FR2805266A1 - A new protein, designated Parkin-Associated Protein 1 (PAP1), is an interaction partner of Parkin and is useful to treat neurodegenerative pathologies including Parkinson's disease - Google Patents

Abstract

An isolated human Parkin-Associated Protein 1 (PAP1) protein, is new. Independent claims are also included for the following: (i) a polypeptide comprising the 344 amino acid sequence fully defined in the specification (I), or its derivative or fragment; (ii) a composition capable of at least partly modifying interaction between PAP1 protein or its homologue and Parkin; (iii) a nucleic acid encoding the above polypeptide or composition (iv) a nucleic acid, particularly a probe, capable of hybridizing with the above nucleic acid; (v) a vector comprising the above nucleic acid; (vi) a recombinant disarmed virus comprising the above nucleic acid; (vii) a nucleic acid having one of the 19-26 nucleotide (nt) sequences fully defined in the specification; (viii) an antibody or its fragment or derivative directed against the above composition, preferably one that recognizes the above polypeptide; (ix) a non-peptide or not naturally exclusively peptide composition likely to at least partially modulate interaction between PAP1 protein or its homologue and Parkin; (x) selecting or characterizing active molecules, comprising selecting molecules capable of binding to sequence I, or the 156, 610 or 313 amino acid sequence fully defined in the specification, or their fragment; (xi) producing the above peptide composition, comprising culturing cells containing the nucleic acid of (iii), or the above vector under expression conditions and recovering the expressed peptide; (xii) a cell containing the nucleic acid of (iii) or the above vector; and (xiii) a non-human mammal whose cells comprise the nucleic acid of (iii). ACTIVITY : Antiparkinsonian. MECHANISM OF ACTION : None given.

Description

<Desc / Clms Page number 1>

COMPOSITIONS FOR USE IN REGULATING ACTIVITY
FROM THE PARK
The present invention relates to compositions and methods useful for regulating the activity of parkin. It relates in particular to a new protein, designated PAP1, a partner for parkin, as well as the peptides or polypeptides derived or homologous to this protein. It also relates to compounds capable of at least partially modulating the activity of Parkin, in particular of interfering with the interaction between parkin and PAP1. The present invention is usable in the therapeutic, diagnostic fields or for the constitution of pharmacological targets allowing the development of new drugs.

 The Parkin gene is mutated in certain familial forms (autosomal recessive juveniles) of Parkinson's disease (Kitada et al., 1998). Parkinson's disease (Lewy, 1912) is one of the most common neurodegenerative diseases, affecting more than 1% of the population over 55 years of age. Patients with this disease have neurological disorders gathered under the term of parkinsonian syndrome, characterized by rigidity, bradykinesia, and a tremor of rest. These symptoms are the consequence of a degeneration of the dopaminergic neurons of the black substance of the brain.

 Most cases with Parkinson's have no family history. However, there are family cases, some of which correspond to a monogenic form of the disease. At present, only three different genes have been identified in certain rare hereditary forms. The first form corresponds to an autosomal dominant form whose responsible gene codes for alpha Synuclein (Polymeropoulos et al., 1997). This protein is an abundant constituent of intracytoplamic inclusions, called Lewy bodies, which serve as a marker for Parkinson's disease (Lewy, 1912). The second form, also autosomal dominant, is linked to a mutation in a gene coding for a hydrolase called ubiquitin carboxy-terminal hydrolase L1 (Leroy et al., 1998). This

<Desc / Clms Page number 2>

 enzyme is believed to hydrolyze polymers or conjugates of ubiquitins to monomers of ubiquitin. The third form is distinguished from the previous ones by an autosomal recessive transmission and an onset often before 40 years as well as by an absence of Lewy bodies. These patients respond more favorably to levodopa, a dopamine precursor used to treat Parkinson's disease. The gene involved in this form codes for a new protein called Parkin (Kitada et al., 1998).

 The Parkin gene consists of 12 exons covering a genomic region of more than 500,000 base pairs on chromosome 6 (6q25. 2-q27). At present, two main types of mutation of this gene, at the origin of the disease, are known, either deletions of variable size in the region which covers exons 2 to 9, or point mutations which produce l premature appearance of a stop codon or change of an amino acid (Kitada et al., 1998; Abbas et al., 1999; Lucking et al., 1998; Hattori et al., 1998). The nature of these mutations and the autosomal recessive mode of transmission suggest a loss of Parkin function leading to Parkinson's disease.

 This gene is expressed in a large number of tissues and in particular in the substantia nigra. There are several transcripts corresponding to this gene which come from different alternative splices (Kitada et al., 1998; Sunada et al., 1998). In the brain, there are two types of messenger RNAs, one of which lacks the part corresponding to exon 5. In leukocytes, parkin messenger RNAs not containing the region coding for exons 3,4 and 5 have been identified. The longest messenger RNA in Parkin, found in the brain, contains 2960 bases and codes for a protein of 465 amino acids.

 This protein has a weak homology in its N-terminal part with ubiquitin. Its C-terminal half contains two ring finger motifs separated by an IBR (In Between Ring) domain corresponding to a region rich in cysteine

<Desc / Clms Page number 3>

 able to bind metals such as zing finger domains (Morett, 1999). By immunocytochemistry, it has been shown that Parkin is localized in the cytoplasm and the Golgi apparatus of black substance neurons which contain melanin (Shimura et al., 1999). In addition, this protein is present in some Lewy bodies of Parkinson's. The cellular function of Parkin is not yet demonstrated, but it could play a role of transporter in the synaptic vesicles, in the maturation or degradation of proteins and in the control of growth, differentiation or cell development. In autosomal recessive juvenile forms, Parkin is absent, thus confirming that the loss of this function is responsible for the disease.

 The elucidation of the exact role of the Parkin protein in the degeneration process of dopaminergic neurons therefore constitutes a major challenge for the understanding and the therapeutic approach of Parkinson's disease and more generally of diseases of the central nervous system.

 The present invention resides in the identification of a parkin partner, interacting with this protein under physiological conditions. This partner represents a new pharmacological target for the manufacture or research of compounds capable of modulating the activity of parkin, in particular its activity on the degeneration of dopaminergic neurons and / or the development of nervous pathologies. This protein, the antibodies, the corresponding nucleic acids as well as the specific probes or primers, can also be used for the detection or the assay of proteins in biological samples, in particular samples of nervous tissue. These proteins or nucleic acids can also be used in therapeutic approaches, to modulate the activity of parkin, as well as any compound according to the invention, capable of modulating the interaction between parkin and the polypeptides of the invention.

<Desc / Clms Page number 4>

 The present invention more particularly results from the discovery by the applicant of a new human protein, designated PAPI (Parkine Associated Protein 1), interacting with parkin. The PAPI protein (sequence SEQ ID NO: 1 or 2) has a certain homology with the synaptotagmins and is capable of interacting more particularly with the central region of parkin (represented in the sequence SEQ ID NO: 3 or 4).

 The present invention also results from the identification and characterization of particular regions of the PAPI protein, involved in the modulation of the function of parkin. The demonstration of the existence of this protein and of regions involved in its function makes it possible in particular to prepare new compounds and / or compositions which can be used as pharmaceutical agents and to develop industrial methods of screening for such compounds.

 A first subject of the invention therefore relates to compounds capable of modulating, at least partially, the interaction between the PAPI protein (or its homologs) and parkin (in particular human parkin), or of interfering at the level of the interaction between these proteins.

 Another subject of the invention resides in the PAP1 protein, its fragments, derivatives and homologs.

 Another aspect of the invention resides in a nucleic acid encoding the PAP1 protein, its fragments, derivatives or homologs, as well as any vector comprising such a nucleic acid and any recombinant cell containing such a nucleic acid or vector.

 The invention also relates to antibodies capable of binding the PAPI protein, its fragments, derivatives and homologs, in particular polyclonal or monoclonal antibodies, more preferably antibodies capable of binding the PAPI protein and of at least partially inhibiting its interaction with parkin. .

 Another aspect of the invention relates to nucleotide probes or primers, specific to PAP1, which can be used to detect or amplify the pap1 gene or a region thereof in any biological sample.

<Desc / Clms Page number 5>

 The invention also relates to pharmaceutical compositions, methods of detecting genetic abnormalities, methods of producing polypeptides as defined above, as well as methods of screening or characterizing active compounds.

 As indicated above, a first aspect of the invention resides in a compound capable of interfering, at least partially, at the level of the interaction between the protein PAP1 (or its homologs) and parkin.

 Within the meaning of the present invention, the name protein PAP1 designates the protein itself as well as all of its homologous forms. The term “homologous form” is intended to denote any protein equivalent to the protein under consideration, of diverse cellular origin and in particular derived from cells of human origin, or of other organisms, and having an activity of the same type. Such homologs also include the natural variants of the PAPI protein of sequence SEQ ID N02, in particular the polymorphic or splicing variants. Such homologs can be obtained by hybridization experiments between the coding nucleic acids (in particular the nucleic acid of sequence SEQ ID NO: 1). Within the meaning of the invention, it is sufficient that a sequence of this type has a significant percentage of identity to lead to physiological behavior comparable to that of the protein PAP 1 as claimed.

 The interference of a compound according to the invention can manifest itself in different aspects. Thus, the compound can slow down, inhibit or stimulate, at least partially, the interaction between the PAP1 protein or one of its homologous forms and Parkin. They are preferably compounds capable of modulating this interaction in vitro, for example in a double-hybrid type system or in any acellular system for detecting an interaction between two polypeptides. The compounds according to the invention are preferably compounds capable of at least partially modulating this interaction, preferably of increasing or

<Desc / Clms Page number 6>

 to inhibit this interaction by at least 20%, more preferably by at least 50%, relative to a control in the absence of the compound.

 In a particular mode of implementation, these are compounds capable of interfering at the level of the interaction between the region of parkin represented on the sequence SEQ ID NO: 4 and the region of the protein PAP 1 represented. on the sequence SEQ ID NO: 2.

 According to a particular embodiment of the invention, the compounds are capable of binding at the level of the interaction domain between the PAPI protein, or one of its homologous forms, and the Parkin.

 The compounds according to the present invention can be of varied nature and origin. In particular, they may be peptide, nucleic acid (i.e., comprising a chain of bases, in particular a DNA or RNA molecule), lipid, saccharide, antibody, etc. type compounds. and, more generally, of any organic or inorganic molecule.

 According to a first variant, the compounds of the invention are of peptide nature. The term peptide designates any molecule comprising a chain of amino acids, such as for example a peptide, a polypeptide, a protein, an antibody (or fragment or derivative of antibody), if necessary modified or associated with other compounds. or chemical groups. In this regard, the term peptide more specifically designates a molecule comprising a chain of 50 amino acids at most, more preferably 40 amino acids at most. A polypeptide (or a protein) preferably comprises from 50 to 500 amino acids, or more.

 According to a first preferred embodiment, the compounds of the invention are peptide compounds comprising all or part of the peptide sequence SEQ ID N 2 or one of its derivatives, in particular all or part of the peptide sequence of the protein PAP1 comprising the sequence SEQ ID N 2.

 Within the meaning of the present invention, the term derivative designates any sequence different from the sequence considered due to a degeneration of the genetic code, obtained by one or more modifications of a genetic and / or chemical nature, as well as any peptide coded by a sequence hybridizing with the sequence

<Desc / Clms Page number 7>

 nucleic acid SEQ ID NO: 1 or a fragment thereof, and having the capacity to interfere at the level of the interaction between the protein PAPI, or one of its homologs, and Parkin. By modification of genetic and / or chemical nature, one can hear any mutation, substitution, deletion, addition and / or modification of one or more residues. The term derivative also includes sequences homologous to the sequence considered, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type.

Such homologous sequences can be obtained by hybridization experiments. Hybridizations can be carried out from nucleic acid libraries, using the native sequence or a fragment thereof as probe, under variable hybridization conditions (Maniatis et al., 1989). Furthermore, the term fragment or part designates any portion of the molecule considered, comprising at least 5 consecutive residues, preferably at least 9 consecutive residues, even more preferably at least 15 consecutive residues. Typical fragments can include at least 25 consecutive residues.

 Such derivatives or fragments can be generated for different purposes, such as in particular that of increasing their therapeutic efficacy or reducing their side effects, or that of imparting to them new pharmacokinetic and / or biological properties.

 As peptide derived from the PAP 1 protein and from the homologous forms, mention may in particular be made of any peptide capable of interacting with Parkin, but carrying an effector region made non-functional. Such peptides can be obtained by deletion, mutation or disruption of this effector region on the PAP 1 protein and of homologous forms. Such modifications can be made, for example, by in vitro mutagenesis, by introduction of additional elements or synthetic sequences, or by deletions or substitutions of the original elements. When a derivative as defined above is produced, its activity as a partial inhibitor of the binding of the PAP 1 protein and of the homologous forms at its binding site on

<Desc / Clms Page number 8>

 Parkine can be highlighted. Any technique known to those skilled in the art can obviously be used for this purpose.

 They can also be fragments of the sequences indicated above. Such fragments can be generated in different ways. In particular, they can be synthesized chemically, on the basis of the sequences given in the present application, using the peptide synthesizers known to those skilled in the art. They can also be synthesized genetically, by expression in a cellular host of a nucleotide sequence coding for the peptide sought. In this case, the nucleotide sequence can be prepared chemically using an oligonucleotide synthesizer, on the basis of the peptide sequence given in the present application and the genetic code. The nucleotide sequence can also be prepared from the sequences given in the present application, by enzymatic cleavages, ligation, cloning, etc., according to techniques known to those skilled in the art, or by screening DNA libraries with elaborate probes from these sequences.

 Furthermore, the peptides of the invention, namely capable of at least partially modulating the interaction between the PAP1 protein and homologous forms and Parkin can also be peptides having a sequence corresponding to the interaction site of the PAP1 protein and homologous forms on Parkine.

 Other peptides according to the invention are peptides capable of competing with the peptides defined above for the interaction with their cell target. Such peptides can be synthesized in particular on the basis of the sequence of the peptide considered, and their capacity to compete with the peptides defined above can be determined.

<Desc / Clms Page number 9>

 A specific object of the present invention relates to the PAPI protein. It is more particularly the PAPI protein comprising the sequence SEQ ID NO: 2 or a fragment or derivative thereof.

 Another object of the invention resides in antibodies or fragments or derivatives of polyclonal or monoclonal antibodies directed against a polypeptide as defined above. Such antibodies can be generated by methods known to those skilled in the art. In particular, these antibodies can be prepared by immunization of an animal against a peptide compound of the invention (in particular a polypeptide or a peptide comprising all or part of the sequence SEQ ID NO: 2), collection of blood, and isolation of antibody. These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art.

 More preferably, the antibodies or antibody fragments of the invention have the ability to at least partially modulate the interaction of the claimed peptides with Parkin.

 Furthermore, these antibodies can also be used to detect and / or measure the expression of PAPI in biological samples, and therefore, to provide information on its state of activation.

 The fragments or derivatives of antibodies are for example fragments Fab, Fab'2, single-chain antibodies (ScFv), etc. These include any fragment or derivative retaining the antigenic specificity of the antibodies from which they are derived.

 The antibodies according to the invention are more preferably capable of binding the PAP 1 proteins comprising the sequence SEQ ID NO: 2, in particular the region of this protein involved in the interaction with parkin. These antibodies (or fragments or derivatives) are more preferably capable of binding an epitope present in the sequence between residues 1 and 344 of the sequence SEQ ID NO: 2.

<Desc / Clms Page number 10>

 The invention also relates to non-peptide or non-exclusively peptide compounds which can be used as a pharmaceutical agent. It is indeed possible, from the active protein motifs described in the present application, to produce molecules modulating the activity of PAP1 which are not exclusively peptide and compatible with pharmaceutical use, in particular by duplicating the active motifs of the peptides with a non-peptide structure or of a non-exclusively peptide nature.

 The present invention also relates to any nucleic acid coding for a peptide compound according to the invention. It may in particular be a nucleic acid comprising all or part of the sequence presented in SEQ ID N 1 or one of its derivatives. By derivative sequence is meant within the meaning of the present invention any sequence hybridizing with the sequence presented in SEQ ID N 1 or with a fragment thereof and coding for a peptide compound according to the invention, as well as the sequences resulting from the latter by degeneration of the genetic code. The different nucleotide sequences of the invention can be of artificial origin or not. They can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained either by screening of DNA libraries (cDNA library, genomic DNA library), or by chemical synthesis, or by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries, either by searching for homology in nucleic or protein databases. The hybridization mentioned above is preferably carried out under the conditions described by Sambrook et al (1989, pages 9.52-9.55).

 A particular nucleic acid 'within the meaning of the invention codes for a polypeptide comprising the sequence SEQ ID NO: 2 or a fragment or derivative thereof, in particular for the human PAPI protein. It is advantageously a nucleic acid comprising the nucleic sequence SEQ ID NO: 1.

<Desc / Clms Page number 11>

 Such nucleic acids can be used for the production of the peptide compounds of the invention. The present application thus relates to a process for the preparation of such peptide compounds according to which a cell containing a nucleic acid according to the invention is cultivated, under conditions of expression of said nucleic acid and the peptide compound produced is recovered. In this case, the part coding for said peptide compound is generally placed under the control of signals allowing its expression in a cellular host. The choice of these signals (promoters, terminators, "leader" secretory sequence, etc.) can vary depending on the cell host used. Furthermore, the nucleic acids of the invention can be part of a vector which can be autonomously replicating or integrative. More particularly, autonomously replicating vectors can be prepared using autonomously replicating sequences in the chosen host. As regards integrative vectors, these can be prepared for example by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector. It can be a vector of plasmid, episomal, chromosomal, viral type, etc.

 The cellular hosts which can be used for the production of the peptide compounds of the invention by the recombinant route are both eukaryotic and prokaryotic hosts. Among suitable eukaryotic hosts, there may be mentioned animal cells, yeasts, or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127, PC12 etc. cells. Among the mushrooms, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferred to use the following bacteria E. coli, Bacillus, or Streptomyces.

 The nucleic acids according to the invention can also be used for the production of antisense oligonucleotides or genetic antisense usable as pharmaceutical agents. The antisense sequences are small oligonucleotides, complementary to the strand coding for a given gene, and therefore capable

<Desc / Clms Page number 12>

 to specifically hybridize with the transcribed mRNA, inhibiting its translation into protein. The subject of the invention is therefore the antisense sequences capable of at least partially inhibiting the interaction of the PAPI proteins on Parkin. Such sequences can consist of all or part of the nucleic acid sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides interacting with Parkin. Such oligonucleotides can be obtained by fragmentation, etc., or by chemical synthesis.

 The claimed sequences can be used in the context of gene therapies, for the transfer and expression in vivo of antisense sequences or of peptides capable of modulating the interaction of the protein PAP1 with Parkin. In this regard, the sequences can be incorporated into viral or non-viral vectors, allowing their administration in vivo (Kahn et al., 1991). By way of viral vectors in accordance with the invention, mention may very particularly be made of vectors of the adenovirus, retrovirus, adenovirus associated virus (AAV) or herpes virus type. The present application also relates to defective recombinant viruses comprising a nucleic acid coding for a polypeptide according to the invention, in particular a polypeptide or peptide comprising all or part of the sequence SEQ ID NO: 2 or a derivative thereof. this.

 The invention also allows the production of nucleotide probes, synthetic or not, capable of hybridizing with the nucleotide sequences defined above or their complementary strand. Such probes can be used in vitro as a diagnostic tool, for the detection of the expression or overexpression of PAP1, or even for the detection of genetic anomalies (poor splicing, polymorphism, point mutations, etc.). These probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for peptides as defined above, from other cellular sources and preferably from cells of human origin. Respondents

<Desc / Clms Page number 13>

 of the invention generally comprise at least 10 bases, and they can for example comprise up to the entirety of one of the abovementioned sequences or of their complementary strand. Preferably, these probes are marked prior to their use. For this, different techniques known to those skilled in the art can be used (radioactive, fluorescent, enzymatic, chemical labeling, etc.).

 The invention also relates to any pharmaceutical composition comprising as active ingredient at least one compound as defined above, in particular a peptide compound.

 It relates in particular to any pharmaceutical composition comprising as active ingredient at least one antibody and / or an antibody fragment as defined above, as well as any pharmaceutical composition comprising as active ingredient at least one nucleic acid or a vector such as defined above.

 It also relates to any pharmaceutical composition comprising as active ingredient a chemical molecule capable of increasing or decreasing the interaction between the PAP1 protein and Parkin.

 Furthermore, it also relates to pharmaceutical compositions in which the peptides, antibodies, chemical molecules and nucleotide sequences defined above are associated with each other or with other active ingredients.

 The pharmaceutical compositions according to the invention can be used to modulate the activity of the Parkin protein and thereby maintain the survival of dopaminergic neurons. More particularly, these pharmaceutical compositions are intended to modulate the interaction between the PAP1 protein and the Parkin. More preferably, they are pharmaceutical compositions intended for the treatment of diseases of the central nervous system such as, for example, Parkinson's disease.

 Another subject of the invention is the use of the molecules described above to modulate the activity of Parkin or the typing of diseases of the nervous system

<Desc / Clms Page number 14>

 central. In particular, the invention relates to the use of these molecules to at least partially modulate the activity of Parkin.

 The invention also relates to a method for screening or characterizing molecules active on the function of parkin, comprising the selection of molecules capable of binding the sequence SEQ ID NO: 2 or the sequence SEQ ID NO: 4, or a fragment (or derivative) of these. The method advantageously comprises bringing into contact, in vitro, the test molecule (s) with a polypeptide comprising the sequence SEQ ID NO: 2 or the sequence SEQ ID NO: 4, or a fragment (or derivative) thereof, and the selection of molecules capable of binding the sequence SEQ ID NO: 2 (in particular the region between residues 1 and 344) or the sequence SEQ ID NO: 4. The molecules tested can be of varied nature (peptide, nucleic, lipid , sugar, etc., or mixtures of such molecules, e.g. combinatorial libraries, etc.). As indicated above, the molecules thus identified can be used to modulate the activity of the Parkin protein, and represent potential therapeutic agents for the treatment of neurodegenerative pathologies.

 Other advantages of the present invention will appear on reading the examples and figures which follow, which should be considered as illustrative and not limiting.

LEGENDS OF THE FIGURE: Figure 1: of the vector pLex9-Parkin (135-290) MATERIALS AND TECHNIQUES IMPLEMENTED 1) Yeast strains:

<Desc / Clms Page number 15>

The strain L40 of the genus S.cerevisiae (Mata, his3D200, trpl-901, leu2-3,112, ade2, LYS2 :: (lexAop) 4-HIS3, URA3: :( lexAop) 8-LacZ, GAL4, GAL80) was used to verify protein-protein interactions when one of the protein partners is fused to the LexA protein. The latter is capable of recognizing the LexA response element controlling the expression of the reporter genes LacZ and His3.

 It was cultivated on the following culture media :.

Complete YPD medium: -Yeast extract (10 g / 1) (Difco) -Bactopeptone (20 g / 1) (Difco) -Glucose (20 g / 1) (Merck) This medium was made solid by addition of 20 g / 1 agar (Difco).

Minimum YNB medium: -Yeast Nitrogen Base (without amino acids) (6.7 g / 1) (Difco) - Glucose (20 g / 1) (Merck) This medium can be made solid by adding 20 g / 1 of agar (Difco). It can also be supplemented with amino acids and / or 3-amino-1,2,4-triazole by addition of CSM media [CSM-Leu, -Trp, -His (620 mg / 1), CSM-Trp (740 mg / 1) or CSM-Leu, -Trp (640 mg / l) (Biol01)] and / or 3-amino-1,2,4-triazole 2.5 mM.

2) Strains of bacteria:
The TG1 strain of Escherichia coli, of genotype supE, hsdA5, thi, A (lac- proAB), F '[tra D36 pro A + B + lacIq lacZAM15], was used for the construction of plasmids, as a means of amplification and isolation of the recombinant plasmids used. It was cultivated on the following medium: LB medium: -NaCI (5 g / 1) (Prolabo) -Bactotryptone (10 g / 1) (Difco) -Yeast extract (5 g / 1) (Difco) This medium is made solid by adding 15 g / l of agar (Difco).

<Desc / Clms Page number 16>

 Ampicillin was used at 100 μg / ml, this antibiotic is used to select the bacteria having received the plasmids carrying as marker the gene of resistance to this antibiotic.

 Escherichia coli strain HB101 of genotype supE44, aral4, galK2, lacYl, A (gpt-proA) 62, rpsL20 (Strr), xyl-5, mtl-1, recA13, A (mcrC-mrr), HsdS (rm ) has been used as a means of amplification and isolation of plasmids from the human lymphocyte cDNA library.

It was cultivated on M9 medium: -Na2HP04 (7 g / 1) (Prolabo) -KH2P04 (3 g / 1) (Prolabo) -NH4C1 (1 g / 1) (Prolabo) -NaCl (0.5 g / 1 ) (Prolabo) -Glucose (20 g / 1) (Sigma) -MgS04 (1 mM) (Prolabo) -Thiamine (0.001%) (Sigma) This medium is made solid by addition of 15 g / 1 of agar (Difco ).

Leucine (50 mg / 1) (Sigma) and proline (50 mg / 1) (Sigma) must be added to the M9 medium to allow the growth of the HB 101 strain.

 When selecting plasmids from the two lymphocyte cDNA hybrid library, leucine was not added to the medium because the plasmids carry a Leu2 selection marker.

3) Plasmids: -
The vector pLex9 (pBTM116) (Bartel et al., 1993) of 5 kb homologous to pGBT10 which contains a multiple cloning site located downstream of the sequence coding for the bacterial repressor LexA and upstream of a terminator to form a protein protein fusion. pLex-HaRasVall2, plasmid pLex9, as described in application WO98 / 21327, which contains the sequence coding for the protein HaRas mutated in position Va112 known to interact with the mammalian Raf protein (Vojtek and

<Desc / Clms Page number 17>

 al., 1993). This plasmid was used to test the interaction specificity of the PAP1 protein in the L40 strain. pLex9-cAPP, plasmid pLex9 which contains the sequence coding for the cytoplasmic domain of the APP protein known to interact with the PTB2 domain of FE65. This plasmid was used to test the interaction specificity of the PAP 1 protein in the L40 strain.

4) Synthetic oligonucleotides: TTAAGAATTC GGAAGTCCAG CAGGTAG (SEQ ID N 5) ATTAGGATCC CTACACACAA GGCAGGGAG (SEQ ID N 6) Oligonucleotides which made it possible to obtain the PCR fragment corresponding to the central region of Parkin bordered by the EcoRI and BamHI sites.

GCGTTTGGAA TCACTACAG (SEQ ID N 7) GGTCTCGGTG TGGCATC (SEQ ID N 8) CCGCTTGCTT GGAGGAAC (SEQ ID N 9) CGTATTTCTC CGCCTTGG (SEQ ID N 10) AATAGCTCGA GTCAGTGCAG GACAAGAG (used as sequence ID) 11 corresponding to the PAP1 gene.

 The oligonucleotides are synthesized on the Applied System ABI 394-08 device. They are detached from the synthesis matrix with ammonia and precipitated twice with 10 volumes of n-butanol and then taken up in water. The quantification is carried out by measuring the optical density (1DO260 corresponds to 30 g / ml).

5) Preparation of the plasmid DNAs.

 The preparations in small quantity and in large quantity of plasmid DNA were carried out according to the protocols recommended by the manufacturer Quiagen of the DNA purification kits:

<Desc / Clms Page number 18>

 - Quiaprep Spin Miniprep kit, ref: 27106 - Quiaprep Plasmid Maxiprep kit, ref: 12163.

6) Enzymatic amplification of DNA by PCR (Polymerase Chain Reaction):
The PCR reactions are carried out in a final volume of 100 l in the presence of the DNA template, dNTP (0.2 mM), PCR buffer (Tris-HCl pH 8.5 10 mM, 1 mM MgCl2, KCl 5 mM, 0.01% gelatin), 10-20 pmol of each of the oligonucleotides and 2.5 IU of Taq DNA polymerase Amplifier (Perkin Elmer). The mixture is covered with 2 drops of paraffin oil to limit the evaporation of the sample. The device used is the "Crocodile II" from Appligene.

 We used a denaturation temperature of the matrix of 94 C, a hybridization temperature of 52 C and a temperature of elongation by the enzyme at 72 C.

7) The ligatures:
All the ligation reactions are carried out at 37 ° C. for one hour in a final volume of 20 l in the presence of 100 to 200 ng of vector, 0.1 to 0.5 g of insert, 40 IU of T4 DNA ligase enzyme. (Biolabs) and a ligation buffer (50 mM Tris-HCl pH 7.8; 10 mM MgCl2; 10 mM DTT; 1 mM). The negative control consists of the ligation of the vector in the absence of an insert.

8) Transformation of bacteria:
The transformation of the bacteria by urrplasmid is carried out according to the following protocol: 10 μl of the ligation volume is used to transform the TG1 bacteria according to the method Chung's method (Chung et al., 1989). After transformation, the bacteria are spread on an LB + ampicillin medium and incubated for 16 h at 37 C.

9) Separation and extraction of DNA:
The DNA is separated according to their size by electrophoresis on agarose gel according to Maniatis (Maniatis et al., 1989): agarose gel

<Desc / Clms Page number 19>

at 1% (Gibco BRL) in TBE buffer (Tris base 90 mM; 90 mM; EDTA 2 mM) 10) Fluorescent sequencing of the plasmid DNAs:
The sequencing technique used is derived from Sanger's method (Sanger et al., 1977) and adapted for fluorescence sequencing developed by Applied Biosystems. The protocol used is that described by the designers of the system (Perkin Elmer, 1997).

11) Yeast transformation:
The plasmids are introduced into the yeast by a conventional yeast transformation technique developed by Gietz (Gietz et al., 1992) and modified as follows:
In the particular case of the transformation of yeast by the lymphocyte cDNA library, the yeast used contains the plasmid pLex9-Parkin (135-290) coding for the central part of Parkin fused with the protein LexA. It is cultivated in 200 ml of YNB minimum medium supplemented with amino acids CSM Trp at 3.0 C with stirring up to a density of 107 cells / ml. To carry out the transformation of the yeasts according to the preceding protocol, the cell suspension was separated into 10 tubes of 50 l in which 5 g of the library were added. The thermal shock was carried out for 20 minutes then the cells were collected by centrifugation and resuspended in 100 ml of YPD medium for 1 h at 30 C and in 100 ml of YNB medium supplemented with CSM-Leu, -Trp for 3 h 30 to 30 C .

The efficiency of the transformation is determined by spreading different dilutions of transformed cells on solid YNB medium supplemented with CSM-Trp, -Leu. After a culture at 30 ° C. for 3 days, the colonies obtained were counted and the transformation rate per g of DNA from the lymphocyte bank was determined.

12) Isolation of plasmids extracted from yeast:

<Desc / Clms Page number 20>

5 ml of a yeast culture incubated for 16 h at 30 ° C. are centrifuged and taken up in 200 l of lysis buffer (1M Sorbitol, KH2PO4 / K2HPO4 O, 1M pH7.4, zymolyase
12.5 mg / ml) and incubated for 1 hour at 37 C. The lysate is then treated according to the protocol recommended by the manufacturer Quiagen of the DNA purification kit, Quiaprep Spin Miniprep kit, ref 27106.

13) Test for ss-galactosidase activity:
A nitrocellulose sheet is previously deposited on the Petri dish containing the individualized yeast clones. This sheet is then immersed in liquid nitrogen for 30 seconds in order to burst the yeasts and thus release the B-galactosidase activity. After thawing, the nitrocellulose sheet is deposited, colonies upwards, in another Petri dish containing Whatman paper previously soaked with 1.5 ml of PBS solution (60 mM Na2HP04, 40 mM NaH2PO4, 10 mM KC1, MgSO4 1 mM, pH7) containing 15 l of X-Gal (5-bromo-4-chloro-3-indoyl-pD-galactoside) at 40 mg / ml of N, N-dimethylformamide.

The dish is then placed in an oven at 37 C. The test is said to be positive when the colonies turn blue on the membrane after 12 hours.

EXAMPLE 1: OF A VECTOR ALLOWING THE EXPRESSION OF A FUSION PROTEIN BETWEEN THE CENTRAL PARK OF THE PARKINE AND THE BACTERIAL REPRESSOR LEXA.

 Screening a library using the double hybrid system requires that the central region of Parkin be fused to a DNA-binding protein such as the bacterial repressor LexA. The expression of this fusion protein is carried out using the vector pLex9 (cf. materials and methods), into which has been introduced, in the same reading frame as the sequence corresponding to the LexA protein, the sequence coding for the central region of the Parkin appearing in the sequence presented in SEQ ID N 3 or 4.

<Desc / Clms Page number 21>

 The DNA fragment of 468 bpd corresponding to the 156 amino acids of the central region of Parkin which begins at amino acid 135 was obtained by PCR from the oligonucleotides (SEQ ID N 5 and N 6) which also allowed to introduce the EcoRI site at the 5 'end and a stop codon and a BamHI site at the 3' end. The PCR fragment was introduced between the EcoRI and BamHI sites of the cloning multisite of the plasmid pLex9 downstream of the sequence coding for the LexA protein to give the vector pLex9-Parkin (135-290) (Fig.l).

 The construction was verified by DNA sequencing. This verification made it possible to show that this fragment did not present mutations generated during the PCR reaction and that it was fused in the same open reading phase as that of the fragment corresponding to LexA.

EXAMPLE 2 SCREENING OF A LYMPHOCYTE FUSION BANK.

 We used the Double-Hybrid method (Fields and Song, 1989).

 The screening of a fusion library makes it possible to identify clones producing proteins fused to the GAL4 transactivating domain, which can interact with the protein of interest described in example 1 (central region of parkin). This interaction makes it possible to reconstitute a transactivator which will then be capable of inducing the expression of the reporter genes His3 and LacZ in the strain L40.

 To carry out this screening, we chose a fusion library produced from cDNA from peripheral human lymphocytes supplied by Richard Benarous (Peytavi et al., 1999). Yeasts were transformed by the lymphocyte bank and positive clones were selected as described below.

 During screening, it is necessary to preserve the probability that each plasmid independent of the fusion library will be present in at least one yeast at the same time as the plasmid pLex9-Parkin (135-290). To preserve this probability, it is important to have a good yeast transformation efficiency. For this we have chosen a yeast transformation protocol

<Desc / Clms Page number 22>

 giving an efficiency of 2.6 × 10 10 transformed cells per g of DNA. In addition, since the cotransformation of the yeast by two different plasmids reduces this efficiency, we preferred to use a yeast previously transformed by the plasmid pLex9-Parkin (135-290). This strain L40 pLex9-Parkin (135-290) of the His-, Lys-, Leu-, Ad- phenotype was transformed with 50 μg of plasmid DNA from the fusion library. This quantity of DNA enabled us to obtain, after estimation, 1.3 107 transformed cells, which corresponds to a number slightly higher than the number of independent plasmids which constitute the library. From this result we can think that almost all of the plasmids from the bank were used to transform yeasts. The selection of the transformed cells, capable of reconstituting a functional transactivator, was made on a YNB medium supplemented with 2.5 mM 3-amino-1,2,4-triazole and 620 mg / 1 of CSM (Bio101) not containing leucine histidine and tryptophan.

 After this selection, many clones with a His + phenotype were obtained. On these transformants, a P-galactosidase activity test was carried out in order to validate by the expression of the other reporter gene, LacZ, this number of clones obtained. 115 clones exhibited the double His + phenotype, p-Gal + possibly corresponding to a protein-protein interaction.

 EXAMPLE 3: BANKING PLASMIDS IN THE SELECTED CLONES.

 In order to identify the proteins that can interact with the central region of Parkin, the plasmids of the fusion bank contained in the yeasts selected during the double hybrid screening were extracted. To be able to obtain large quantities, this isolation requires a transformation of E. coli beforehand with a DNA extract of the positive yeast strains. As the bank plasmid contained in this extract is a yeast / E. coli shuttle plasmid, it can easily replicate in bacteria. The bank plasmid was selected by

<Desc / Clms Page number 23>

 complementation of the bacterium HB101 auxotrophic for leucine on medium lacking leucine.

 The plasmid DNAs of the bacterial colonies obtained after transformation with yeast DNA extracts were analyzed by digestion with restriction enzymes and separation of the DNA fragments on agarose gel.

Among the 115 clones analyzed, a clone containing a plasmid from the library having a profile different from the others was obtained. This plasmid, called pGADLyl 1 lb, has been more specifically studied.

EXAMPLE 4: OF THE SEQUENCE OF THE INSERT CONTAINED IN THE IDENTIFIED PLASMID.

 The sequencing of the insert contained in the identified plasmid was carried out firstly from the oligonucleotide SEQ ID N 7 complementary to the sequence of GAL4TA near the EcoRI site of insertion of the cDNA library of lymphocytes. Then in a second step from the oligonucleotides SEQ ID N 8 to SEQ ID N 11, corresponding to the sequence of the insert obtained during the progress of the sequencing. The sequence obtained is presented on the sequence SEQ ID NO1. The protein thus identified was designated PAP1 (Parkin-Associated Protein 1).

 Comparison of the sequence of this insert with the sequences contained in the GENBank and EMBL (European Molecular Biology Lab) databases showed 25% protein homology with different members of the Synaptotagmins family. Synaptotagmins are part of a family of membrane proteins encoded by at least eleven different genes expressed in the brain and other tissues. They contain a unique transmembrane domain and two calcium-regulated domains called C2. It is in this area that we find the homology between Synaptotagmins and the PAP protein 1. No other significant homology was observed.

<Desc / Clms Page number 24>

EXAMPLE 5: ANALYSIS OF THE SPECIFICITY OF INTERACTION BETWEEN THE CENTRAL PARKIN REGION AND THE PAPI PROTEIN.

 In order to determine the specificity of interaction between the fragment corresponding to the PAP1 protein and the central region of Parkin, a specific interaction test between two hybrids with other non-relevant proteins was carried out. To carry out this test, we transformed the L40 strain by the control plasmids plex9-cAPP or pLex9-HaRasVa112 in place of the plasmid pLex9-Parkin (135-290) coding respectively for the cytoplasmic domain of APP or the protein HaRasVall2 to the domain, the binding to the DNA of LexA and by the plasmid isolated during the screening of the library of two hybrids. A P-Gal activity test on cells transformed by the various plasmids was carried out in order to determine a protein-protein interaction. According to the test result, only the yeasts transformed by the plasmid isolated during the screening of the two hybrid library and by the plasmid pLex9-Parkin (135-290) exhibited an ss-Gal + activity, thus showing an interaction between the central region. Parkin and PAPI protein. This interaction therefore appears to be specific since this fragment of PAP1 does not seem to interact with the cAPP or HaRasVa112 proteins.

 These results therefore show the existence of a new protein, designated PAP1, capable of interacting specifically with Parkin. This protein, related to synaptotagmins, has no significant homology with known proteins, and can be used in therapeutic or diagnostic applications, for the production of antibodies, probes or peptides or for the screening of active molecules.

<Desc / Clms Page number 25>

 BIBLIOGRAPHICAL REFERENCES Abbas, N. et al. (1999). A wide variety of mutations in the parkin gene are responsible for autosomal recessive parkinsonism in Europe. Hum Mol Genet.

 8, 567-574.

Bartel, P.L. et al. (1993). D.A Hartley Ed, Oxford University press, 153 Chung, CT. et al. (1989). One-step preparation of competent Escherichia coli: transformation andstorage of bacterial cells in the same solution. Proc. Natl.

 Acad. Sci. USA. 86.2172-2175.

Fields, S. and Song, 0. (1989). A novel genetic system to detect protein-protein interactions. Nature. 340.245-246.

Gietz, RD. et al. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res., 20,1425.

Hattori, N. et al. (1998). Molecular genetic analysis of a novel Parkin gene in
Japanese families with autosomal recessive juvenile parkinsonism: evidence for variable homozygous deletions in the Parkin gene in affected individuals. Ann
Neurol. 6, 935-941.

Kahn, A. et al., (1991) Gene therapy: hopes and limits. Medicine and Sciences. 7,
705-714.

Kitada, T. et al. (1998). Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature. 392.605-608.

Leroy, E. et al. (1998). The ubiquitin pathway in Parkinson's disease. Nature. 395, 451-452. - Lewy, FH (1912). in Handbuch der Neurologie (Lewandowski, M., ed) pp 920 -
933, Springer, Berlin Lucking, C. et al. (1998). Homozygous deletions in parkin gene in European and
North African families with autosomal recessive juvenile parkinsonism. Lancet.

 352.1355-1356.

Maniatis, T. et al. (1989). Molecular cloning, scond edition. Cold spring harbor
Laboratory. Cold Spring Harbor, NY

<Desc / Clms Page number 26>

Morett, E. (1999). A novel transactivation domain in parkin. Trends Biochem Sci. 24,
229-231.

Peytavi, R. et al. (1999). HEED, the product of the human homolog of the murine eed gene, binds to the matrix protein of HIV-1. J. Biol. Chem. 274, 1635-1645.

Polymeropoulos, M.H. et al. (1997). Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science. 276.2045-2047.

Sanger, F. et al. (1977). DNA sequencing with chain terminating inhibitors. Proc.

 Natl. Acad. Sci. USA. 74.5463-5467.

Shimura, H. et al. (1999). Immunohistochemical and subcellular localization of
Parkin protein: absence ofprotein in autosomal recessive juvenile parkinsonism patients. Ann Neurol. 45,668-672.

Sunada, Y. et al. (1998). Differential expression of the parkin gene in the human brain and peripheral leukocytes. Neurosci Lett. 254,180-182.

Vojtek, AB. et al., (1993). Mammalian Ras interacts directly with the serine / threonine kinase Raf. Cell. 74, 205-214.

Claims (25)

1. Compound capable of modulating, at least partially, the interaction between the PAP1 protein or a homolog thereof, and Parkin.  2. Compound according to claim 1, characterized in that it slows down, inhibits or stimulates, at least partially, said interaction.  3. Compound according to one of claims 1 or 2, characterized in that it is capable of binding the interaction domain between the PAPI protein or a homolog thereof, and the Parkin.  4. Compound according to one of claims 1 to 3, characterized in that it is a compound of peptide, nucleic, lipid, saccharide type or of an antibody.  5. Compound according to claim 4, characterized in that it is a peptide compound comprising all or part of the peptide sequence SEQ ID N 2 or one of its derivatives.  6. Compound according to claim 4, characterized in that it is a peptide compound comprising a region whose sequence corresponds to all or a functional part of the site of interaction of the protein PAP1 with Parkin.  7. Compound according to claim 4, characterized in that it is a peptide compound derived from the PAP1 protein (and / or homologous forms) carrying an effector region made non-functional.  8. Polypeptide comprising the sequence SEQ ID NO: 2 or a derivative or fragment thereof.  9. The polypeptide according to claim 8, comprising at least 5 consecutive residues of the sequence SEQ ID NO: 2, preferably at least 9, more preferably at least 15.  10. Nucleic acid coding for a peptide compound according to one of claims 4 to 9. <Desc / Clms Page number 28>  11. Nucleic acid according to claim 10, characterized in that it comprises all or part of the sequence SEQ ID NO: 1 or of a sequence derived therefrom.  12. Nucleic acid encoding a polypeptide according to claim 8.  13. Nucleic acid, in particular a nucleotide probe, capable of hybridizing with a nucleic acid according to one of claims 10 to 12 or with their complementary strand.  14. Vector comprising a nucleic acid according to one of claims 10 to 13.  15. Defective recombinant virus comprising a nucleic acid according to one of claims 10 to 13.  16. Antibody or antibody fragment or derivative, characterized in that it is directed against a peptide compound according to one of claims 4 to 9.  17. Antibody according to claim 16, characterized in that it recognizes a polypeptide according to claim 9.  18. Pharmaceutical composition comprising at least one compound or polypeptide according to one of claims 1 to 9 or an antibody according to claim 16 or 17.  19. A non-peptide compound or a compound of a non-exclusively peptide nature capable of modulating, at least partially, the interaction of the protein PAP1 or a homolog thereof, with Parkin.  20. Compound according to claim 19 characterized in that the active units of a peptide according to one of claims 5 to 7 have been duplicated with a non-peptide structure or of a nature which is not exclusively peptide. <Desc / Clms Page number 29>  21. Pharmaceutical composition comprising at least one nucleic acid according to one of claims 10 to 13 or a vector according to claim 14.  22. Pharmaceutical composition comprising a peptide compound according to one of claims 4 to 9.  23. Composition according to claim 20, 21 or 22, intended for the treatment of neurodegenerative pathologies.  24. A method for screening or characterizing active molecules, comprising a step of selecting molecules capable of binding the sequence SEQ ID N02 or the sequence SEQ ID N04, or a fragment thereof.  25. A method of producing a peptide compound according to one of claims 4 to 9, comprising culturing a cell containing a nucleic acid according to one of claims 10 to 13 or a vector according to claim 14, in conditions for expression of said nucleic acid, and recovery of the peptide compound produced.