U.S. patent application number 12/352016 was filed with the patent office on 2010-01-14 for peptides binding the phosphatase 2a protein.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Xavier Cayla, Frederic Dessauge, Alphonse Garcia, Angelita Rebollo.
Application Number | 20100009909 12/352016 |
Document ID | / |
Family ID | 32097367 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100009909 |
Kind Code |
A1 |
Garcia; Alphonse ; et
al. |
January 14, 2010 |
PEPTIDES BINDING THE PHOSPHATASE 2A PROTEIN
Abstract
The invention relates to novel synthetic or natural E4orf4 or
Bcl-2 peptides particularly useful in antitumoral, antiviral and
antiparasitic treatments, said peptides being less than 30 amino
acids long and binding in vitro to a phosphatase 2A protein
holoenzyme or one of its subunits. The invention also relates to
polynucleotides encoding the novel peptides, vectors expressing
same, as well as antibodies identifying same and probes identifying
transcripts thereof.
Inventors: |
Garcia; Alphonse; (Paris,
FR) ; Dessauge; Frederic; (Paris, FR) ; Cayla;
Xavier; (Rochecorbon, FR) ; Rebollo; Angelita;
(Paris, FR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
Paris
FR
|
Family ID: |
32097367 |
Appl. No.: |
12/352016 |
Filed: |
January 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11107814 |
Apr 18, 2005 |
7498306 |
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12352016 |
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PCT/FR03/03018 |
Oct 13, 2003 |
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11107814 |
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Current U.S.
Class: |
514/1.1 ;
435/320.1; 530/324 |
Current CPC
Class: |
A61P 33/00 20180101;
A61P 31/12 20180101; A61P 35/00 20180101; C07K 14/4747
20130101 |
Class at
Publication: |
514/12 ; 530/324;
435/320.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; C12N 15/74 20060101
C12N015/74; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 16, 2002 |
CA |
2,408,207 |
Claims
1-23. (canceled)
24. An isolated peptide that is at least 90% identical with a
complete amino acid sequence of TABLE-US-00006 VKKKKIKREIKI SMLE
SPMEFLFDTI DDVT. (SEQ. ID NO: 6) AS.
25. A composition comprising the isolated peptide of claim 24 and a
pharmaceutically acceptable carrier.
26. A composition comprising the isolated peptide of claim 24
coupled to a vector which can transfer said peptide into a
eukaryotic cell.
27. The isolated peptide of claim 24, wherein the peptide
specifically binds a phosphatase 2A protein holoenzyme or one of
its subunits in vitro.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Ser. No.
11/107,814 filed Apr. 18, 2005, allowed, which is a continuation of
PCT/FR03/03018 filed Oct. 13, 2003 and claims the benefit of
Canadian application 2,408,207 filed Oct. 16, 2002.
FIELD OF THE INVENTION
[0002] The present invention concerns peptides binding a
phosphatase 2A protein, an important target for the control of
apoptosis, in particular in cancer cells, as well as for the
control of viral and parasitic infections.
BACKGROUND OF THE INVENTION
[0003] Given the role of the peptides of the invention in
modulating the activity of the cellular protein phosphatase 2A, it
is important to mention the present state of the art on protein
phosphatases 2A, their physiological role and their interactions
with some cellular, viral or parasitic proteins.
[0004] Cell physiology is controlled in part by modulation of the
phosphorylation state of proteins. The phosphorylation state of
cellular proteins depends on the antagonistic action of the protein
kinases that phosphorylate them and the protein phosphatases that
dephosphorylate them.
[0005] Protein phosphatases are divided into two main groups:
tyrosine phosphatases and serine/threonine phosphatases.
Serine/threonine phosphatases are classified into two categories
depending on the specificity of their substrate and their
sensitivity to certain inhibitors, the type 1 and type 2
phosphatases (PP1 and PP2). The type 2 phosphatases are again
divided into different classes, including phosphatase 2A (PP2A),
phosphatase 2B (PP2B) or calcineurin which activity is regulated by
calcium, and phosphatase 2C (PP2C) which activity is
magnesium-dependent.
[0006] In vivo, the serine/threonine protein phosphatases PP1 and
PP2A form two families of many ubiquitously expressed holoenzymes.
These holoenzymes are produced by the specific interaction between
their catalytic subunits (PP1c and PP2Ac) and a wide variety of
regulatory subunits. Moreover, these holoenzymes are involved in
targeting and/or regulation of phosphatase activity (for recent
review, see Garcia A. et al., PP1 et PP2A, des ser/thr phosphatases
au coeur de l'apoptose (2001) Med/Sci 17, 1214-1216).
[0007] It is now known that type 2A phosphatases are very conserved
throughout evolution and are potentially activated during
regulation of various biological processes. The PP2A enzymes have
clearly been involved in transcription control, cell cycle control,
and viral transformation. Moreover, the PP2As are the targets of
different viral or parasitic proteins, thus suggesting a role for
PP2As in host-pathogen interactions.
[0008] The PP2As are oligomeric complexes (holoenzymes), each of
said complexes comprising a catalytic subunit C and one or two
regulatory subunits, (A) and (B). The structure of subunit (A)
consists of 15 imperfect repeats of a conserved 38 to 40-amino acid
sequence, some subunits (A) interacting with subunits (B) and (C).
Subunits (A) and (C), conserved throughout evolution, form the base
structure of the enzyme and are constitutively expressed. By
contrast, subunits (B) form a family of regulatory proteins
differentially expressed and with no common structure between one
another (Cohen P. The structure and regulation of protein
phosphatases. Annu. Rev. Biochem. 1989; 58:453-508). Therefore, the
protein phosphatases 2A are present in vivo under two different
forms: a dimeric form (AC) and a trimeric form (ABC). Subunits (B)
regulate the phosphatase activity and the specificity towards the
substrate. The existence of multiple forms of PP2A correlates with
distinct and varying functions of the PP2As in vivo.
[0009] Recently, it has been found that different non cellular
proteins, and in particular viral and parasitic proteins, are
involved in the modulation of some specific activities of protein
phosphatases 2A.
[0010] Different strategies involving the PP2A are adopted by the
viruses to facilitate their replication and survival in the host
cell. For example, the parainfluenza virus incorporates, in its
viral particle the PKC protein, a protein of cellular origin under
the control of the PP2A. This virus can thus perturb the host
proteins' phosphorylation and facilitate its own replication (B. P.
Gupta et al. Cellular protein kinase C .zeta. regulates human
parainfluenza virus type 3 replication. Proc. Natl. Acad. Sci. USA
1995; 92:5204-8).
[0011] Many DNA viruses with a transforming potential, such as
papovaviridae or adenoviruses, as well as some retroviruses, such
as the type 1 human immuno-deficiency virus (HIV-1), code for
proteins that interact directly with some host PP2As. All these
viruses include proteins that, even though they are structurally
different from one another, interact with some holoenzymes and
modify the phosphatase activity thereof.
[0012] More particularly, the E4orf4 protein of adenoviruses binds
to a heterotrimeric PP2A and, more precisely, to a regulatory
subunit (B), thus leading to a decrease in transcription of JunB in
the infected cell. This effect could play an important role during
viral infection by regulating the apoptotic response in infected
cells. Interestingly, the interaction between E4orf4 and PP2A
induces apoptosis of transformed cells in a p53-independent manner
(Shtrichman R. et al. Adenovirus type 5 E4 open reading frame 4
protein induces apoptosis in transformed cells. J. Virol. 1998;
72:2975-82).
[0013] The oncogenic DNA viruses of the Papovaridae family,
including SV40 and the polyoma virus, induce cell transformation.
PP2A interacts with the "small T" antigens of SV40 and the "small
T" and "middle T" of the polyoma virus. The interactions between
these viral proteins and the PP2A are clearly involved in viral
transformation. Finally, the transcriptional control, a process
normally led in the cell by the different factors which
specifically bind to promoter regulatory sequences represents
probably the most important mechanism in viral expression control
by PP2A. Therefore, PP2A is a negative regulator of numerous
transcription factors, namely involved in the cell growth and
proliferation processes, including APl/SRE, NF-kB, Spl and CREB
(Waszinski, B. E. et al. Nuclear protein phosphatase 2A
dephosphorylates protein kinase A-phosphorylated CREB and regulates
CREB Transcriptional stimulation. Mol. Cell Biol. 1993:13,
2822-34). The viral control of these transcription factors could
allow to modulate viral transcription.
[0014] Vpr, the viral protein of HIV-1, interacts in vitro with
PP2A and stimulates the catalytic activity thereof (Tung L, et al.
Direct activation of protein phosphatase 2A0 by HIV-1 encoded
protein complex Ncp7:vpr. FEBS Lett 1997; 401: 1997-201). Vpr can
induce a G2 arrest in infected cells by inhibiting the activation
of the p34cdc2-cyclin B complex. In addition, Vpr interacts with
the Spl transcription factor and is a weak trans-activator of the
Spl-dependent transcription of HIV-1. Therefore, the Vpr protein of
HIV-1, which is incorporated within the virion, would be involved
in vivo in the initiation of viral transcription, an essential step
in regulating the expression of the Tat transcription factor (a
major regulator in the transcription coded by the HIV-1 virus).
[0015] Unlike the protein kinases that have a well-established role
in parasitic infections, serine/threonine phosphatases have only
recently been recognized as potentially important regulators in
parasitology.
[0016] The absence of common motifs for the bulk of proteins that
interact with PP2A impedes the simple identification by
bio-informatics of peptide motifs directly involved in the binding
of these proteins with PP2A.
[0017] However, given the major role of protein phosphatases 2A in
virus-host or parasite-host interactions as detailed hereinabove,
it will be understood that there is a great interest in identifying
the binding sites of viral or parasitic proteins with PP2A
holoenzymes or one of their subunits, to identify new therapeutic
targets for these viral or parasitic pathogens.
[0018] The type 1 and type 2A serine/threonine phosphatases (PP1
and PP2A) represent new potentially important targets for apoptotic
control, namely in cancer cells, as well as for the control of
viral or parasitic infections (for review, see A. Garcia et al.
(2000) Protein Phosphatase 2A: a definite player in Viral and
parasitic regulation. Microbes Inf. 2,401-407; Et X. Cayla et al.
(2000). La Proteine Phosphatase 2A: une nouvelle piste pour l'etude
des virus et des parasites. Med/Sci 16, 122-127). More
particularly, PP1/PP2A would play a crucial role in the regulation
of anti-apoptotic Bcl-2 proteins and cell survival (Garcia A et
al., PP1 et PP2A des ser/thr phosphatases au coeurde l'apoptose
(2001). Med/Sci 17, 1214-1416; Ayllon, V. et al. (2000). Protein
phosphatase 1--is a Ras-activated Bad phosphatase that regulates
IL-2 deprivation-induced apoptosis. EMBO J. 19, 2237-2246, Ayllon,
V. et al. (2000) Bcl-2 targets protein phosphatase 1 alpha to Bad.
J. Immunol. 15; 166:7345-7352). Identification of peptides that
interact with PP2A could help to produce new drugs susceptible to
block, by competitive inhibition, cellular mechanisms induced by
viral or parasitic proteins, by their interaction with PP2A and, in
particular, the mechanisms of infection, pathogen proliferation and
neoplastic cell transformation.
[0019] In particular, PP2A activation after interaction with the
E4orf4 adenoviral protein induces apoptosis in transformed cells
(Shtrichman R, et al. (2000) Oncogene. 19, 3757-3765). This
specific effect requires an interaction with the B alpha (B.alpha.)
subunit of PP2A (Marcellus et al. J Virol. (2000)
74:7869-7877)/Goedert et al. J. Neurochem (2000) 75,2155-2162)).
All of the above-mentioned observations suggest the hypothesis that
the interaction of peptides mimicking the ABC1 and/or A3 site with
PP2A could lead to apoptosis of transformed cells.
[0020] WO9801563 and WO0104329 A1 describe the human E4orf4 protein
and its role in induction of apoptosis in tumour cells,
particularly when this protein is expressed via an adenoviral
vector. WO0104629 A1 relates to the modulating and mimetic
polypeptides E4orf4 and PP2A, capable to induce a selective cell
death. This document discloses an invention which relates to the
ability of the E4orf4 adenoviral protein to induce the death of
neoplastic cells but not of non neoplastic cells. Moreover,
WO9801563 A2 relates to the E4orf4 and E4orf6 adenoviral proteins,
destined to induce cell death. Finally, the patent application No.
FR 0110139 describes peptidic compounds that can bind to PP2A.
[0021] Also, the Bcl-2 protein family which, in mammals, comprises
about twenty members, can be divided into three sub-families
including: [0022] the anti-apoptotic members (of the Bcl-2 type
itself) which all present at least four conserved motifs, called
"BH1 to BH4" for "Bcl-2 Homology domain", are necessary to the
function of cell survival. The BH4 motif comprises the interaction
domain with the Raf and Apaf-1 proteins, and with calcineurine;
[0023] the pro-apoptotic members of the Bax type do not present a
BH4 domain; and [0024] the pro-apoptotic members of the Bad type
only present one BH3 domain.
[0025] Mutagenesis experiments show that the anti-apoptotic
activity of Bcl-2 requires its phosphorylation at the specific
residue serine 70 that, when replaced by alanine, inhibits
survival. Moreover, the work from Dr. May's team (USA) (2001, Vol.
15, No. 4, pp 515-522) suggests that, in the presence of IL-3, PP2A
can transitorily associate itself with Bcl-2. The use of a point
mutant (wherein one alanine residue replaces the serine 70)
indicates that the binding of PP2A to Bcl-2 requires the presence
of serine 70 which, consequently, could belong to the binding site.
The authors thus suggest a dynamic regulatory mechanism whereby
PP2A would be a Bcl-2 phosphatase, antagonistic to Bcl-2 kinases,
for example the PKCs (for discussion, see Garcia A. et al., PP1 et
PP2A des ser/thr phosphatases au Coerde I'apoptaose (2001). Med/Sci
17, 1214-1216).
[0026] The presence of these Bcl-2 peptides inside the cell could
thus regulate the phosphorylation, consequently the activity, of
Bcl-2 which in turn could block the development of Bcl-2-dependent
tumours.
[0027] There is thus a need, at the level of antitumour, antiviral
and antiparasitic treatments, for peptides derived from E4orf4 and
Bcl-2 sequences which bind to PP2A or one of its subunits.
SUMMARY OF THE INVENTION
[0028] The present invention takes an interest in E4orf4 and Bcl-2
peptides of small dimension that bind a PP2A holoenzyme or one of
its sub-units. Contrary to E4orf4 and Bcl-2 native proteins or
polypeptidic domains of greater dimensions, peptides of small
dimensions have the advantage of being easily synthesized, by
chemical way or in cellular systems, with a high yield and a
reduced cost. Peptides of the invention present, moreover, the
advantage of being more accessible and of being more easily
transferred in the cytoplasm or in the nucleus of the cells using
appropriate vectors, for a therapeutic use.
[0029] The invention follows from the identification of E4orf4 and
Bcl-2 peptides, which have a dimension less than 30 amino acids,
and especially peptides having a dimension less than 20 amino
acids, interacting in vitro with a purified phosphatase 2A protein
holoenzyme or one of its subunits.
[0030] In particular, the inventors have identified by the "SPOT
synthesis" technique described by Frank and Overwing (Methods in
Molecular Biology, 1996, vol. 66: 149-169, Epitope Mapping
Protocols, G.E. Morris Humana Press Inc., Totowa, N.J.) the binding
sites of the E4orf4 (canine adenovirus type 2) and Bcl-2 proteins
interacting with a PP2A holoenzyme or one of its subunits.
[0031] On the one hand, the peptides of the invention are peptides
having a dimension less than 30 amino acids, interacting in vitro
with a purified PP2A holoenzyme or one of its subunits, said
peptides being derived from the E4orf4 protein (canine adenovirus
type 2) and Bcl-2 protein. Antagonists derived from these peptides
and selected because they inhibit the interaction of viral or
parasitic proteins with a specific PP2A holoenzyme could thus
constitute new antitumor, antiviral or antiparasitic agents
[0032] On the other hand, the invention also concerns a peptide
comprising a 12 amino acid sequence of the Theileria parva
Ck.alpha. 2 protein (FD6) capable of interacting with the PP2A
sub-unit A and to penetrate in a Hela cell (Garcia, A. et al.
(2000). The invention is further concerned with the sequence
corresponding to the interaction site of the PP2A with the Ba
sub-unit of the PP2A 1-B site (cf. table 1). This peptide could
penetrate in cells and, like the E4orf4 protein of the human
adenovirus, could interact with PP2A and provoke apoptosis in
cancer cells without affecting the normal cells.
[0033] The identification method described in application FR no.
0110139 filed on Jul. 27, 2001 in the name of the Applicant,
comprises the following steps which consist of: [0034] a) spotting,
on a support, E4orf4 or Bcl-2 peptides which there sequence derived
respectively from the viral E4orf4 protein or the cellular Bcl-2
protein, each spot corresponding to a deposit of a peptide with a
defined sequence, [0035] b) contacting the solid support with a
solution containing a phosphatase 2A holoenzyme or one of its
subunits under conditions allowing said peptides present onto the
support, to bind the holoenzyme or one of its subunits, and, [0036]
c) identifying on the solid support the E4orf4 peptide or the Bcl-2
peptide onto which said phosphatase 2A holoenzyme or one of its
subunits is bound.
[0037] According to step a), different peptides are spotted on a
support at defined positions (spot), each position corresponding to
a specific peptide sequence and the whole then forming a two
dimension peptide array.
[0038] Different preparation methods of such arrays have been
recently described (for review, see Figeys et Pinto, 2001
Electrophoresis 22: 208-216; and Walter et al., 2000 Curr Opin
Microbiol 3: 298-302). These methods comprise in general the
covalent binding of peptides on a support, in particular with the
aid of chemical linkers. For instance, one skilled in the art could
refer to the "SPOT synthesis" technique which consists of directly
synthesize on a cellulosic membrane, peptides comprising up to 20
residues (Frank and Overwing, Methods in Molecular Biology, 1996,
vol. 66: 149-169, Epitope Mapping Protocols, G.E. Morris Humana
Press Inc., Totowa, N.J.).
[0039] Generally any method could be used from the moment that such
a method allows the production of such a peptide array, E4orf4 or
Bcl-2, spotted on a solid support, useful for detecting specific
interaction between spotted peptides and the PP2A holoenzyme or one
of its subunits.
[0040] The totality of the spotted E4orf4 and Bcl-2 peptide
sequences cover the complete sequence of the corresponding viral or
cellular protein. Therefore, the process allows to test in a single
step the complete sequence, the latter being <<split>>
into a number of defined peptides, generally of overlapping
sequences.
[0041] The spotted peptides have a dimension less than 20 amino
acids, and preferably, less than 15 amino acids.
[0042] The peptides may also be spotted on a cellulosic
membrane.
[0043] The array thus obtained is contacted at step b), with a
phosphatase 2A protein holoenzyme or one of its subunits.
[0044] By "phosphatase 2A protein holoenzyme", it is meant any
dimeric (AC) or heterotrimeric (ABC) complex, purified from a
cellular extract or reconstituted following purification of the two
subunits (A) and (C) of a phosphatase type (2A) protein or, if
necessary, from the subunit (B), the phosphatases type (2A)
proteins are preferably derived from mammals.
[0045] The supports are incubated, for instance, in a buffered
solution comprising the purified phosphatase proteins or one of
their purified subunits. A usable buffered solution is TBS (TRIS
BUFFER SALINE) containing 5% of skim milk powder and 3% of BSA.
[0046] The peptide onto which the phosphatase 2A protein holoenzyme
is bound, is identified generally by direct or indirect labelling
of the phosphatase protein and identification of spots where the
labelled protein is bound. The binding of the PP2A or one of its
subunits on one of the peptidic spots may be shown, in particular,
with antisera, according to known techniques with regards to
Western Blot or ELISA, following the incubation of the support
containing the peptide array with an antibody raised against the
subunits (A) or (B) or (C) or a mixture of antibodies raised
against the PP2A subunits (A), (B) or (C).
[0047] The use of the <<SPOT synthesis>> method
described in FR no. 0110139 has lead to the identification E4orf4
and Bcl-2 peptides, useful especially in anti-tumor, antiviral and
antiparasitic treatments, having a dimension less than 30 amino
acids, even less than 20 amino acids, these peptides being able to
bind in vitro a phosphatase 2A protein holoenzyme or one of its
subunits.
[0048] The invention is thus concerned with a E4orf4 or Bcl-2
peptide, natural or synthetic, with a dimension less than 30 amino
acids, preferably of dimension less than 20 amino acids,
characterized in that said peptide specifically binds in vitro a
phosphatase 2A protein holoenzyme or one of its subunits. By
specifically bind, it is understood that the peptide is able to
inhibit in a competitive way the binding of a viral or parasitic
protein with PP2A peptides.
[0049] The invention is also concerned with a peptide comprising a
12 amino acid sequence of the Theileria parva Ck.alpha. 2 protein
(FD6) capable of interacting with the PP2A sub-unit A and to
penetrate in a Hela cell (Garcia, A. et al. (2000) Inhibitions de
processus tumoraux ou infectieux par transfert intracellulaire de
peptides mimant des sites d'interaction avec la serine/threonine
phosphatase PP2A). The invention is further concerned with the
sequence corresponding to the interaction site of the PP2A with the
B.alpha. sub-unit of the PP2A 1-B site (cf. table 1). This peptide
could penetrate in cells and, like the E4orf4 protein of the human
adenovirus, could interact with PP2A and provoke apoptosis in
cancer cells without affecting the normal cells.
[0050] The identified E4orf4 and Bcl-2 peptides, and the proposed
FD6-E4orf4 peptides are particularly useful in the treatment of
certain tumors, viral or parasitic infections. A person of the art
may select, with the aid of competition binding assays, novel
peptides, derived from identified sequences of the invention, such
peptides inhibiting in a competitive way the binding of the native
protein to which they derive with a PP2A holoenzyme or one of its
subunits.
[0051] Thus, the invention also concerns a natural or synthetic
peptide, as defined above, characterized in that it inhibit in a
competitive way, the interaction of the native protein to which it
derives with a PP2A holoenzyme or one of its subunits.
[0052] The peptides of the invention, on order to be efficient in
vivo in the treatment of certain tumors or certain viral or
parasitic infections, may be coupled to a vector capable of
transferring said peptide in a eukaryotic cell. The
small-dimensioned peptides of the invention allow them to pass
through the cell membrane. The use of appropriate vectors further
allow to target certain tissue-related peptide, certain cells, even
certain specific cellular compartments, and particularly, the
cytoplasm or the cell nucleus, in accordance with the desired
therapeutic effect.
[0053] The invention is naturally concerned with the means allowing
synthesis of the peptides of the invention. Particularly, the
invention is concerned with a polynucleotide characterized in that
its sequence codes for a peptide of the invention. Preferred
polynucleotides are polynucleotides which their sequence is chosen
from one of the following sequences:
TABLE-US-00001 E4 orf4 (375 bp) atggcccacc atcgtctgcc ccgcgtttgt
gtaaagggca ttattcattt tgaggaagat 60 tttgttagag agcttggatc
aatgctggag tctcctatgg agtttctctt cgacaccatt 120 gatgatgtta
ctgcttctat attttgtgaa agcatgttta aggctgttga taaaaacaag 180
cctgggatta cttttaaggt ggtcttttac tctcagcttg gttttgagta tgatgatgct
240 cttgcacatt ttaaaggcac tttaattaaa gaaatttctg acgttgttaa
taatcaccct 300 aatgtaaaca atgcttttag aggaagggag attgtcactg
tatctttgtt agaagtgttt 360 agtttttgtt cataa BcI-2 (618 bp)
atggcgcacg ctgggagaac ggggtacgac aaccgggaga tagtgatgaa gtacatccat
60 tataagctgt cgcagagggg ctacgagtgg gatgcgggag atgtgggcgc
cgcgcccccg 120 ggggccgccc ccgcaccggg catcttctcc tcccagcccg
ggcacacgcc ccatccagcc 180 gcatcccgcg acccggtcgc caggacctcg
ccgctgcaga ccccggctgc ccccggcgcc 240 gccgcggggc ctgcgctcag
cccggtgcca cctgtggtcc acctggccct ccgccaagcc 300 ggcgacgact
tctcccgccg ctaccgcggc gacttcgccg agatgtccag ccagctgcac 360
ctgacgccct tcaccgcgcg gggacgcttt gccacggtgg tggaggagct cttcagggac
420 ggggtgaact gggggaggat tgtggccttc tttgagttcg gtggggtcat
gtgtgtggag 480 agcgtcaacc gggagatgtc gcccctggtg gacaacatcg
ccctgtggat gactgagtac 540 ctgaaccggc acctgcacac ctggatccag
gataacggag gctgggtagg tgcatctggt 600 gatgtgagtc tgggctga
[0054] It may be advantageous to synthesize a polypeptide
comprising the repetition of the identified peptide motifs of the
invention. Consequently, the invention concerns a polynucleotide
characterized in that it consists of a multimer of said
polynucleotide coding for a peptide of the invention. The invention
also concerns a polypeptide characterized in that it is obtained
from the repetition of a peptide of the invention.
[0055] The invention is further concerned with a cellular
expression vector, characterized in that it comprises a
polynucleotide as defined above and regulatory sequences allowing
expression of a peptide of the invention in a host cell.
[0056] The invention is also concerned with a preparation method of
a peptide according to the invention, comprising the transformation
of a cell with the aid of a cellular expression vector as defined
above, followed by the culture of the transformed cellular host,
and the recovery of the peptide in the culture media.
[0057] The invention is further concerned with a purified
monoclonal or polyclonal antibody characterized in that it is able
to specifically bind to a peptide of the invention.
[0058] Antibodies specifically raised against identified peptides
of the invention are obtained, for instance, by the immunization of
an animal by injecting a peptide of the invention, and the recovery
of the produced antibodies. A monoclonal antibody may be obtained
according to methods known by one skilled in the art, such as the
method described by Kohler and Milstein (1975).
[0059] The obtained antibodies, specifically raised against
phosphatase 2A protein targets find their use in particular in
immunotherapy. They can, for instance, serve as antagonists of
viral or parasitic proteins raised against the phosphatase 2A
protein so as to block the viral or parasitic development.
[0060] Furthermore, the polynucleotides coding the peptides of the
invention may be directly transferred in the nucleus of the target
cells, if necessary with the aid of appropriate vectors, so as to
allow the in vivo expression of corresponding peptides, said
peptides being likely able to block by competitive inhibition a
specific interaction between the phosphatase 2A protein and the
viral or cellular protein which they derive from.
[0061] Thus, the invention is concerned with a pharmaceutical
composition comprising one of the elements chosen from a
polynucleotide of the invention or an antibody of the
invention.
[0062] The invention also concerns a pharmaceutical composition
comprising one of the peptides of the invention in combination with
a pharmaceutically acceptable vehicle.
[0063] Moreover, the invention is concerned with the use of a
peptide of the invention, in the preparation of a medicament in the
treatment of a viral or parasitic infection.
[0064] The peptides of the invention may be advantageously chosen
so as to stimulate the induction of apoptosis linked to the
activation of the cellular phosphatase 2A protein. Thus, the
invention also concerns the use of a peptide of the invention in
the preparation of a medicament suited to induce apte apoptosis in
target cells, such as tumoral cells.
[0065] Cancer results in the specific expression of proteins which
their activity is regulated by the sequences of the peptides of the
invention. The sequences coding the peptides of the invention may
be used as probe for detecting, in a specific manner, from RNA
extracts of a patient's biological sample, the tumoral
development.
[0066] Furthermore, an antibody of the invention may be used for
specifically recognizing peptidic sequences contained in viral or
cellular proteins expressed during the tumoral development.
[0067] Thus, the invention is concerned with the use of a
polynucleotide of the invention or an antibody of the invention in
the in vitro diagnosis of cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1: Identification of binding sites of E4orf4 protein
with PP2A.
[0069] FIG. 2: Identification of binding sites of Bcl-2 protein
with PP2A.
DETAILED DESCRIPTION OF THE INVENTION
[0070] According to a preferred embodiment of the invention, the
peptide of the invention is characterized in that it consists of a
fragment of a E4orf4 viral protein, a fragment of a Bcl-2 cellular
protein or a fragment of the proposed Ck.alpha.2-E4orf4 peptide,
said protein binding in vitro the phosphatase 2A protein, or a
sequence distinguishable from said protein fragment by amino acid
substitution or deletion, said sequence retaining the binding
properties to the phosphatase 2A protein holoenzyme or one of its
subunits.
[0071] In particular, a distinct sequence is a peptide sequence
increasing the binding affinity to the phosphatase 2A protein
holoenzyme or one of its subunits compared with the native sequence
to which said distinct sequence derives. Another distinct sequence
as defined above is a peptide sequence homologous to a peptide
sequence originally identified, i.e. a sequence derived from a
protein of another species than the one of the peptide sequence
originally identified, and of which the primary sequence can be
aligned with the peptide sequence originally identified with the
aid of an optimal alignment program commonly used, such as BESTFIT
(Wisconsin Genetics Software Package, Genetics Computer Group,
GCG). In particular, a sequence A will be considered as being
homologous to a sequence B if the sequences A and B present a
homology of at least 50%, preferably 75%, after alignment of the
sequences with the aid of a program such as BESTFIT. More
preferably, two sequences are also considered homologous if the
sequence are almost identical with the exception of some residues
which may represent 10 to 20% of variability over the total
sequence. Moreover, similar amino acids having similar by their
chemical function (such as Arg and Lys) are considered as
equivalents.
[0072] A particular preferred peptide of the invention is a
fragment of the adenovirus type 2 E4orf4 protein, and more
specifically a fragment of the canine adenovirus type 2 E4orf4
protein, or a sequence distinguishable from said protein fragment
by amino acid substitution or deletion, said sequence retaining the
binding properties to the phosphatase 2A protein holoenzyme or one
of its subunits.
[0073] In a more preferred embodiment, a peptide of the invention
is characterized in that it includes one of the following
sequences:
TABLE-US-00002 A) RELGSMLE SPMEFLFDTI DDVTAS (SEQ. ID NO: 1) b)
LGSMLE SPMEFLFDTI DDVTAS (SEQ. ID NO: 2) c) SMLE SPMEFLFDTI DDVTAS
(SEQ. ID NO: 3) d) HFKGTLIKEISDVVNN (SEQ. ID NO: 4) E) AFRGRE
VTVSLLEVFSFC (SEQ. ID NO: 5) f) VKKKKIKREIKI SMLE SPMEFLFDTI D
(SEQ. ID NO: 6) DVTAS g) ARTSPLQTPAAPGAAAGPAL (SEQ. ID NO: 7) h)
RFATVVEELFRDGVNW (SEQ. ID NO: 8) i) RPLFDFSWLSLKTLLSLALVGA (SEQ. ID
NO: 9) j) PLQTPAAPGAAAGPAL (SEQ. ID NO: 10) k) LFDFSWLSLKTLLSLALVGA
(SEQ. ID NO: 11) l) a sequence distinguishable from SEQ ID NO: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 by amino acid subtitution or
deletion, said sequence retaining the binding properties to the
phosphatase 2A pro- tein holoenzyme or one of its subunits.
[0074] Among the peptide sequences distinguishable from SEQ ID NO:
1, 2, 3, 4, 5 or 6 by amino acid substitution or deletion, and
being contemplated by the present invention, are more particularly
the peptides which their sequence is enclosed in one of the
sequence of the E4orf4 protein, different variants of canine and
human adenovirus type 2, and corresponding to the homologous
sequences of these variants of SEQ ID NO: 1, 2, 3, 4, 5 or 6.
[0075] Among the peptide sequences distinguishable from SEQ ID NO:
7, 8, 9, 10 or 11 by amino acid substitution or deletion, and being
contemplated by the present invention, are more particularly the
peptides which their sequence is enclosed in one of the sequence of
the Bcl-2 protein, and corresponding to the homologous sequences of
these variants of SEQ ID NO: 7, 8, 9, 10 or 11.
[0076] A preferred peptide according to the invention is a peptide
chosen from one of the sequences SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or 11 and characterized in that its administration induces
apoptosis of cells, in particular apoptosis of tumoral cells.
[0077] The invention preferably aims at the use of a peptide which
its sequence derives from a fragment of the E4orf4 protein, as
defined above, in the preparation of a medicament suited to inhibit
a viral infection.
Examples
[0078] PP2A Purified Proteins
[0079] The trimeric PP2A1 protein has been purified to homogeneity
from porcine brain.
[0080] Recombinant protein A (structural sub-unit of PP2A)
expressed in bacteria.
[0081] Recombinant protein B55 (regulatory sub-unit of PP2A)
expressed in bacteria.
[0082] Identification of E4orf4 and Bcl-2 Binding Sites with
PP2A
[0083] We have mapped the binding sites between the E4orf4 proteins
(coded by the canine adenovirus type 2) and the anti-apoptotic
Bcl-2 protein with PP2A by using the <<peptides spot>>
technique previously described (Frank et Overwing. (1996). Meth.
Mol. Biol. 66, 149-169).
[0084] This approach is based on the use of membranes containing
thereon dodecapeptides representing the whole protein of interest
(E4orf4 and Bcl-2) with a two amino acid gap per peptide.
[0085] Each membrane is first saturated one hour at room
temperature with TBS containing 5% skim milk powder and 3% BSA,
then incubated overnight in the same buffer in the of 4 .mu.g/ml of
purified protein (sub-unit A of PP2A and PP2A1 holoenzyme): Fifty
six dodecapeptides covering the whole sequence of the E4orf4
protein and hundred-fifteen dodecapeptides covering the whole
sequence of the Bcl-2 protein were synthesized and covalently bound
to cellulosic membranes.
TABLE-US-00003 E4orf4:
MAHHRLPRVCVKGIIHFEEDFVRELGSMLESPMEFLFDTIDDVTAFTSIF
CESMFKAVDKNKPGITFKVVFYSQLGFEYDDALAHFKGTLIKEISDVVNN
HPNVNNFTAFRGREIVTVSLLEVFSFCS. Bcl-2:
MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAFTPGI
FSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLALR
QAGDDFFTSRRYRGDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGR
IVAFFEFGGVMCVESVNFTREMSPLVDNIALWMTEYLNRHLHTWIQDNGG WVGASGDVSLG.
[0086] The specific interaction of each purified protein
(respectively the structural sub-unit A, the regulatory sub-unit of
B55(B) or the trimeric ABC holoenzyme (named PP2A1) with a peptide
sequence is shown by western blot, following incubation of the
membrane with an antibody raised against the structural protein A
(A), the regulatory sub-unit B (B) and with a pool of antibodies
recognising the A, B, and C proteins of PP2A (ABC) (FIGS. 1 and
2).
Example 1
[0087] Identification of Peptidic Sequences Containing Binding
Sites of the E4orf4 Protein Coded by the Canine Adenovirus Type 2
with Proteins of the PP2A Family.
[0088] Screening of a membrane containing peptides covering the
E4orf4 sequences with different forms of purified PP2A (FIG. 1)
allowed us to identify five amino acid sequences containing
interaction sites of E4orf4 with PP2A (cf. table 1).
[0089] We were able to respectively determine:
[0090] a peptidic sequence containing a binding site of E4orf4 with
the structural sub-unit B ("site B1" corresponding to peptides 12
to 18).
[0091] Three peptidic sequences corresponding to three binding
sites of E4orf4 with the sub-unit A ("site A 1" corresponding to
peptides 13 to 18 and "site A2" corresponding to peptides 41 to 43
and "site A3" corresponding to peptides 52 to 55).
[0092] Two peptidic sequences corresponding to two binding sites of
E4orf4 with the PP2A1 protein ("site B1" corresponding to peptides
14 to 18 and "site A3" corresponding to peptides 52 to 55).
[0093] It is interesting to note that (table 1) the B1, A1 and ABC1
sites partially overlap one another thus suggesting:
[0094] that the sub-units A and B can interact on the same site
which has never been established in the PP2A system. Moreover, the
interaction of the trimeric PP2A with this site requires a shorter
sequence thus suggesting a conformational regulation.
TABLE-US-00004 TABLE 1 Binding sites of E4orf4 with different PP2A
proteins Site B1 23-RELGSMLE SPMEFLFDTI DDVTAS-46 Site A1 25-LGSMLE
SPMEFLFDTI DDVTAS-46 Site ABC1 28-SMLE SPMEFLFDTI DDVTAS-4 Site A2
83-HFKGTLIKEISDVVNN-98 Site A3 106-AFRGRE VTVSLLEVFSFC-124
Example 2
[0095] Identification of Peptidic Sequences Containing Binding
Sites of Bcl-2 Protein with Proteins of PP2A Family.
[0096] Screening of a membrane containing peptides covering the
Bcl-2 sequences with different forms of purified PP2A (FIG. 2)
allowed us to identify five amino acid sequences containing
interaction sites of Bcl-2 with PP2A (cf. table 2).
[0097] We were able to respectively determine:
[0098] a peptidic sequence containing a binding site of Bcl-2 with
the regulatory sub-unit B ("site 1-B" corresponding to peptides 33
to 37).
[0099] two peptidic sequences containing two binding sites of Bcl-2
with the structural sub-unit A (site A1 corresponding to peptides
64-67 and site A2 corresponding to peptides 104-109).
[0100] two peptidic sequences containing a binding site of Bcl-2
with the PP2A1 holoenzyme (site 1-ABC corresponding to peptides 35
to 37 and site 2-ABC corresponding to peptides 105-109).
[0101] It is interesting to note that the 1-ABC and 2-ABC sites
correspond respectively to the 1-B and A2 sites with respectively
two and four amino acid gaps probably linked to a different
conformation thus resulting in the interaction of proteins A and B
within the holoenzyme.
[0102] Moreover it is noteworthy that the 1-B/1-ABC site is located
at the ser-70 level where the phosphorylation regulated the PP2A
activity while the 2-ABC/A2 site is located at the C-terminus end
of the protein. Contrary to the interaction sites with PP1, these
two sites do not interfere with the BH domains of PP2A (see Garcia
A. et al. PP1 et PP2A des ser/thr phosphatases au coeur de
I'apoptose (2001). Med/Sci 17, 1214-1216 for general
discussion).
TABLE-US-00005 TABLE 2 Binding sites of Bcl-2 with different PP2A
proteins Site 1-B 67-ARTSPLQTPAAPGAAAGPAL-86 Site A1
129-RFATVVEELFRDGVNW-144 Site A2 207-RPLFDFSWLSLKTLLSLALVGA- 228
Site 1-ABC (site 1B) 71-PLQTPAAPGAAAGPAL-86 Site 2-ABC (site A2)
209-LFDFSWLSLKTLLSLALVGA-228
Sequence CWU 1
1
15124PRTArtificial SequenceSynthetic Peptide 1Arg Glu Leu Gly Ser
Met Leu Glu Ser Pro Met Glu Phe Leu Phe Asp1 5 10 15Thr Ile Asp Asp
Val Thr Ala Ser 20222PRTArtificial SequenceSynthetic Peptide 2Leu
Gly Ser Met Leu Glu Ser Pro Met Glu Phe Leu Phe Asp Thr Ile1 5 10
15Asp Asp Val Thr Ala Ser 20320PRTArtificial SequenceSynthetic
Peptide 3Ser Met Leu Glu Ser Pro Met Glu Phe Leu Phe Asp Thr Ile
Asp Asp1 5 10 15Val Thr Ala Ser 20416PRTArtificial
SequenceSynthetic Peptide 4His Phe Lys Gly Thr Leu Ile Lys Glu Ile
Ser Asp Val Val Asn Asn1 5 10 15518PRTArtificial SequenceSynthetic
Peptide 5Ala Phe Arg Gly Arg Glu Val Thr Val Ser Leu Leu Glu Val
Phe Ser1 5 10 15Phe Cys632PRTArtificial SequenceSynthetic Peptide
6Val Lys Lys Lys Lys Ile Lys Arg Glu Ile Lys Ile Ser Met Leu Glu1 5
10 15Ser Pro Met Glu Phe Leu Phe Asp Thr Ile Asp Asp Val Thr Ala
Ser 20 25 30720PRTArtificial SequenceSynthetic Peptide 7Ala Arg Thr
Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala1 5 10 15Gly Pro
Ala Leu 20816PRTArtificial SequenceSynthetic Peptide 8Arg Phe Ala
Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp1 5 10
15922PRTArtificial SequenceSynthetic Peptide 9Arg Pro Leu Phe Asp
Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Ser1 5 10 15Leu Ala Leu Val
Gly Ala 201016PRTArtificial SequenceSynthetic Peptide 10Pro Leu Gln
Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu1 5 10
151120PRTArtificial SequenceSynthetic Peptide 11Leu Phe Asp Phe Ser
Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala1 5 10 15Leu Val Gly Ala
2012375DNACanine adenovirus type-2 12atggcccacc atcgtctgcc
ccgcgtttgt gtaaagggca ttattcattt tgaggaagat 60tttgttagag agcttggatc
aatgctggag tctcctatgg agtttctctt cgacaccatt 120gatgatgtta
ctgcttctat attttgtgaa agcatgttta aggctgttga taaaaacaag
180cctgggatta cttttaaggt ggtcttttac tctcagcttg gttttgagta
tgatgatgct 240cttgcacatt ttaaaggcac tttaattaaa gaaatttctg
acgttgttaa taatcaccct 300aatgtaaaca atgcttttag aggaagggag
attgtcactg tatctttgtt agaagtgttt 360agtttttgtt cataa
37513128PRTCanine adenovirus type-2 13Met Ala His His Arg Leu Pro
Arg Val Cys Val Lys Gly Ile Ile His1 5 10 15Phe Glu Glu Asp Phe Val
Arg Glu Leu Gly Ser Met Leu Glu Ser Pro 20 25 30Met Glu Phe Leu Phe
Asp Thr Ile Asp Asp Val Thr Ala Phe Thr Ser 35 40 45Ile Phe Cys Glu
Ser Met Phe Lys Ala Val Asp Lys Asn Lys Pro Gly 50 55 60Ile Thr Phe
Lys Val Val Phe Tyr Ser Gln Leu Gly Phe Glu Tyr Asp65 70 75 80Asp
Ala Leu Ala His Phe Lys Gly Thr Leu Ile Lys Glu Ile Ser Asp 85 90
95Val Val Asn Asn His Pro Asn Val Asn Asn Phe Thr Ala Phe Arg Gly
100 105 110Arg Glu Ile Val Thr Val Ser Leu Leu Glu Val Phe Ser Phe
Cys Ser 115 120 12514618DNAArtificial SequenceSynthetic DNA
14atggcgcacg ctgggagaac ggggtacgac aaccgggaga tagtgatgaa gtacatccat
60tataagctgt cgcagagggg ctacgagtgg gatgcgggag atgtgggcgc cgcgcccccg
120ggggccgccc ccgcaccggg catcttctcc tcccagcccg ggcacacgcc
ccatccagcc 180gcatcccgcg acccggtcgc caggacctcg ccgctgcaga
ccccggctgc ccccggcgcc 240gccgcggggc ctgcgctcag cccggtgcca
cctgtggtcc acctggccct ccgccaagcc 300ggcgacgact tctcccgccg
ctaccgcggc gacttcgccg agatgtccag ccagctgcac 360ctgacgccct
tcaccgcgcg gggacgcttt gccacggtgg tggaggagct cttcagggac
420ggggtgaact gggggaggat tgtggccttc tttgagttcg gtggggtcat
gtgtgtggag 480agcgtcaacc gggagatgtc gcccctggtg gacaacatcg
ccctgtggat gactgagtac 540ctgaaccggc acctgcacac ctggatccag
gataacggag gctgggtagg tgcatctggt 600gatgtgagtc tgggctga
61815211PRTArtificial SequenceSynthetic Peptide 15Met Ala His Ala
Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met1 5 10 15Lys Tyr Ile
His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala 20 25 30Gly Asp
Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Phe Thr Pro 35 40 45Gly
Ile Phe Ser Ser Gln Pro Gly His Thr Pro His Pro Ala Ala Ser 50 55
60Arg Asp Pro Val Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro65
70 75 80Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val
His 85 90 95Leu Ala Leu Arg Gln Ala Gly Asp Asp Phe Phe Thr Ser Arg
Arg Tyr 100 105 110Arg Gly Asp Phe Ala Glu Met Ser Ser Gln Leu His
Leu Thr Pro Phe 115 120 125Thr Ala Arg Gly Arg Phe Ala Thr Val Val
Glu Glu Leu Phe Arg Asp 130 135 140Gly Val Asn Trp Gly Arg Ile Val
Ala Phe Phe Glu Phe Gly Gly Val145 150 155 160Met Cys Val Glu Ser
Val Asn Phe Thr Arg Glu Met Ser Pro Leu Val 165 170 175Asp Asn Ile
Ala Leu Trp Met Thr Glu Tyr Leu Asn Arg His Leu His 180 185 190Thr
Trp Ile Gln Asp Asn Gly Gly Trp Val Gly Ala Ser Gly Asp Val 195 200
205Ser Leu Gly 210
* * * * *