrial molecular
complex containing ANT and the VDAC. Yeast strains lacking ANT or
VDAC are less susceptible to Vpr-induced killing than are control
cells. Thus, Vpr induces apoptosis via a direct effect on the
mitochondrial PTPC. In analogy to Vpr, the p13 (II) protein derived
from the X-II open reading frame of HTLV-1 is targeted to
mitochondria and can cause a dissipation of the .DELTA..PSI..sub.m
and mitochondrial swelling. Mitochondrial targeting of this protein
has been mapped to a decapeptide sequence that contains several Arg
residues that are asymmetrically distributed in the .alpha.-helix.
However, Arg-Ala substitutions within the mitochondriotoxic domain
of p13 (II) did not abolish the mitochondrial targeting of p13.
[0017] Lethal peptides may be targeted to mitochondria and more
specifically, at least in the case of Vpr, to the PTPC. Ellerby et
al. recently have fused the mitochondriotoxic (KLAKLAK).sub.2 motif
to a targeting peptide that interacts with endothelial cells. Such
a fusion peptide is internalized and induces mitochondrial membrane
permeabilization in angiogenic endothelial cells and kills
MDA-MD-435 breast cancer xenografts transplanted into nude mice.
Similarly, a recombinant chimeric protein containing interleukin 2
(IL-2) protein fused to Bax selectively binds to and kills IL-2
receptor-bearing cells in vitro. Thus, specific cytotoxic agents
that target surface receptors, translocate into the cytoplasm, and
induce apoptosis via mitochondrial membrane permeabilization might
be useful in treating cancer.
[0018] A recurrent problem with conventional chemotherapeutic
agents is that they exploit endogenous apoptosis-induction pathways
that may be compromised by alterations such as mutations of p53,
increased antioxidant activity, blockade of CD95/CD95L pathway,
overexpression of Bcl-2-like proteins, etc. One possible strategy
to enforce cell death is to trigger downstream events of the common
apoptotic pathway. Thus, adenovirus-mediated transfer of caspases
has been proposed as one strategy to induce cell death beyond any
regulation. An alternative strategy is to use mitochondriotoxic
agents that induce cell death irrespective of the upstream control
mechanisms and irrespective of the status of caspases and
endogenous caspase inhibitors. As an example, LND, arsenite, or
CD437 induce cell death independently of the p53 status via a
pathway that is not affected by caspase inhibitors. Similarly,
betulinic acid and Vpr trigger CD95 (Apo-1/Fas)- and
p53-independent apoptosis, and both permeabilize mitochondrial
membranes in a caspase-independent fashion. As a result, these
types of agents may prove to be highly useful in killing normally
resistant cells. Moreover, the future of tumor therapy may profit
from the design of agents that overcome the Bcl-2-mediated
stabilization of mitchondrial membranes as well as from targeting
amphipathic peptides or peptidomimetics to defined cellular
populations or tissues.
[0019] Selective eradication of transformed cells by use of
mitochondrion-specific agents should be effective. One strategy is
to target a toxic agent to selected cell types on the basis of the
specific expression of surface receptors. Another, yet to be
developed, strategy would aim at exploiting difference in the
composition or regulation of the PTPC between normal and tumor
cells. Future research will tell to which extent cell targeting (by
use of retroviral or adenoviral vectors, use of integrin-specific
domains, etc.) and/or targeting of tumor-specific alterations in
the PTPC will prove to be useful in cancer therapy, and also in the
treatment of neurodegenerative diseases hypothetically linked to
mitochondrial dysfunction (i.e., Friedrich ataxia, Hereditary
spastic paraplegia, Huntington disease, Amyotrophic lateral
sclerosis, Parkinson disease, Alzheimer disease) and treatment of
acute organ failure that may involve regulatory events acting at
the level of MMP (i.e., ischemia) (Kroemer, G. et al., Mitchondrial
control of cell death, Nature Med., vol. 6, no. 5, 513-519
(1999)).
[0020] Thus, there exist a need in the art for methods and reagents
for regulating mitochondrial permeabilization and apoptosis.
SUMMARY OF THE INVENTION
[0021] The present invention relates to the physical and functional
interactions between Vpr and the adenine nucleotide translocator
(ANT), which function to permeabilize mitochondrial membranes and
result in the death of cells by apoptosis. In a preferred
embodiment, the present invention relates to the physical and
functional interactions between Vpr and the three human isoforms of
ANT also designated ANT1, ANT2, and ANT3. The invention encompasses
methods of exploiting this novel mechanism to permeabilize
mitochondrial membranes. The invention further encompasses methods
of causing cell death by apoptosis.
[0022] The invention also encompasses methods of altering or
preventing binding of Vpr to ANT. The invention further encompasses
methods of altering or preventing channel formation due to the
association of Vpr with ANT. The invention also encompasses methods
of causing or preventing permeabilization of mitchondrial
membranes. The invention also encompasses methods of causing or
preventing cell death by apoptosis.
[0023] The invention also encompasses methods of screening for
molecules that alter or prevent binding of Vpr to ANT. The
invention further encompasses methods of screening for molecules
that alter or prevent channel formation due to the association of
Vpr with ANT. The invention also encompasses methods of screening
for molecules that cause or prevent permeabilization of
mitochondrial membranes. The invention also encompasses methods of
screening for molecules that cause or prevent cell death by
apoptosis.
[0024] The invention also encompasses methods of screening for
molecules that compete with the binding of the C-terminal moeity of
Vpr (vpr52-96 for HIV-1) to ANT. The invention also encompasses
methods of screening for molecules that promote the binding of the
C-terminal moeity of Vpr (vpr52-96 for HIV-1) to ANT. The invention
also encompasses methods of screening for molecules that alter or
prevent binding of the C-terminal moeity of Vpr (vpr52-96 for
HIV-1) to ANT. The invention further encompasses methods of
screening for molecules that alter or prevent permeabilization of
mitochondrial membranes due to the association of the C-terminal
moeity of Vpr (vpr52-96 for HIV-1) with ANT. The invention further
encompasses methods of screening for molecules that alter or
prevent apoptosis due to the association of the C-terminal moeity
of Vpr (vpr52-96 for HIV-1) with ANT.
[0025] The invention also encompasses peptidic or non-peptidic
molecules that alter or prevent binding of Vpr to ANT. The
invention also encompasses peptidic or non-peptidic molecules that
mimic Vpr or Vpr fragment in its capacity to interact physically or
functionally with ANT. The invention further encompasses peptidic
or non-peptidic molecules that alter or prevent channel formation
due to the association of Vpr with ANT. The invention also
encompasses peptidic or non-peptidic molecules that cause or
prevent permeabilization of mitochondrial membranes. The invention
also encompasses peptidic or non-peptidic molecules that cause or
prevent cell death by apoptosis. The invention further encompasses
pharmaceutical and diagnostic compositions comprising these
molecules and the use of these compositions to cause or prevent
permeabilization of mitochondrial membranes or apopotosis.
[0026] The invention further encompasses peptidic or non-peptidic
molecules that mimic the C-terminal moeity of Vpr (vpr52-96 for
HIV-1) and modulate the permeabilization of mitochondrial
membranes. The invention also encompasses peptidic or non-peptidic
molecules that compete with the binding of the C-terminal moeity of
Vpr (vpr52-96 for HIV-1) to ANT. The invention also encompasses
peptidic or non-peptidic molecules that promote the binding of the
C-terminal moeity of Vpr (vpr52-96 for HIV-1) to ANT. The invention
further encompasses pharmaceutical and diagnostic compositions
comprising these molecules and the use of these compositions to
cause or prevent permeabilization of mitochondrial membranes or
apopotosis.
[0027] The invention also encompasses methods for screening for
genetic or epigenetic alterations in the expression or structure of
the three ANT isoforms in humans. The invention further encompasses
screening and diagnosis for differences in the ability of the three
ANT isoforms in different patients to interact with Vpr and to
promote mitochondrial membrane permeabilization, channel formation
and/or apopotosis.
[0028] The invention also encompasses methods for specific cell
killing by induction of apoptosis.
[0029] The invention also encompasses methods for screening
molecules modifying channel properties of ANT.
[0030] The invention further encompasses methods of screening of
active molecules able to alter or prevent ANT-Bcl2 interaction.
[0031] By studying the cytotoxic properties of the Vpr protein of
HIV-1, the inventors discovered that Vpr interacts direct with ANT
to trigger the permeabilization of mitochondrial membranes, as well
as apoptosis. First, Vpr goes through an external mitochondrial
membrane using the mitchondrial protein (also called
"voltage-dependent anion channel": VDAC) and then attaches itself
to the ANT, its primary target, with strong affinity (KD=1 nM). The
ANT/Vpr complex forms high-conductance channels that trigger the
permeabilization of the internal mitochondrial membrane, the
swelling of the mitochondrial matrix and, finally, the breakage of
the external membrane, and thus the release of factors that
implement apoptosis (e.g., AIF, cytochrome c and some
pro-caspases). The inventors have identified interaction sites
between ANT and Vpr: for Vpr (14 Kd; 96 aa), the binding site to
ANT brings into play the pattern 71HFRIGCRHSRIG82 (minimal toxic
pattern) in the heart of the linear structure (.alpha.-helicoidal
between amino-acids 52 and 83) of Vpr 52-96. For ANT1 (30 Kd; 298
aa), the binding site to Vpr brings into play the pattern
104DRHKQFWRYFAGN116 in the middle of the second ANT ring (aa
92-116).
[0032] The inventors' discovery of the physical and functional
interaction between Vpr and ANT, and of at least one of the
interaction sites, led them to build analogs of said toxic pattern
of Vpr that can interact with the protein complex (permeability
transition pore; PTPC) that contains ANT. These molecules can serve
to imitate the pro-apoptotic effect of Vpr in order to destroy
cancerous cells in vitro or in vivo. These molecules are either
peptide or non-peptide molecules and are acquired in isolated or
purified form.
[0033] The present invention pertains to a novel protein/protein
interaction between the retroviral HIV regulatory protein Vpr and
the mitochondrial adenine nucleotide translocator (ANT) a membrane
associated receptor implicated in the control of cell death by
apoptosis. The invention also concerns peptidic or non-peptidic
molecules having the ability to alter and/or to prevent the binding
(or the chanel formation due to this binding) of Vpr to ANT.
Another aspect of the invention concern peptidic or non-peptidic
molecules having the ability to mimic the C-terminal moiety of Vpr
(Vpr52-96 for HIV-1) in its capacity to bind ANT and cooperate with
ANT to permeabilise mitochondrial membranes (and consequently kill
cells). The invention is also directed to pharmaceutical and
diagnostic compositions containing an effective amount of the
molecules altering and/or preventing the binding (and/or
conformational consequences of this binding such as chanel
formation) of Vpr to ANT (consequently such compositions will be
cytoprotectives), as well as to therapeutic or diagnostic methods
using such pharmaceutical or diagnostic composition. Moreover, the
invention is also directed to pharmaceutical and diagnostic
compositions containing an effective amount of the molecules able
to mimic the C-terminal part of Vpr in its capacity to bind and
cooperate with ANT to permeabilise mitochondrial membranes (and
consequently kill cells), as well as to therapeutic or diagnostic
methods using such pharmaceutical or diagnostic composition. The
invention also deals with methods of screening new active molecules
(endogenous or xenobiotics) having the ability to alter and/or to
prevent the binding (or the chanel formation due to this binding)
of Vpr to ANT, or having the ability to mimic the C-terminal moiety
of Vpr (Vpr52-96 for HIV-1) in its capacity to bind ANT and
cooperate with ANT to permeabilise mitochondrial membranes (and
consequently kill cells). Finally the invention is directed to
methods of screening genetics or epigenetics (such as specific
modifications in cancer affected individuals) alterations in the
expression or structure of the three ANT isoforms in humans.
[0034] Thus, the present invention concerns a protein-to-protein
interaction between Vpr and ANT, and potentially between Vpr and
VDAC, which can exploited to screen therapeutic molecules active as
cytoprotectors (an inhibitor of ANT/Vpr interaction) or active as
cytotoxics (analogs of Vpr with respect to interaction with ANT
and/or VDAC). In this regard, the inventors have established a new
ELISA screening test for Vpr ligands and molecules than can inhibit
the attachment of ANT to Vpr.
[0035] Consequently, one of the objectives of the present invention
concerns peptide or non-peptide molecules that can imitate Vpr by
attaching themselves to ANT in the cells (or certain cells) of an
individual; specifically, a person afflicted with cancer.
[0036] The invention also concerns structural or functional
inhibitors effective in blocking Vpr/ANT interaction or Vpr/VDAC
interaction and thus 1) that inhibit in vitro or in vivo infection
by HIV and 2) that inhibit the cytotoxic effect of any ANT ligand
(natural, endogenous, xenobiotic) and thus produce a cytoprotective
effect in patients afflicted with a disease associated with excess
apoptosis.
[0037] Thus, the present invention also covers components that can
modify the interaction between, on the one hand, Vpr (found in the
cells, extracellular fluids, or HIV particles of an individual
infected by a retrovirus) or an analog (endogenous [e.g., Bcl-2 or
a sub-region of this protein] or xenobiotic) of Vpr and, on the
other hand, at least one of the isoforms of ANT found in cells at
the mitochondrial membrane level. Molecules derived from ANT or
from an interaction pattern (for example, ANT104-116) with Vpr are
also considered as active molecules forming a part of the present
invention.
[0038] The invention also concerns the use of the compounds and
inhibitors defined earlier as active principles of pharmaceutical
compounds. One possible specific application might be the coupling
of Vpr, from a Vpr pattern (e.g., pattern 52-96, 71-82, or 71-96)
or from an analog of Vpr, with a molecule that can screen a tumor
in vivo. Thus, the invention includes the use of a Vpr pattern (for
example, pattern 52-96, 71-82, or 71-96).
[0039] The invention also includes the means to screen molecules
that can imitate the cytotoxic and/or mitochondrial effect of Vpr
(particularly its interaction with ANT and/or VDAC) and the means
to screen molecules (cytoprotective) that can modify the
interaction between, on the one hand, Vpr (found in the cells,
extra-cellular fluids, or HIV particles of individuals infected by
a retrovirus) or an analog (endogenous or xenobiotic) of Vpr and,
on the other hand, at least one of the isoforms of ANT found in the
cell at the mitochondrial membrane level.
[0040] Thus, the invention includes the methods to screen agonists
(structural or functional analogs of Vpr) or antagonists
(inhibitors of Vpr/ANT interaction, or the adoption of a "lethal
pore" conformation in response to Vpr, or a structural or
functional analog of Vpr) of ANT. Hence, the invention includes at
least two screening tests:
[0041] a binding test of ANT (or an ANT derived peptide containing,
for example, the pattern 104-116 of ANT) on Vpr (or a peptide
derived from Vpr containing, for example, the pattern 71-82). The
protocol of this test has already been established in the case of
binding Vpr 52-96 at the bottom of a plate with 96 wells and to
which an ANT or a peptide of ANT containing the pattern 104-116 is
attached and then exposed.
[0042] a test called a "double test" functional for ANT and that
simultaneously evaluates the specific antiport function and the
non-specific lethal pore function of ANT. The principle of this
test and the detailed protocol are described in Example 5.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 presents physical and functional interaction between
Vpr and ANT.
[0044] A Plasma surface resonance sensorgrams of the interaction of
ANT with Vpr52-96, Vpr52-96[R73A, 80A] or an irrelevant control
(Co). Only the sensorgram of the interaction with Vpr52-96 exhibits
an increase of binding as a function of time and a positive signal
at the start of the dissociation phase (off). The calculated
K.sub.D (K.sub.D=k.sub.on/k.sub.- off) of the interaction is
9.7.+-.6.4 nM (X.+-.SD, n=5).
[0045] B. Langmuir isotherm determined at different concentrations
of ANT on sensorgrams corrected by substraction of the blank
(sensorgrams obtained with Vpr52-96[R73A, 80A]).
[0046] C. Modulation of the Vpr52-96-ANT interaction by ANT ligands
and ANT-derived peptides. Measurements were performed as in A, in
the absence (.O slashed.) or presence of bongkrekic acid (BA, 250
.mu.M), atractyloside (Atr, 50 .mu.M), the ANT104-116 peptide, or
three control peptides (all at 5 .mu.M). ANT-2-derived peptide
ANT104-116 [DKRTQFWRYFAGN] and control peptides (Co. I: scrambled
ANT104-116 [FQNYWGHKRFRDA]; Co. II: mutated ANT104-116
[DGHKQFWGYFAGN]; Co. III: topologically equivalent peptide (aa
149-161) from the ANT-related human phosphate carrier protein
[SNMLGEENTYLWR]. Activation or inhibition was calculated as
(1-k.sub.0a/k.sub.0).times.100, in which k.sub.0a and k.sub.0 are
the initial velocity in the presence or absence of the agent,
respectively.
[0047] D. Langmuir isotherm for the binding of ANT104-116 to
biotinylated Vpr52-96 (as determined in A). The calculated K.sub.D
of the interaction is 35 .mu.M.
[0048] E. Schematic diagram showing the topology of ANT and the
sequence of the ANT-2-derived peptide ANT104-116.
[0049] FIG. 2 presents physical (A, B) and functional (C)
interaction between Vpr and liposomes containing ANT.
[0050] A. Dose-response curve of FITC-labeled Vpr52-96 binding onto
ANT-liposomes and plain liposomes.
[0051] B. Binding of FITC-Vpr52-96 (2 .mu.M) to plain liposomes,
ANT-proteoliposomes, in the presence or absence of BA (50
.mu.M).
[0052] C. Permeabilization of ANT proteoliposomes by Vpr (X.+-.SD,
n=3). Liposomes were loaded with 4-methylumbelliferylphosphate
(4-MUP) and exposed for 60 min to Atr (200 .mu.M) or the indicated
Vpr-derived peptides (1 .mu.M), in the presence or absence of BA
(50 .mu.M), ADP (800 .mu.M), and/or the indicated peptides (same as
in B, 0.5 .mu.M, pre-incubated with Vpr52-96 for 5 min). Then,
alkaline phosphatase was added to convert liposome-released 4-MUP
into the fluorochrome 4-methylumbelliferone (4-MU) and the
percentage of 4-MUP release induced by Vpr-derived peptides was
calculated as described in Material and Methods".
[0053] FIG. 3 presents electrophysiological properties of Vpr52-96
and ANT in planar lipid bilayers. Current fluctuations of Vpr52-96
(80 nM, +150 mV), Vpr52-96(0.4 nM, +100 mV), ANT (1 nM, +110 mV)
and Vpr52-96+ANT (0.4:1 nM, +115 mV) and associated histograms
(right) of conductance levels are shown.
[0054] A. Cooperative effect between ANT and Vpr52-96 at the single
channel level. Current fluctuations of Vpr52-96 (80 nM, +150 mV),
Vpr52-96 (0.4 nM, +100 mV), ANT (1 nM, +110 mV) and Vpr52-96+ANT
(0.4:1 nM, 115 mV) after incorporation into synthetic membranes.
Single channel recordings were performed using the "Tip-Dip"
technique. The recordings shown are representative for at least
three independent determinations.
[0055] B. Statistical analysis of conductances obtained in A.
Results were expressed as current distributions at different
voltages. Conductances (.gamma.; in picosiemens, pS) are calculated
by division of current by voltage.
[0056] FIG. 4 presents oxidative properties of purified
mitochondria exposed to Vpr.
[0057] A. Oxygen consumption curves after addition of the indicated
agents. Trace a: control mitochondria (no pretreatment). Trace b:
mitochondria pretreated for 10 min with 1 .mu.M Vpr52-96. Numbers
along the traces are nmol of O.sub.2 consumed min.sup.-1 mg.sup.31
1 protein.
[0058] B. Respiratory control (RC) values calculated by dividing
oxygen consumption in the presence of CCCP by that measured with
oligomycin (determined as in A), 10 min after addition of 1 .mu.M
of Vpr-derived peptides (mean values of 3 determinations).
[0059] FIG. 5 presents inner versus outer mitochondrial membrane
permeabilization.
[0060] A. Respirometry performed after addition of NADH and
Vpr52-96 (1 .mu.M). Numbers along the traces are nmol of O.sub.2
consumed min.sup.-1 mg.sup.-1 protein. Note that the
Vpr-stimulated, NADH-dependent O.sub.2 consumption was fully
sensitive to rotenone.
[0061] B. Kinetics of Vpr52-96 induced inner membrane
permeabilization to NADH and outer membrane permeabilization to
reduced cytochrome c. Oxygen consumption was determined in the
presence of 2 mM NADH (full squares) as in A (traces C-D) and
cytochrome c (15 .mu.M) oxidation (open circles) was
spectrofluorometrically measured, as described Rustin et al.,
1994). The 100% value of cytochrome c oxidation was determined by
addition of 2.5 mM laurylmaltoside.
[0062] C. Kinetics of Vpr52-96-induced .DELTA..PSI..sub.m loss and
cytochrome c release. Purified mitochondria were treated with 1
.mu.M Vpr52-96 subjected to cytofluorometric determination of the
percentage of mitochondria having a low .DELTA..PSI..sub.m using
the .DELTA..PSI..sub.m-sensitive fluorochrome JC-1. In parallel,
cytochrome c was immunodetected in the supernatant of
mitochondria.
[0063] FIG. 6 presents Bcl-2-mediated inhibition of Vpr effects on
mitochondria.
[0064] A. Vpr52-96-induced .DELTA..PSI..sub.m dissipation induced
in intact cells. COS cells were microinjected with recombinant
human Bcl-2 (10 .mu.M), Konigs polyanion (PA10, 2 .mu.M), or PBS
only, then incubated in the absence (Co.) or presence of 1 .mu.M
Vpr52-96 for 3 hours, and stained with the
.DELTA..PSI..sub.m-sensitive dye JC-1 (2 .mu.M; red fluorescence of
mitochondria with a high .DELTA..PSI..sub.m, green fuorescence of
mitochondria with a low .DELTA..PSI..sub.m).
[0065] B. Effect of Bcl-2 on the Vpr-induced inner MMP to NADH.
Mitochondria were left untreated (Co.) or pretreated (10 min) with
Bcl-2 (0.8 .mu.M) or BA (10 .mu.M). Oxygen consumption of purified
mitochondria was measured as in FIG. 5 after addition of
succinate+CCCP or NADH, as indicated.
[0066] C. Ultrastructural effects of Vpr on isolated mitochondria.
Electron micrographs were obtained after incubation of mitochondria
for 5 or 15 min with 3 .mu.M Vpr52-96, after pre-incubation (5 min)
wuh 0.8 .mu.M Bcl-2 or 2 .mu.M PA10.
[0067] D. Effect of Bcl-2 and PA-10 on Vpr52induced
.DELTA..PSI..sub.m dissipation in purified mitochondria. Isolated
mitochondria (200 .mu.g protein per ml) were pre-incubated with the
indicated inhibitors (5 .mu.M CsA, 50 .mu.M BA, 0.8 .mu.M Bcl-2, 2
.mu.M PA10; 5-10 min), washed (10 min, 6800 g, 20.degree. C.),
incubated with the .DELTA..PSI..sub.m-sensit- ive dye JC-1 (200 nM,
10 min), exposed to Vpr52-96 (3 .mu.M, 5 min), and subjected to
flow cytometric determination of the fluorescence (570-595 nm) and
the particle size (FSC). Numbers indicate the percentage of
JC-1.sup.high and JC-1.sup.low mitochondria among .about.10.sup.4
events.
[0068] E. Quantitation of the frequency of JC-1.sup.low
mitochondria (X.+-.SD, n=5) after incubation with different
Vpr-derived peptides. Purified mitochondria were preincubated 10
min with or without Bcl-2 (0.8 .mu.M), Bcl-2.DELTA..alpha..sub.5/6
(0.8 .mu.M) or BA (10 .mu.M) in PT buffer, incubated with the
.DELTA..PSI..sub.m-sensitive dye JC-1 (200 nM, 10 min), and then
treated 5 min with 3 .mu.M of Vpr52-96 (wt, biotinylated, or
modified as indicated), or 10 min. with 5-10 .mu.M of Vpr-1-96,
Vpr1-51, Vpr71-96, Vpr71-82 (wt or modified as indicated), and
finally subjected to flow cytometric analysis as in D.
[0069] FIG. 7 presents differential effect of Bcl-2 and PA-10 on
Vpr52-96 binding to mitochondria.
[0070] A. Vpr52-96 binds mitochondria before inducing
.DELTA..PSI..sub.m loss. Mitochondria were left unstained (insert
in Co.) or exposed to the .DELTA..PSI..sub.m-insensitive
mitochondrial dye MitoTracker.RTM. Green (75 nM), alone (MTG) or
with 0.5 .mu.M of FITC-Vpr52-96; green fluorescence), in
combination with the .DELTA..PSI..sub.m-sensitive mitochondrial dye
MitoTracker.RTM. Red (CMXRos; red Fluorescence) followed by
cytofluorometric two-color analysis. Numbers indicate the
percentage of mitochondria in each quadrant.
[0071] B. PA-10 but not Bcl-2 inhibit Vpr52-96 binding to
mitochondria. Mitochondria were pre-incubated 10 min. with the
indicated inhibitors and the percentage of FITC-Vpr52-96-labeled
mitochondria is determined as in A. C. inhibitory effect of Bcl-2
on affinity purification of ANT by biotinylated Vpr52-96.
Mitochondria were incubated with the indicated inhibitors, and then
exposed for 30 min at RT with 5 .mu.M biotinylated Vpr52-96.
Mitochondria were lysed either after incubation with biotinylated
Vpr52-96 (upper panel) or lysed before (lower panel) with Tris/HCl
as described in materials and methods. Biotinylated Vpr52-96
complexed with its mitochondrial ligands was retained on
avidin-agarose and subjected to immunoblot detection of ANT.
[0072] FIG. 8 presents Bcl-2-mediated inhibition of the Vpr-ANT
interaction.
[0073] A. Plasmon surface resonance determination of the
Bcl-2-mediated inhibition of interaction between Vpr52-96 and
native purified ANT. The interaction was measured after addition of
the indicated concentrations of recombinant Bcl-2,
Bcl-2.DELTA..alpha..sub.5/6, or recombinant Bid, and data (X.+-.SD,
n=3) were calculated as in FIG. 1.
[0074] B. Effect of Bcl-2 on Vpr binding to ANT proteoliposomes.
The retention (X.+-.SD, n=3) of FITC-labeled Vpr52-96 on ANT
proteoliposomes preincubated with 800 nM of Bcl-2 or 2 .mu.M PA10
was assessed as in FIG. 2A.
[0075] C. Effect of Bcl-2 on the formation of Vpr-ANT channels in
planar lipid bilayers. Single channel recordings (+75 mV) of
Vpr52-96+ANT+Bax (0.4:1:0.3 nM) and Vpr52-96+ANT+Bcl-2 (0.4:1:1 nM)
and corresponding amplitude histograms are displayed. Control
values for Vpr52-96+ANT alone are similar as in FIG. 1e (not
shown). c, closed; o, open.
[0076] FIG. 9 presents a model of the Vpr/PTPC interactions. Vpr
crosses the outer membrane through VDAC, which is inhibited by
Koenig's polyanion. Vpr then interacts with ANT. Bcl-2 and the ANT
ligand bongkrekate inhibit the binding of Vpr to ANT, whereas CsA
indirectly affects the pore forming function of ANT via its effect
on cyclophilin D (Cyp-D).
DESCRIPTION OF THE EMBODIMENTS
[0077] The invention relates to discovery that the proapoptotic
HIV-1-encoded protein Vpr induces mitochondrial membrane
permeabilization via its physical and functional interaction with
the mitochondrial inner membrane protein ANT (adenine nucleotide
translocator, also called ADP/ATP carrier). This is shown using a
variety of different techniques: surface plasmon resonance,
electrophysiology, synthetic proteoliposomes, studies on purified
mitochondria (respirometry, electron microscopy,
organellofluometry), as well as microinjection of intact cells. The
mode of action of Bcl-2 acts on ANT and to prevent Vpr-mediated
mitochondrial effects.
[0078] This invention relates to the discovery that Vpr primarily
affects IM and not OM permeability in vitro. Vpr binds ANT in an
ANT conformation-dependent fashion (FIGS. 1 and 2) and cooperates
with ANT to form channels (FIG. 3) which permeabilize IM before OM
becomes permeable to cytochrome c (FIGS. 4 and 5). Bcl-2
antagonizes this effect, based on two independent observations.
First, its mode of action clearly differs from that of the VDAC
inhibitor PA10 (FIGS. 6 and 7). Second, Bcl-2 can affect the
physical and functional ANT-Vpr interaction in a synthetic,
VDAC-free system (FIG. 8). Although these data do not exclude the
possibility that Bcl-2 and other members of Bcl-1 family modulate
the permeability of VDAC to relatively large, globular proteins
(14.5 kDa for cytochrome c, as opposed to the linear, mostly
.alpha. helical structure of Vpr52-96 resolved by NMR, W. Schuler,
et al., J. Mol. Biol. 285, 2105-2117 (1999)), they indicate that,
at least in this particular model, Bcl-2 exerts its
membrane-protective mitochondrial effect via ANT.
[0079] HIV-1 Viral protein R (Vpr) interacts with the permeability
transition pore complex (PTPC) to trigger mitochondrial membrane
permeabilizaton (MMP) and consequent apoptosis. Vpr binds to the
adenine nucleotide translocator (ANT), an inner mitochondrial
membrane protein. E. Jacotot, et al., J. Exp. Med. 191, 33-45
(2000). When Vpr binds to ANT, it cooperatively forms large
conductance channels in synthetic membranes. When added to purified
mitochondria, Vpr uncouples the respiratory chain and induces a
rapid inner MMP which precedes outer MMP to cytochrome c,
Vpr-induced matrix swelling and inner MMP to protons and NADH are
prevented by preincubation of purified mitochondria with
recombinant Bcl-2 protein. In contrast to Konig's polyanion, a
specific inhibitor of the voltage-dependent anion channel (VDAC),
Bcl-2 fails to prevent Vpr from crossing the outer mitochondrial
membrane. Bcl-2 reduces the ANT-Vpr interaction and abolishes
channel formation by the ANT-Vpr complex. Hence, both Vpr and Bcl-2
modulate MMP through a direct interaction with ANT.
[0080] Methods of Altering or Preventing Binding of Vpr to ANT
[0081] The discovery of the physical and functional interaction of
Vpr with ANT enables methods of altering or preventing binding of
Vpr to ANT. As illustrated in Examples 1-4, the interaction of Vpr
to ANT can be detected and modulated in a variety of different
assay systems. For example, Bcl-2 modulates the physical and
functional interaction of Vpr with ANT. Likewise, a peptide,
ANT104-116, corresponding to the overlap between the Bcl-2 binding
motif and the second ANT loop inhibits ANT-Vpr binding. Thus, these
molecules can be used to alter or prevent binding of Vpr to ANT.
Other peptidic or non-peptidic molecules can be designed to
similarly inhibit this binding.
[0082] The identification of Vpr-ANT binding allows the generation
of molecules that can modulate apoptosis. The methods presented in
the Examples, and other conventional techniques, can be adapted to
screen for Vpr, Bcl-2, or ANT variants, or other polypeptides or
molecules that affect Vpr-ANT binding. This allows for the
generation of molecules capable of enhancing or inhibiting Vpr-ANT
binding. The activity of these molecules can be assessed by
competitive binding assays. For example, molecules can be assessed
for there ability to inhibit ANT-Vpr binding using the binding
assays described in the Examples. The, skilled artisan understands
that many other techniques could similarly be used. The identified
molecules can be further assessed for apoptotic activity by
conventional techniques. Furthermore, based on the structure of Vpr
molecules determined to bind to ANT, structurally similar molecules
can be designed to mimic Vpr activity or to inhibit this
activity.
[0083] In one embodiment, soluble versions of Vpr or ANT
polypeptides can be incubated with cells to enhance or inhibit the
induction of apoptosis. In one embodiment these polypeptides
contain mutations that interfere with apoptosis. In another
embodiment, these polypeptides contain mutations that enhance
apoptosis. In one embodiment, these polypeptides are synthetic. In
another embodiment, these polypeptides are produced by recombinant
techniques.
[0084] Vpr and ANT polypeptides and peptides of greater than 9
amino acids that inhibit or augment Vpr-ANT binding, mitochondrial
membrane permeabilization, or apoptosis are an embodiment of the
invention, as well as peptides that are at least 10-20, 20-30,
30-50, 50-100, and 100-365 amino acids in size. DNA fragments
encoding these polypeptides and peptides are encompassed by the
invention.
[0085] Synthetic polypeptides and peptides can be generated by a
variety of conventional techniques. Such techniques include those
described in B. Merrifield, Methods Enzymol. 289:3-13, 1997, H.
Ball and P. Mascagni, Int. J. Pept. Protein Res. 48:31-47, 1996; F.
Molina et al., Pept. Res. 9:151-155, 1996; J. Fox, Mol. Biotechnol.
3:249-258, 1995; and P. Lepage et al., Anal. Biochem. 213: 40-48,
1993.
[0086] In another embodiment, peptides can be prepared by
subcloning a DNA sequence encoding a desired peptide sequence into
an expression vector for the production of the desired peptide. The
DNA sequence encoding the peptide is advantageously fused to a
sequence encoding a suitable leader or signal peptide.
Alternatively, the DNA fragment may be chemically synthesized using
conventional techniques. The DNA fragment can also be produced by
restriction endonuclease digestion of a done of, for example HIV-1,
DNA using known restriction enzymes (New England Biolabs 1997
Catalog, Stratagene 1997 Catalog, Promega 1997 Catalog) and
isolated by conventional means, such as by agarose gel
electrophoresis.
[0087] In another embodiment, the well known polymerase chain
reaction (PCR) procedure can be employed to isolate and amplify a
DNA sequence encoding the desired protein fragment.
Oligonucleotides that define the desired termini of the DNA
fragment are employed as 5' and 3' primers. The oligonucleotides
can contain recognition sites for restriction endonucleases, to
facilitate insertion of the amplified DNA fragments into an
expression vector. PCR techniques are described in Saiki et al.,
Science 239:487 (1988); Recombinant DNA Mythology, Wu et al., eds.,
Academic Press, Inc., San Diego (1989), pp. 189-196: and PCR
Protocols: A Guide to Methods and Applications, Innis et al., ed.,
Academic Press, Inc., (1990). It is understood of course that many
techniques could be used to prepare polypeptide and DNA fragments,
and that this embodiment in no way limits the scope of the
invention.
[0088] Screening Methods with Vpr and ANT
[0089] Vpr or ANT polypeptides can be assessed for their ability to
mediate apoptosis, as well as to block Vpr mediated apoptosis. For
example, fragments of Vpr can be assessed for their ability to
block native Vpr binding to ANT by conventional titration
experiments.
[0090] In one embodiment, surface plasmon resonance is used to
assess binding of Vpr to ANT as described herein. In another
embodiment, electrophysiology is used to assess binding of Vpr to
ANT as described herein. In another embodiment, purified
mitochondria are used to assess binding of Vpr to ANT as described
herein. In another embodiment, synthetic proteoliposomes are used
to assess binding of Vpr to ANT as described herein. In another
embodiment, microinjection of live cells is used to assess binding
of Vpr to ANT as described herein. It is understood of course that
many techniques could be used to assess binding of Vpr to ANT, and
that these embodiments in no way limit the scope of the
invention.
[0091] In another embodiment the yeast two-hybrid system developed
at SUNY (described in U.S. Pat. No. 5,283,173 to Fields et al.; J.
Luban and S. Goff., Curr Opin. Biotechnol. 6:59-64, 1995; R.
Brachmann and J. Boeke, Curr Opin. Biotechnol. 8:561-568, 1997; R.
Brent and R. Finley, Ann. Rev. Genet. 31:663-704, 1997; P. Bartel
and S. Fields, Methods Enzymol. 254:241-263, 1995) can be used to
screen for a inhibitors of the Vpr-ANT interaction as follows. Vpr,
or portions thereof responsible for interaction, can be fused to
the Gal4 DNA binding domain and introduced, together with an ANT
molecule fused to the Gal 4 transcriptional activation domain, into
a strain that depends on Gal4 activity for growth an plates lacking
histidine. Interaction of the Vpr polypeptide with an ANT molecule
allows growth of the yeast containing both molecules and allows
screening for the molecules that inhibit or alter this interaction
(i.e., by inhibiting or augmenting growth).
[0092] In an alternative embodiment a detectable marker (e.g.
.beta.-galactosidase) can be used to measure binding in a yeast
hybrid assay.
[0093] In addition, the identification of Vpr as an ANT-binding
molecule allows methods of detecting and quantifying ANT expression
in cells. For example, by contacting a labeled Vpr polypeptide with
a biological sample comprising ANT and detecting the Vpr-ANT
complex, the level of ANT can be determined.
[0094] Purified ANT polypeptides (including proteins, polypeptides,
fragments, variants, oligomers, and other forms) may be tested for
the ability to bind Vpr in any suitable assay, such as a
conventional binding assay. Similarly, Vpr polypeptides (including
proteins, polypeptides, fragments, variants, oligomers, and other
forms) may be tested for the ability to bind ANT. To illustrate,
the Vpr polypeptide may be labeled with a detectable reagent (e.g.,
a radionucleotide, chromophore, enzyme that catalyzes a
colorimetric or fluorometric reaction, and the like). The labeled
polypeptide is contacted with cells expressing ANT. The cells then
are washed to remove unbound labeled polypeptide, and the presence
of cell-bound label is determined by a suitable technique, chosen
according to the nature of the label.
[0095] Alternatively, the binding properties of Vpr polypeptides
and polypeptide fragments can be determined by analyzing the
binding of Vpr polypeptides and polypeptide fragments to
ANT-expressing cells by FACS analysis and/or immunofluorescence.
This allows the characterization of the binding of Vpr and ANT
polypeptides and polypeptide fragments, and the discrimination of
relative abilities of Vpr polypeptides and polypeptide fragments to
bind to ANT. In vitro binding assays with Vpr and ANT can similarly
be used to characterize Vpr-ANT binding activity.
[0096] Another type of suitable binding assay is a competitive
binding assay. To illustrate, biological activity of a variant may
be determined by assaying for the variant's ability to compete with
the native protein for binding to Vpr or ANT.
[0097] Competitive binding assays can be performed by conventional
methodology. Reagents that may be employed in competitive binding
assays include radiolabeled Vpr and intact cells expressing ANT
(endogenous or recombinant). For example, a radiolabeled Vpr
fragment can be used to compete with a soluble Vpr variant for
binding to ANT in cells, instead of intact cells, one could
substitute ANT protein bound to a solid phase.
[0098] Another type of competitive binding assay utilizes
radiolabeled Vpr and isolated mitochondria. Qualitative results can
be obtained by competitive autoradiographic plate binding assays,
while Scatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660,
1949) may be utilized to generate quantitative results.
[0099] Peptidic or Non-Peptidic Molecules that Affect Interaction
of Vpr to ANT or Mimic Its Capacity to Interact with ANT
[0100] Variants
[0101] The invention encompasses variants of Vpr or ANT that are
altered in their binding activity. Among the variant polypeptides
provided herein are variants of native polypeptides that retain the
native biological activity or the substantial equivalent thereof.
One example is a variant that binds with essentially the same
binding affinity as does the native form. Binding affinity can be
measured by conventional procedures, e.g., as described in U.S.
Pat. No. 5,512,457 and as set forth below.
[0102] Variants may also bind with increased affinity. In one
embodiment, a variant is an agonist of the native Vpr for ANT's
biological activity. In another embodiment, a variant is an
antagonist of the native Vpr for ANT's biological activity.
Agonistic or antagonistic activity can be readily determined by the
procedures described herein.
[0103] Variants include polypeptides that are substantially
homologous to the native form, but which have an amino acid
sequence different from that of the native form because of one or
more deletions, insertions or substitutions. Particular embodiments
include, but are not limited to, polypeptides that comprise from
one to ten deletions, insertions or substitutions of amino acid
residues, when compared to a native sequence.
[0104] A given amino acid may be replaced, for example, by a
residue having similar physiochemical characteristics. Examples of
such conservative substitutions include substitution of one
aliphatic residue for another, such as Ile, Val, Leu, or Ala for
one another, substitutions of one polar residue for another, such
as between Lys and Arg, Glu and Asp, or Gln and Asn; or
substitutions of one aromatic residue for another, such as Phe,
Trp, or Tyr for one another. Other conservative substitutions,
e.g., involving substitutions of entire regions having similar
hydrophobicity characteristics, are well known. Variants can be
generated using conventional techniques including random or
site-directed mutagenesis.
[0105] Antibodies
[0106] Within an aspect of the invention, Vpr and ANT polypeptides,
and peptides based on the amino acid sequence of Vpr and ANT, can
be utilized to prepare antibodies that specifically bind to Vpr and
ANT polypeptides. Antibodies that are immunoreactive with the
polypeptides of the invention are provided herein. In this aspect
of the invention, the polypeptides based on the amino acid sequence
of Vpr and ANT can be utilized to prepare antibodies that
specifically bind to Vpr and ANT. Such antibodies specifically bind
to the polypeptides via the antigen-binding sites of the antibody
(as opposed to non-specific binding). Thus, the polypeptides,
fragments, variants, fusion proteins, etc., as set forth above may
be employed as immunogens in producing antibodies immunoreactive
therewith. More specifically, the polypeptides, fragment, variants,
fusion proteins, etc. contain antigenic determinants or epitopes
that elicit the formation of antibodies.
[0107] These antigenic determinants or epitopes can be either
linear or conformational (discontinuous). Linear epitopes are
composed of a single section of amino acids of the polypeptide,
while conformational or discontinuous epitopes are composed of
amino acids sections from different regions of the polypeptide
chain that are brought into close proximity upon protein folding
(C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland
Publishing Inc., 2nd ed. 1996)). Because folded proteins have
complex surfaces, the number of epitopes available is quite
numerous; however, due to the conformation of the protein and
steric hinderances, the number of antibodies that actually bind to
the epitopes is less than the number of available epiopes (C. A
Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland
Publishing Inc., 2nd ed. 1996)). Epitopes may be identified by any
of the methods known in the art.
[0108] Thus, one aspect of the present invention relates to the
antigenic epitopes of the polypeptides of the invention. Such
epitopes are useful for raising antibodies, in particular
monoclonal antibodies, as described in detail below. Additionally,
epitopes from the polypeptides of the invention can be used as
research reagents, in assays, and to purify specific binding
antibodies from substances such as polyclonal sera or supernatants
from cultured hybridomas. Such epitopes or variants thereof can be
produced using techniques well known in the art such as solid-phase
synthesis, chemical or enzymatic cleavage of a polypeptide, or
using recombinant DNA technology.
[0109] As to the antibodies that can be elicited by the epitopes of
the polypeptides of the invention, whether the epitopes have been
isolated or remain part of the polypeptides, both polyclonal and
monoclonal antibodies may be prepared by conventional techniques as
described below.
[0110] The term "antibodies" is meant to include polyclonal
antibodies, monoclonal antibodies, fragments thereof, particular
antigen binding fragments such as F(ab')2 and Fab fragments, as
well as any recombinantly produced binding partners. Antibodies are
defined to be specifically binding if they bind with a K.sub.a of
greater than or equal to about 10.sup.7 M.sup.-1. Affinities of
binding partners or antibodies can be readily determined using
conventional techniques, for example those described by Scatchard
et al., Ann. N.Y Acad. Sci., 51:660 (1949).
[0111] Polyclonal antibodies can be readily generated from a
variety of sources, for example, horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, or rats, using procedures that are well
known in the art. In general, purified Vpr (or ANT) or a peptide
based on the amino acid sequence of Vpr (or ANT) that is
appropriately conjugated is administered to the host animal
typically through parenteral injection. The immunogenicity of Vpr
(or ANT) can be enhanced through the use of an adjuvant, for
example, Freund's complete or incomplete adjuvant. Following
booster immunizations, small samples of serum are collected and
tested for reactivity to Vpr or ANT polypeptide. Examples of
various assays useful for such determination include those
described in Antibodies: A Laboratory Manual, Harlow and Lane
(eds.) Cold Spring Harbor Laboratory Press, 1988; as well as
procedures, such as countercurrent immuno-electrophoresis (CIEP),
radioimmunoassay, radio-immunoprecipitation, enzyme-linked
immunosorbent assays (ELISA), dot blot assays, and sandwich assays.
See U.S. Pat. Nos. 4,376,110 and 4,486,530.
[0112] Monoclonal antibodies can be readily prepared using well
known procedures. See, for example, the procedures described in
U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993;
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.). 1980.
Briefly, the host animals, such as mice, are injected
intraperitoneally at least once and preferably at least twice at
about 3 week intervals with isolated and purified Vpr (or ANT),
conjugated Vpr (or ANT) peptide, optionally in the presence of
adjuvant. Mouse sera are then assayed by conventional dot blot
technique or antibody capture (ABC) to determine which animal is
best to fuse. Approximately two to three weeks later, the mice are
given an intravenous boost of Vpr (or ANT) or conjugated Vpr (or
ANT) peptide. Mice are later sacrificed and spleen cells fused with
commercially available myeloma cells, such as Ag8.653 (ATCC),
following established protocols. Briefly, the myeloma cells are
washed several times in media and fused to mouse spleen cells at a
ratio of about three spleen cells to one myeloma cell. The fusing
agent can be any suitable agent used in the art, for example,
polyethylene glycol (PEG). Fusion is plated out into plates
containing media that allows for the selective growth of the fused
cells. The fused cells can then be allowed to grow for
approximately eight days. Supernatants from resultant hybridomas
are collected and added to a plate that is first coated with goat
anti-mouse Ig. Following washes, a label, such as .sup.125I-Vpr (or
.sup.125I-ANT), is added to each well followed by incubation.
Positive wells can be subsequently detected by autoradiography.
Positive clones can be grown in bulk culture and supernatants are
subsequently purified over a Protein A column (Pharmacia).
[0113] The monoclonal antibodies of the invention can be produced
using alternative techniques, such as those described by
Alting-Mees et al., "Monoclonal Antibody Expression Libraries; A
Rapid Alternative to Hybridomas", Strategies in Molecular Biology
3:1-9 (1990), which is incorporated herein by reference. Similarly,
binding partners can be constructed using recombinant DNA
techniques to incorporate the variable regions of a gene that
encodes a specific binding antibody. Such a technique is described
in Larrick et at., Biotechnology, 7:394 (1989).
[0114] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., humanized versions of murine monoclonal
antibodies. Such humanized antibodies may be prepared by known
techniques, and offer the advantage of reduced immunogenicity when
the antibodies are administered to humans. In one embodiment, a
humanized monoclonal antibody comprises the variable region of a
murine antibody (or just the antigen binding site thereof) and a
constant region derived from a human antibody. Alternatively, a
humanized antibody fragment may comprise the antigen binding site
of a murine monoclonal antibody and a variable region fragment
(lacking the antigen-binding site) derived from a human antibody.
Procedures for the production of chimeric and further engineered
monoclonal antibodies include those described in Riechmann et al.
(Nature 332:323, 1988), Liu et al. (PNAS 84:34-39, 1987), Larrick
et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS
14:139, May, 1993). Procedures to generate antibodies
transgenically can be found in GB 2,272,440, U.S. Pat. Nos.
5,569,825 and 5,545,806 and related patents claiming priority
therefrom, all of which are incorporated by reference herein.
[0115] Once isolated and purified, the antibodies against Vpr and
ANT, and other Vpr and ANT binding proteins, can be used to detect
the presence of Vpr and ANT in a sample using established assay
protocols. Further the antibodies of the invention can be used
therapeutically to bind to Vpr or ANT and inhibit its activity in
vivo.
[0116] Antibodies directed against Vpr or ANT and other Vpr or ANT
binding proteins can be used to modulate the biological activity of
Vpr and ANT. One class of these antibodies produce mitochondrial
membrane permeabilization and apoptosis. In contrast, another class
of these antibodies can inhibit mitochondrial membrane
permeabilization and apoptosis.
[0117] Those antibodies that additionally can block Vpr-ANT binding
of may be used to inhibit a biological act that results from such
binding. Such blocking antibodies may be identified using any
suitable assay procedure, such as by testing antibodies for the
ability to inhibit binding of Vpr to ANT. Alternatively, blocking
antibodies may be identified in assays for the ability to inhibit a
biological effect that results from binding of Vpr to ANT in
cells.
[0118] Such an antibody may be employed in an in vitro procedure,
or administered in vivo to inhibit a biological activity mediated
by the entity that generated the antibody. Disorders caused or
exacerbated (directly or indirectly) by the interaction of Vrp with
ANT. A therapeutic method involves in vivo administration of a
blocking antibody to a mammal in an amount effective in inhibiting
ANT-mediated apoptosis. Monoclonal antibodies are generally
preferred for use in such therapeutic methods. In one embodiment,
an antigen-binding antibody fragment is employed.
[0119] Antibodies may be screened for agonistic (i.e.,
ligand-mimicking) properties. Such antibodies, upon binding to ANT,
induce biological effects (e.g., apoptosis) similar to the
biological effects induced when Vpr binds to ANT. Agonistic
antibodies may be used to induce ANT-mediated apoptosis of
cells.
[0120] Compositions comprising an antibody that is directed against
Vpr or ANT, and a physiologically acceptable diluent, excipient, or
carrier, are provided herein. Suitable components of such
compositions are as described above for compositions containing Vpr
or ANT.
[0121] Also provided herein are conjugates comprising a detectable
(e.g., diagnostic) or therapeutic agent, attached to the antibody.
Examples of such agents are presented above. The conjugates find
use in in vitro or in vivo procedures.
[0122] Other Molecules
[0123] The invention also encompasses molecules that compete for or
enhance the binding of Vpr to ANT, which can be identified through
the screening assays described herein or by structure-based design
using, for example, molecular modeling of Vpr-ANT binding.
[0124] Pharmaceutical and Diagnostic Compositions
[0125] Compositions of the present invention may contain a peptidic
or non-peptidic molecules in any form, such as native proteins,
variants, derivatives, oligomers, and biologically active
fragments. In particular embodiments, the composition comprises a
soluble polypeptide or an oligomer comprising soluble Vpr, Bcl-2,
or ANT polypeptides or fragments.
[0126] Compositions comprising an effective amount of a molecule of
the present invention, in combination with other components such as
a physiologically acceptable diluent carrier, or excipient, are
provided herein. The molecules can be formulated according to known
methods used to prepare pharmaceutically useful compositions. They
can be combined in admixture, either as the sole active material or
with other known active materials suitable for a given indication,
with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl,
acetate, and phosphate buffered solutions), preservatives (e.g.,
thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers,
adjuvants and/or carriers. Suitable formulations for pharmaceutical
compositions include those described in Remington's Pharmaceutical
Sciences, 16th ed. 1980, Mack Publishing Company, Easton, Pa.
[0127] In addition, such compositions can be complexed with
polyethylene glycol (PEG), meal ions, or incorporated into
polymeric compounds such as polyacetic acid, polyglycolic acid,
hydrogels, dextran, etc., or incorporated into liposomes,
microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts or spheroblasts. Such compositions will
influence the physical state, solubility, stability, rate of in
vivo release, and rate of in vivo clearance, and are thus chosen
according to the intended application.
[0128] The compositions of the invention can be administered in any
suitable manner, e.g., topically, parenterally, or by inhalation.
The term "parenteral" includes injection, e.g., by subcutaneous,
intravenous, or intramuscular routes, also including localized
administration, e.g., at a site of disease or injury. Sustained
release from implants is also contemplated. One skilled in the
pertinent art will recognize that suitable dosages will vary,
depending upon such factors as the nature of the disorder to be
treated, the patient's body weight, age, and general condition, and
the route of administration. Preliminary doses can be determined
according to animal tests, and the scaling of dosages for human
administration is performed according to art-accepted
practices.
[0129] Compositions comprising nucleic acids in physiologically
acceptable formulations are also contemplated. DNA may be
formulated for injection, for example.
[0130] Methods for Screening for ANT Alterations
[0131] The invention also provides methods for screening for
genetic or epigenetic alterations in the expression or structure of
the three ANT isoforms in humans.
[0132] The invention provides for diagnosis of diseases associated
with aberrant ANT expression. For example, a particular cancer may
have a specific modification of ANT associated with it. Diagnosis
of that cancer can be achieved by using Vpr or a fragment of Vpr
capable of retaining binding to ANT in a binding assay, for
example, as described herein. The expression or structure of the
three ANT isoforms in patients can thereby be determined and a
diagnosis achieved.
[0133] Methods for Specific Cell Killing
[0134] Vpr, or a biologically active fragment thereof such as
vpr52-96, can be used to induce apoptosis in cells. In one
embodiment, a vpr52-96 peptide is fused to molecule for targeting
to a specific cell type and induces apoptosis in that cell type. In
a further embodiment, a Vpr-targeting molecule conjugate
specifically kills cancer cells. The methodology can be similar to
the successful use of a recombinant chimeric protein containing
interleukin 2 (IL-2) protein fused to Bax to selectively kill IL-2
receptor-bearing cells in vitro R. Aqeilan, S. Yarkoni, and H.
Lorberbourn-Galski, FEBS Lett 457:271-6 (1999).
[0135] In other embodiments, biologically active Vpr-targeting
molecule conjugates can be used to specifically target and kill
other cell types involved in disease.
[0136] Double Test
[0137] To study ANT's role in apoptosis and, more specifically,
ANT's role in the permeabilization of mitochondrial membranes
(Brenner et al., Oncogene, 2000; Costantini at al., Oncogene,
2000), the inventors have developed a functional double test that
makes it possible to measure simultaneously the antiport (vital)
function and the pore (lethal) function of ANT in artificial lipid
double-layers or liposomes.
[0138] The principle of the functional double test is based on the
reconstitution of ANT in liposomes, the encapsulation of different
substrates (fluorescent substrate and ATP) in the interior of
proteoliposomes, the addition of enzymes and ADP to the exterior of
the liposomes, and then measurement by fluorescence of the salting
out of a substrate through the pore formed by ANT and, at the same
time, the measurement by luminescence of ATP translocated in
response to erogenous ADP. Any peptide or non-peptide compound can
be reconstituted with ANT during the formation of liposomes (e.g.,
Bax, Bcl-2, Bcl-x(L)), encapsulated in liposomes or added in an
external manner to proteoliposomes (e.g., addition of peptide
molecules [e.g., Vpr, Vpr52-96], Bid, etc.) or not (atractyloside,
calcium, t-BHP, diamide, BA. cyclosporine A., verteporfin, etc.) to
determine its impact on the two functions of ANT. Quantitative
measurements can be performed in a fixed point or kinetic manner.
This test is operation in 96 well microplates and can be adapted to
HTS (high throughput screening).
[0139] This functional double test enables the screening of
molecules that induce or inhibit apoptosis, of ANT partner
molecules capable of transforming or of facilitating the
ANT-to-pore transformation, or the diagnosis of particular
functional forms of ANT (alteration of vital and/or lethal
functions, alterations of the ratio of ANT isoforms). The antiport
function test makes it possible to evaluate the toxicity of
molecules vis--vis the vital function of ANT.
[0140] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification
which are hereby incorporated by reference. The embodiments within
the specification and the examples provide an illustration of
embodiments of the invention and should not be construed to limit
the scope of the invention. The skilled artisan readily recognizes
that many other embodiments are encompassed by the invention.
EXAMPLE 1
[0141] Physical and Functional Interaction between Vpr and ANT
[0142] Surface plasmon resonance measurements indicate that
purified detergent-solubilized ANT protein binds to the immobilized
Vpr C-terminal moiety biotin-Vpr52-96 (but to a far lesser extent
to mutated biotin-Vpr52-96[R73,80A]) with an affinity in the
nanomolar range (FIGS. 1A and B). This interaction is modulated by
two ANT ligands which differentially affect ANT conformation (M.
Klingenberg, J. Membrane Biol. 56, 97-105 (1980)), namely the PTPC
activator atractyloside (Atr). which favors Vpr binding, and the
PTPC inhibitor bongkrekic acid (BA), which reduces Vpr binding
(FIG. 1C). Vpr52-96 binding to membranes is greatly facilitated in
liposomes in which ANT has been reconstituted as compared to
protein-free liposomes (FIG. 2A). This ANT effect is inhibited by
BA (FIG. 2B). Vpr52-96 (but not the N-terminal moiety of Vpr
[Vpr1-51] nor mutated Vpr52-96. in which arginine 77 is replaced by
alanine, Vpr52-96[R77A]) also causes permeabilization of ANT
proteoliposomes (FIG. 2C), yet has no effect on plain
liposomes.
[0143] Bcl-2-like proteins bind to a motif of ANT (aa 105-155), I.
Marzo. et al., Science 281, 2027-2031 (1998), whose implication in
apoptosis control has been confirmed by deletion mapping of ANT. M.
K. A. Bauer. A Schubert O. Rocks, S. Grimm, J. Cell Biol. 147,
1493-1501 (1999). This motif partially overlaps with the second ANT
loop (aa 92-116), a regulatory domain exposed to the intermembrane
space. G. Brandolin, A Le-Saux, V. Trezeguet, G. J. Lauquinn, P. V.
Vignais. J. Bioenerg. Biomembr. 25, 493-501 (1993). M. Klingenberg.
J. Bioenerg. Biomembr. 25, 447-457 (1993). A peptide corresponding
to the overlap between the Bcl-2 binding motif and this loop
(ANT104-116) inhibited the ANT Vpr interaction (FIG. 1C),
presumably via direct association with Vpr52-96 (insert in FIG.
1D).
[0144] Neither a topologically-related peptide motif derived from
the human phosphate carrier nor mutated and scrambled versions of
ANT104-116 (control peptides in FIG. 1C) had such inhibitory
effects. ANT104-116 (but not the control peptides) also prevented
Vpr52-96-induced membrane permeabilizion of ANT proteoliposomes
(FIG. 2C), indicating that in the context of the lipid bilayer, me
effect of Vpr involved a direct interaction with ANT. In planar
lipid bilayers, low doses of Vpr52-96 (<1 nM) were incapable of
forming channels, unless ANT was present.
[0145] ANT and Vpr52-96 cooperated to form discrete channels whose
conductance (190.+-.2 pS) (FIG. 3) was much larger than those
formed by high doses (80 nM) of Vpr52-96 alone (55.+-.2 pS) (FIG. 3
and S. C. Piller, G. D. Ewart, A. Prekumar, G. B. Cox, P. W. Gage,
Proc. Natl. Acad. Sci. USA 93, 11-1-115 (1996)), yet was in the
range of those formed by Ca.sup.2+-treated ANT (N. Brustovetsky, M.
Klingenberg, Biochemistry 35, 8483-8488 (1996). C. Brenner. et al.,
Oncogene 19, 329-336 (2000). These biophysical experiments
demonstrate that ANT and Vpr directly interact in membranes to form
functionally competent channel-forming hetero(poly)mers.
EXAMPLE 2
[0146] Oxidative Properties of Purified Mitochondria Exposed to
Vpr.
[0147] As compared to untreated organelles (FIG. 4A trace a),
purified mitochondria preincubated with Vpr52-96 (FIG. 4A, trace b)
exhibited a gross deficiency in respiratory control (RC). Vpr
increased succinate oxidation preceding ADP addition and abolished
both the inhibitory effect of oligomycin (a specific ATPase
inhibitor) and the stimulatory effect of uncoupling by the
protonophore carbamoyl cyanide m-chlorophenylhydrazone (CCCP).
Thus, Vpr52-96 (but not Vpr1-51) reduced the RC (ratio of oxygen
consumption with oligomycin versus CCCP) to a value of 1.1, as
compared to 5.3 in control mitochondria (FIG. 4B). The entire Vpr
protein (Vpr1-96), and a short peptide corresponding to the minimum
"mitochondriotoxic" motif of Vpr (Vpr71-82), (L G. Macreadie, et
al., Proc. Natl. Acad. Sci. USA 92, 2770-2774 (1995). I. G.
Macreadie, et al., FEBS Lett. 410, 145-149 (1997); E. Jacotot, et
al., J. Exp. Med. 191, 33-45 (2000)) also reduced the RC values
(FIG. 4B) Noticeably, the Vpr-induced loss of RC was not associated
with a significant decrease of the oxidation rate (FIG. 4A),
suggesting that no major loss of membrane-bound cytochrome c
occurred upon short-term incubation with Vpr52-96. Accordingly,
adding cytochrome c to Vpr52-96-treated mitochondria oxidizing
succinate did not stimulate the rate of oxygen uptake (FIG. 4A,
trace b). The observation of Vpr-mediated uncoupling of the
respiratory chain prompted us to test its capacity to induce IM
permeabilization. The IM being essentially impermeable to NADH (P.
Rustin, et al., J. Biol. Chem. 271, 14785-14790 (1996), no
significant oxygen uptake could be measured when NADH was added to
control mitochondria (FIG. 5A, trace a). However, addition of
Vpr52-96 prompted a significant, NADH-stimulated oxygen consumption
(FIG. 5A, trace b). This indicates that Vpr permeabilized IM both
to protons (leading to uncoupling, FIG. 4A trace b) and to NADH
(FIG. 5A, trace b).
[0148] The differential kinetics of inner and outer MMP to NADH and
cytochrome c, respectively, were assessed FIG. 5B). NADH oxidation
by mitochondria added with Vpr52-96 was found maximal after 10 min
(FIG. 5B). Under similar conditions, Vpr52-96 only induced a
marginal access of cytochrome c to cytochrome c oxidase (FIG. 5B).
Moreover, the .DELTA..PSI..sub.m loss occurred well before
cytochrome c release can be detected by immunoblot (FIG. 5C).
Hence, Vpr52-96 causes inner MMP well before OM becomes permeable
to exogenous cytochrome c. Accordingly, at the ultrastructural
level (see below. FIG. 6C), Vpr52-96 treated mitochondria exhibited
matrix swelling before OM rupture became apparent.
EXAMPLE 3
[0149] Bcl-2-Mediated Inhibition of Vpr Effects on
Mitochondria.
[0150] Extracellular addition of Vpr to intact cells induced a
rapid .DELTA..PSI..sub.m loss, before nuclear condensation
occurred. These effects were prevented by microinjection of
recombinant Bcl-2 into the cytoplasm (FIG. 6A). Preincubation of
purified mitochondria with recombinant Bcl-2 (or the ANT ligand BA)
also prevented the Vpr-mediated inner MMP to NADH (FIG. 6B).
Concomitantly, both the Vpr-induced matrix swelling (FIG. 6C) and
.DELTA..PSI..sub.m loss (FIGS. 6D and E) were inhibited by Bcl-2
(but not by Bcl-2.DELTA..alpha.5/6, a deletion mutant lacking the
putative pore-forming .alpha.5 and .alpha.6 helices, S. Schendel,
M. Montal, J. C. Reed, Cell Death Differ. 5, 372-380 (1998)), by
two pharmacological inhibitors of the PTPC (BA and cyclosporin A;
CsA), as well as by the specific VDAC inhibitor Koenig's polyanion
(PA10; S. Stanley, J. A. Dias, D. D'Arcangelis, C. A. Mannella, J.
Biol. Chem. 270, 16694-16700 (1995)). Microinjected PA10 also
inhibits the effect of Vpr52-96 on intact cells (FIG. 6A). Binding
of Vpr52-96 to purified mitochondria was completely abolished by
pre-incubation of the organelles with PA10, partially reduced by
BA, but not affected by CsA (FIG. 7B). Thus, Vpr must access
mitochondria through VDAC.
[0151] Bcl-2 may be expected to prevent Vpr from crossing OM via
VDAC (based on the Bcl-2 mediated closure of VDAC) (S. Shimizu M.
Narita, Y. Tsujimoto, Nature 399, 483-487 (1999). S. Shimizu, A.
Konishi, T. Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA 97,
3100-3105 (2000)) and/or to inhibit the Vpr effect on ANT (based on
its physical and functional interaction wit ANT) (I. Marzo, et al.,
Science 281, 2027-2031 (1998); C. Brenner, et al., Oncogene 19,
329-336 (2000); M. Narita, et al., Proc., Natl. Acad. Sci. USA 95,
14681-14686 (1998)). Although recombinant Bcl-2 strongly reduced
the Vpr52-96-induced matrix swelling (FIG. 6C) and .DELTA..PSI.m
loss (FIGS. 6D and E), it failed to impair the binding of Vpr52-96
to purified mitochondria (FIG. 7B). The differential inhibitory
effects of PA10 and Bcl-2 on the Vpr-ANT interaction was confirmed
in a distinct experimental system. PA10 fully abolished the
affinity-mediated purification of ANT using biotinylated Vpr52-96
(FIG. 7C), provided that its effect was assessed on intact
mitochondria (in which Vpr52-96 has to cross OM to reach ANT). In
contrast, PA10 did not affect the Vpr52-96-mediated purification of
ANT from triton-solubilized mitochondria (in which ANT is readily
accessible to Vpr52-96). In the same conditions, Bcl-2 reduced the
Vpr52-96-mediated recovery of ANT, irrespective of its addition to
intact or solubilized mitochondria (FIG. 7C). Thus, Bcl-2 does not
interfere with the (PA10 inhibited) VDAC-mediated conduit allowing
Vpr52-96 to pass OM.
EXAMPLE 4
[0152] Bcl-2Mediated Inhibition of the Vpr-ANT Interaction.
[0153] A further series of experiments indicated that Bcl-2
modulated the physical and functional interaction of Vpr with ANT.
Recombinant Bcl-2 (but not Bcl-2 .DELTA..alpha.5/6) reduced
Vpr52-96 binding to soluble (FIG. 8A) or membrane-associated (FIG.
8B) ANT. Since Bcl-2 did not bind Vpr52-96, inhibition of the
Vpr/ANT binding is likely due to a direct Bcl-2/ANT interaction (I.
Marzo, et al., Science 281, 2027-2031 (1998); C. Brenner, et al.,
Oncogene 19, 329-336 (2000)). Accordingly, Bcl-2 abolished the
formation of Vpr52-96 induced channels in ANT-containing lipid
bilayers. In contrast, in the same conditions Bax exacerbates the
conductance of Vpr52-96-ANT channels to a mean value of 245.+-.2S
(as compared to 190.+-.2 for Vpr52-96-ANT without any further
addition) (FIG. 8C).
EXAMPLE 5
[0154] Double Test Protocol
[0155] 1. Purification of ANT. ANT is purified from rat heart and
reconstituted in liposomes according to the protocol described by
Brenner et al., Oncogene, 2000.
[0156] 2. Reconstitution of ANT in liposomes. ANT (0.03 mg/ml) is
incorporated in liposomes composed of phosphatidylcholine and
cardiolipine (PC:CL; 45:1; W: w; and 300 ng ANT per mg of lipids)
in the presence of 0.3% n-octyl-.beta.-D-pyranoside for 2 min. at
room temperature. If need be, other proteins or compounds are added
at this stage during incorporation (e.g., Bcl-2, Bax). Then, the
detergent is eliminated by dialyzing, overnight, the liposomes
against the buffer 10 mM HEPES, 125 mM sucrose, pH7.4 at 4.degree.
C. (e.g.: about 1 l of buffer per 1 ml liposomes).
[0157] 3. Pore function test. The liposomes are charged with 1 mM
of 4-UMP (4methylumbelliferylphosphate) in 10 mM KCl by sonication
(50 W, 22 sec). The proteoliposomes are then separated on a
Sephadex G25 column to eliminate the non-encapsulated compounds,
the elution being performed using the buffer 10 mM HEPES, 125 mM
sucrose, pH7.4 at room temperature. In a microplate with 96 wells.
25 .mu.l of liposomes are put in each well.
[0158] 25 .mu.l of product to be tested are added and incubated for
30-60 min. with different compounds (e.g.: 30 min. for Vpr 52-96;
60 min. for a non-peptide compound) at room temperature. Then,
alkaline phosphatase (5 U/ml) and 150 .mu.l of the reaction buffer
10 mM HEPES, 125 mM sucrose, 0.5 mM MgCl2, pH 7.4 are added. The
plate is incubated for 15 min. while being shaken at 37.degree. C.
to allow the enzymatic conversion of 4-UMP to 4-umbelliferone
(4-UM). The reaction is stopped by adding 150 .mu.l Stop buffer (10
mM HEPES-NaOH, 200 mM EDTA, pH 10). The fluorometric determination
of 4-UM is performed (excitation: 365 nm; emission 450+-5 nm). In
each experiment, samples of non-treatment of liposomes, of
encapsulation of 4-UMP, and of maximum salting out of 4-UMP are
created and permit the results to be expressed as a percentage of
salted 4-UMP.
[0159] 4 Antiport function test. The liposomes are charged with 1
mM of ATP (4-methylumbelliferylphosphate) in some 10 mM KCl by
sonication (50 W, 22 sec). The proteoliposomes are then separated
on a Sephadex G25 column to eliminate the non-encapsulated
compounds, with elution being performed with some 10 mM HEPES
buffer solution, 125 mM sucrose, pH7.4 at room temperature. In a
microplate with 96 wells, 25 .mu.l of liposomes are put in each
well.
[0160] 25 .mu.l of product to be tested are added and incubated for
30-60 min. (e.g.: 30 min. for Vpr 52-96; 60 min for a non-peptide
compound) at room temperature. Then, 25 .mu.l luciferase (HS II
Boerhinger kit) are added, and the emitted light is immediately
measured. The results are expressed as a percentage by comparison
to the maximum ATP exported in response to an addition of 400 .mu.l
the exterior of the liposomes.
[0161] 5. Note: Proteoliposomes charged with 4-UMP and KCl with the
objective of determining the pore function will freeze at
-20.degree. C. but those charged with ATP and KCl to determine the
translocator function will not.
References
[0162] The following references are specifically incorporated by
reference in their entirety.
[0163] Brenner C, Cadiou H, Vieira H L, Zamzami N. Marzo I, Xie Z,
Leber B. Andrews D, Duclohier H, Reed J C, Kroemer G (2000) Bcl-2
and Bax regulate the channel activity of the mitochondrial adenine
nucleotide translocator. Oncogene, 19:329-36.
[0164] Costantini P, Belzacq A S, Vieira H L, Larochette N, de
Pablo M A, Zamzami N. Susin S A, Brenner C, Kroemer G (2000). The
critical oxidation of a thiol residue of the adenine nucleotide
translocator triggers the opening of a Bcl-2 independent
permeability transition pore and apoptosis. Oncogene. 19:
307-14.
[0165] Zamzami N, E I Hamel C, Maisse C, Brenner C, Munoz-Pinedo C,
Belzacq A S, Costantini P, Vieira H, Loeffler M, Molle G, Kroemer G
(2000) Bid acts on the permeability transition pore complex to
induce apoptosis. Oncogene, 19(54):6342-50.
[0166] Jacotot E, Ferri K F, E I Hamel C. Brenner C, Druillennec S,
Hoebecke J, Rustin P, Metivier D, Lenoir C, Geukens M, Vieira H L,
Loeffler M, Belzacq A S, Briand J P, Zamzami N, Edelman L, Xie Z H,
Reed J C, Roques B P, Kroemer G (2001) Control of mitochondrial
membrane permeabilization by adenine nucleotide translocator
interacting with HIV-1 viral protein rR and Bcl-2. J Exp Med,
193(4):509-19.
[0167] Belzacq A S, Vieira H L, Xie Z H, Reed J C, Kroemer G,
Brenner C. ANT as a vital antiporter and a lethal pore. Regulation
by Bcl-2 like proteins. In preparation
[0168] Belzacq A S, Dallaporta B., E I Hamel C., Vieira H L,
Marchetti P., Reed J C, Kroemer G, Brenner C. Three
chemotherapeutic agents act on ANT to permeabilize mitochondrial
membranes during apoptosis. In preparation
[0169] Ferri K F, Jacotot E, Blanco J, Este J A, Kroemer G (2000).
Mitochondrial control of cell death induced by HIV-1-encoded
proteins. Ann N Y Acad Sci. 926:149-64.
[0170] Marchetti P, Zamzami N, Joseph B, Schraen-Maschke S,
Mereau-Richard C, Costantini P, Metivier D, Susin S A, Kroemer G,
Formstecher P (1999). The novel retinoid
6-[3-(1-adamantyl)-4-hydrophenyl]-2-naphthalene carboxylic acid can
trigger apoptosis through a mitochondrial pathway independent of
the nucleus. Cancer Res. December 15; 59(24):6257-66.
[0171] Larochette N, Decaudin D, Jacotot E, Brenner C, Marzo I,
Susin S A, Zamzami N, Xie Z, Reed J, Kroemer G (1999). Arsenite
induces apoptosis via a direct effect on the mitochondrial
permeability transition pore. Exp Cell Res. June
15;249(2):413-21.
[0172] Ravagnan L, Marzo I, Costantini P, Susin S A, Zamzami N,
Petit P X, Hirsch F, Goulbern M, Poupon M F, Miccoli L, Xie Z, Reed
J C, Kroemer G (1999). Lonidamine triggers apoptosis via a direct,
Bcl-2-inhibited effect on the mitochondrial permeability transition
pore. Oncogene. April 22;18(16):2537-46.
[0173] Fulda S, Scaffidi C, Susin S A, Krammer P H, Kroemer G,
Peter M E, Debatin K M (1998). Activation of mitochondria and
release of mitochondrial apoptogenic factors by betulinic acid. J.
Biol. Chem. December 18;273(51):33942-8.
[0174] Belzacq A-S, Jacotot E, Vieria H L A, Mistro D, Granville D
J, Xie Z, Reed J C, Kroemer G, Brenner C (2001). Apoptosis
induction by the photosensitizer verteporfin: identification of
mitochondrial adenine nucleotide translocator as a critical target
Cancer Research. February 15;61:1260-1264.
[0175] Vieira H L A Haouzi D, E I Hamel C, Jacotot E, Belzacq A-S,
Brenner C, Kroemer G (2000). Permeabilization of the mitochondrial
inner membrance during apoptosis; impact of the adenine nucleotide
translocator. Cell Death and Differentiation. 7:1146-1154.
[0176] Vieira H L, Belzacq A S, Haouzi D, Bernassola F, Cohen I,
Jacotot E, Ferri K F, E I Hamel C, Bartle L M, Melino G, Brenner C,
Goldmacher V, Kroemer G (2001). The adenine nucleotide
translocator: a target of nitric oxide, peroxynitriate, and
4-hydroxynonenal. Oncogene. July 19;20(32):4305-16.
Sequence CWU 1
1
7 1 14 PRT Artificial Sequence Mitochondrial membrane
permeabilizing peptide 1 Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala
Lys Leu Ala Lys 1 5 10 2 12 PRT Artificial Sequence HIV-1 Vpr
peptide 2 His Phe Arg Ile Gly Cys Arg His Ser Arg Ile Gly 1 5 10 3
13 PRT Artificial Sequence ANT-1 peptide 3 Asp Arg His Lys Gln Phe
Trp Arg Tyr Phe Ala Gly Asn 1 5 10 4 13 PRT Artificial Sequence
ANT-2-derived peptide 4 Asp Lys Arg Thr Gln Phe Trp Arg Tyr Phe Ala
Gly Asn 1 5 10 5 13 PRT Artificial Sequence Scrambled ANT-1 peptide
5 Phe Gln Asn Tyr Trp Gly His Lys Arg Phe Arg Asp Ala 1 5 10 6 13
PRT Artificial Sequence Mutated ANT peptide 6 Asp Gly His Lys Gln
Phe Trp Gly Tyr Phe Ala Gly Asn 1 5 10 7 13 PRT Artificial Sequence
Human phosphate carrier protein peptide 7 Ser Asn Met Leu Gly Glu
Glu Asn Thr Tyr Leu Trp Arg 1 5 10
* * * * *