PT1259606E - Compositions useful for regulating parkin gene activity - Google Patents

Description

DESCRIPTION " COMPOSITIONS USED TO REGULATE THE ACTIVITY OF PARKINE " The present invention relates to compositions and methods usable for regulating parkin activity. This relates in particular to a novel protein, designated PAP1, partner of the parkin, as well as the peptides or polypeptides derived or homologues of this protein. This also relates to compounds capable of modulating, at least partially, Parkine's activity, namely to interfere with the interaction between parkin and PAP1. The present invention is useful in the therapeutic, diagnostic or pharmacological domains for the development of new drugs. The Parkine gene is mutated in certain family 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 suffering from this disease have neurological disorders grouped under the term Parkinson's syndrome, characterized by stiffness, bradykinesia and tremor at rest. These symptoms are the consequence of a degeneration of the dopaminergic neurons of the substantia nigra of the brain. Most cases of Parkinson's disease have no family history. However, there are familial cases where 1 some correspond to a monogenic form of the disease.

At present, only three different genes have been identified in some rare hereditary forms. The first form corresponds to an autosomal dominant form, in which the gene responsible encodes for Synuclein alfa (Polymeropoulos et al., 1997). This protein is an abundant constituent of intracytoplasmic inclusions, termed Lewy bodies, which serve as markers of Parkinson's disease (Lewy, 1912). The second form, also autosomal dominant, is related to a mutation in a gene coding for a hydrolase designated carboxyl-terminal hydrolase of ubiquitin L1 (Leroy et al., 1998). This enzyme is supposed to hydrolyze the ubiquitin polymers or conjugates to ubiquitin monomers. The third form is distinguished from previous ones by an autosomal recessive transmission and an appearance often before the age of 40 as well as by an absence of Lewy bodies. These patients respond more favorably to levodopa, a precursor of dopamine used as a treatment for Parkinson's disease. The gene related to this form codes for a new protein called Parkina (Kitada et al., 1998). The Parkine gene consists of 12 exons covering a genomic region of more than 500,000 base pairs on chromosome 6 (6q25.2-q27). Currently, two major types of mutation of this gene at the origin of the disease are known, either deletions of variable size in the region covering exons 2 to 9, or point mutations that produce the premature appearance of a stop codon or the modification of a 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 mode of autosomal recessive transmission suggest a loss of Parkine's 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 that come from different alternative processes (Kitada et al., 1998; Sunada et al.

1998). In the brain, two types of messenger RNA are found in which one is devoid of the part corresponding to exon 5. RNAs from Parkina containing the coding region for exons 3, 4 and 5 were identified in the leucocytes. Messenger RNAs of the longer Parkina, present in the brain, contain 2960 bases and encode a protein with 465 amino acids.

This protein has a low homology to its N-terminal part with ubiquitin. Its part of the C terminal contains two " ring finger " separated by an IBR (In Between Ring) domain corresponding to a cysteine-rich region that can bind to metals such as " ring finger " (Morett, 1999). It has been shown by immunocytochemistry that parkin is located in the cytoplasm and Golgi apparatus of melanin-containing black matter neurons (Shimura et al., 1999). In addition, this protein is present in certain Lewy bodies of patients with Parkinson's. Parkina's cellular function has not yet been demonstrated, but it may play a role as a carrier in synaptic vesicles, in maturation or protein degradation, and in growth control, cell differentiation or development. Parkina is absent in juvenile autosomal recessive forms, thus confirming that the loss of this function is responsible for the disease. The elucidation of the exact role of the Parkine protein in the process of dopaminergic neuron degeneration therefore constitutes a significant advantage for the understanding and therapeutic approach of Parkinson's disease and more generally of diseases of the central nervous system. The present invention is based on the identification of a parkine partner that interacts with this protein under physiological conditions. This partner represents a novel pharmacological target for the production or investigation of compounds capable of modulating parkin activity, namely its activity in the degeneration of dopaminergic neurons and / or the development of nerve pathologies. This protein, the antibodies, the corresponding nucleic acids, as well as the specific probes or primers, are also usable in the detection or assay of proteins in biological samples, especially in nerve tissue samples. These proteins or nucleic acids are also usable in therapeutic approaches to modulate the activity of the parkin, as well as any compound according to the invention, capable of modulating the interaction between the parkin and the polypeptides of the invention. The present invention results more particularly from the applicant's identification of a novel human protein, designated PAP1 (Parkine Associated Protein 1) or LY111, which interacts with parkin. The PAP1 protein (sequence SEQ ID NO: 1 or 2) shows some homology with the synaptotagmines and is able to interact more particularly with the central region of the parkin (represented in the sequence SEQ ID NO: 3 or 4). The PAP1 protein was also cloned, sequenced and characterized from different tissues of human origin, namely the lung (SEQ ID NO: 12, 13) and the brain (SEQ ID NO: 42, 43), as well as forms corresponding to the processing variants (SEQ ID NO: 14, 15, 44, 45). The present invention also results from the identification and characterization of particular regions of the PAP1 protein involved in the modulation of parkin function. Detecting the existence of this protein and regions involved in its function enables in particular to prepare new compounds and / or compositions usable as pharmaceutical agents and to develop industrial methods of screening for such compounds.

A first object of the invention relates to a polypeptide which interacts with the protein having, over its entire length, at least 95% identity with the sequence of SEQ ID NO: 2.

A further aspect of the invention resides in a nucleic acid encoding the PAP1 protein, to its fragments, derivatives or homologues, as well as any vector comprising said nucleic acid, any recombinant cell containing said nucleic acid or vector and any non-human mammal comprising, in its cells , this nucleic acid. The invention also relates to antibodies capable of binding to the PAP1 protein, to its fragments, derivatives and homologs, in particular polyclonal or monoclonal antibodies, more preferably antibodies capable of binding to the PAP1 protein and inhibiting, at least partially, its interaction with parkina. 5

A further aspect of the invention relates to probes or nucleotide primers, specific for PAP1, useful for detecting or amplifying the PAP1 gene or a region thereof in any biological sample. The invention further relates to pharmaceutical compositions, methods of detecting genetic defects, methods of producing polypeptides, as defined hereinbefore, as well as methods of separating or characterizing active compounds.

Within the scope of the present invention, the designation protein PAP1 designates the protein, as well as all its homologous forms. By homologous means, it is meant to designate all proteins equivalent to the protein in question, of diverse cellular origin and, in particular, coming from cells of human or other organism origin and having an activity of the same type. These homologues also comprise the natural variants of the PAP1 protein with the sequence SEQ ID NO: 2, in particular the polymorphic or processing variants. Such homologues may be obtained by hybridization experiments between coding nucleic acids (namely the nucleic acid of the sequence SEQ ID NO: 1). Within the scope of the invention, it is sufficient for such a sequence to exhibit a significant percentage of identity to give a physiological behavior comparable to that of the PAP1 protein as claimed. In this regard, variants and / or homologs of the sequence SEQ ID NO: 2 are described in sequences SEQ ID NO: 13, 15, 43 and 45, identified from tissues of human origin. The term PAP1 therefore also encompasses these polypeptides. The " identity percentage " between two nucleotide or 6 amino acid sequences within the scope of the present invention can be determined by comparing two optimally aligned sequences through a comparison window. The portion of the nucleotide or polypeptide sequence in the comparison window may thus comprise additions or deletions (e.g. " spaces ") in comparison with the reference sequence (which does not comprise these additions or deletions), so as to obtain a optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which a nucleic base or an amino acid residue identical to the two compared (nucleic or peptidic) sequences is observed, then dividing the number of positions in which identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of the sequences for comparison can be accomplished computerized with the aid of known algorithms contained in the software of the Company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN. By way of illustration, the percentage of sequence identity may be realized with the aid of BLAST software (BLAST versions 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 and BLAST 2.0.6 of September 1998), using exclusively the default parameters (Altschul et al., J. Mol. Biol., (1990) 215: 403-410; Altschul et al., Nucleic Acids 7

Res. (1997) 25: 3389-3402). Blast looks for sequences homologous to an " reporting " using the algorithm of Altschul et al. (Supra). The reporting sequence and the databases used may be peptidic or nucleic, any combination being possible.

Within the scope of the present invention, the term derivative indicates any sequence other than the sequence considered due to a degeneracy of the genetic code, obtained by one or more modifications of a genetic and / or chemical nature, as well as any peptide encoded by a sequence hybridizing to the sequence nucleic acid SEQ ID NO: 1 or a fragment thereof, for example with the nucleotide sequence SEQ ID NO: 12, 14, 42 or 44 or a fragment thereof and having the ability to interfere with the level of interaction between the PAP1 protein, or one of its counterparts, and Parkina. By genetic and / or chemical modification, any mutation, substitution, elimination, addition and / or modification of one or more residues can be understood. The term derivative also comprises sequences homologous to the sequence envisaged from other cell sources and in particular from cells of human or other organism origin and having an activity of the same type. Such homologous sequences may be obtained by hybridization experiments. Hybridizations can be performed from nucleic acid libraries using the native sequence or a fragment thereof as probe under variable hybridization conditions (Maniatis et al., 1989). In addition, the term " fragment " or " part " refers to any portion of the molecule in question, comprising at least 5 consecutive residues, preferably at least 9 consecutive residues, even more preferably at least 15 consecutive residues. Typical fragments may comprise at least 25 consecutive residues. 8

Such derivatives or fragments may be produced for different purposes, such as in particular to increase their therapeutic efficacy or to reduce their side effects or to impart new pharmacokinetic and / or biological properties.

A specific object of this invention relates to the PAP1 protein of the sequence SEQ ID NO: 13 or 43.

A further object of the invention resides in antibodies or fragments or derivatives of polyclonal or monoclonal antibodies directed against a polypeptide as hereinbefore defined. Such antibodies may be produced by methods known to the person skilled in the art. In particular, such antibodies may be prepared by immunizing an animal against a peptidic compound of the invention (in particular a polypeptide or a peptide comprising all or part of the sequence of SEQ ID NO: 2), collection of blood and isolation of antibodies. These antibodies may also be produced by preparing hybridomas according to techniques known to the person skilled in the art.

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

In addition, such antibodies may also be used to detect and / or dose the expression of PAP1 in biological samples and for this reason to report on their activation state. 9

Fragments or derivatives of antibodies are, for example, Fab, Fab'2 fragments, single chain antibodies (ScFv), etc. These are in particular any fragment or derivative retaining the antigenic specificity of the antibodies from which they originate.

Antibodies according to the invention are more preferably capable of binding to PAP1 proteins comprising the sequence of SEQ ID NO: 2, 13, 15, 43 or 45, namely the region of this protein involved in the interaction with the parkina. These antibodies (or fragments or derivative) are more preferably capable of binding to an epitope present in the sequence comprised between residues 1 and 344 of the sequence SEQ ID NO: 2. The invention also relates to compounds non-peptidic or non-peptidic exclusively usable as pharmaceutical agents. It is in fact possible, from the active protein motifs described in the present application, to prepare non-peptidic peptidic activity modulating molecules of PAP1, in particular by doubling the active motifs of the peptides with a non-peptidic or nature structure not exclusively peptidic. The present invention also relates to any nucleic acid encoding a peptidic compound according to the invention. It may in particular be a nucleic acid comprising all or part of the sequence SEQ ID NO: 1, 12, 14, 42 or 44 or a derivative thereof. By derived sequence, within the scope of the present invention is meant any sequence hybridizing to the sequence set forth in SEQ ID No. 1 or a fragment thereof and encoding a peptidic compound according to the invention, as well as the resulting sequences by the degeneracy of the genetic code. The nucleic acids according to the invention comprise, for example, all or part of the nucleic sequence SEQ ID NO: 12, 14, 42 or 44. The present invention further relates to sequences having a significant percentage of identity with the sequence set forth in SEQ ID No. 1 or a fragment thereof and coding for a peptide compound exhibiting a physiological behavior comparable to that of the PAP1 protein. By percentage of significant identity is meant a percentage of at least 60%, preferably 80%, more preferably 90%, and even more preferably 95%.

The different nucleotide sequences of the invention may be of artificial origin or not. These may be genomic sequences, cDNAs, RNAs, hybrid sequences or synthetic or semisynthetic sequences. These sequences may be obtained by screening of DNA libraries (cDNA library, genomic DNA library) or by chemical synthesis or by various methods including the chemical or enzymatic modification of sequences obtained by library screening either by homology research on bases nucleic or protein data. The above-mentioned hybridization is preferably performed under the conditions described by Sambrook et al. (1989, pp. 9.52-9.55). It is advantageously carried out under stringency hybridization conditions. By " stringency hybridization conditions " in the context of the present invention, the following conditions will be understood: 1- Membrane competition and PRE-HYBRIDIZATION:

Mix: 40 μl salmon sperm DNA (10 mg / ml) + 40 μl human placental DNA (10 mg / ml)

Denature for 5 min at 96 ° C, then dip the mixture on ice.

Discard the 2X SSC buffer and pour 4 mL formamide blend into the hybridization tube containing the membranes.

Add the mixture of the two denatured DNAs.

Incubation at 42 ° C for 5 to 6 hours with rotation. 2- Competition of the marked probe:

Add 10 to 50 μl of Cot I I DNA to the labeled and purified probe, according to the amount of non-specific hybridizations.

Denature for 7 to 10 min at 95 ° C.

Incubate at 65 ° C for 2 to 5 hours. 12 3- Hybridization:

Discard the prehybridization mixture.

Mix 40 μl salmon sperm DNA + 40 μl human placental DNA; denature for 5 min at 96 ° C, then dip onto ice.

Add 4 ml of the formamide mixture, the mixture of the two DNAs and the labeled probe / DNA denatured with Cot I to the hybridization tube.

Incubate for 15-20 hours at 42 ° C with rotation. 4- Washing:

A wash at room temperature in 2X SSC, to rinse. 2 times 5 minutes in 2X SSC and 0.1% SDS at room temperature. 2 x 15 minutes 0.1 X SSC and 0.1% SDS at 65 ° C.

Wind the membranes in adherent film and expose.

The hybridization conditions described above are adapted to hybridization under conditions of strong severity, of a common acid nucleic acid variable length from 20 nucleotides to several hundreds of nucleotides. 13

It will be apparent that the hybridization conditions described above can be adapted according to the length of the nucleic acid whose hybridization is required or the type of labeling selected, according to techniques known to the person skilled in the art.

Suitable hybridization conditions may, for example, be adapted according to the teachings contained in the work of HAMES and HIGGINS (1985) (Nucleic acid Hybridization Approach Approach, Hames and Higgins Ed., IRL Press, Oxford) or in the work of F AUSUBEL et al. (1999) (Currents Protocols in Molecular Blology, Green Publishing Associates and Wiley Interscience, N.I).

A particular nucleic acid within the scope of the invention codes for a polypeptide comprising the sequence SEQ ID NO: 2 or a fragment or derivative thereof, namely for the human protein PAP1. It is advantageously a nucleic acid comprising the nucleic sequence SEQ ID NO: 1, 12, 14, 42 or 44.

Such nucleic acids may be used for the production of the peptidic compounds of the invention. The present application thus relates to a method of preparing such peptide compounds according to which a cell containing a nucleic acid according to the invention is cultured under conditions of expression of said nucleic acid and recovering the produced peptidic compound . In this case, the coding portion for said peptidic compound is generally placed under the control of signals, allowing expression in a cellular host. Selection of these signals (promoters, terminators, secretion leader, sequence, etc.) may vary according to the cellular host used. In addition, the nucleic acids of the invention may form part of a vector which may be autonomous or integrative replication. More particularly, vectors with autonomous replication can be prepared using sequences with autonomous replication in the selected host. As integrative vectors, they may be prepared for example using sequences homologous to certain regions of the host genome, allowing integration of the vector by homologous recombination. It may be a vector of the plasmid, episomal, chromosomal, viral, etc. type.

The cell hosts usable for the production of the peptide compounds of the invention recombinantly are both eukaryotic and prokaryotic hosts. Among suitable eukaryotic hosts, animal cells, yeast or fungi may be referred to. Namely, in the case of yeasts, yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be mentioned. By treating animal cells, COS, CHO, Cl 2 7, PCI 2, etc. cells can be referred to. Among the fungi, more particularly Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferred to use the following bacteria E. coli Bacillus or Streptomyces. The present invention furthermore has as object non-human mammals comprising in their cells a nucleic acid or a vector according to the invention.

These mammals (rodents, canines, rabbits, etc.) are particularly useful in the study of the properties of PAP1 and in the identification of compounds with a therapeutic perspective. Modification of the genome of that transgenic animal may result in a modification or modification of one or more genes by " knock-in " or " knock-out ". This modification may be carried out using normal altering or mutagenic agents or, then, by site-directed mutagenesis. Modification of the genome may also result from an insertion of gene (s) or replacement of gene (s) in its wild or mutated form. Modifications of the genome are advantageously performed on reproductive stem cells and, advantageously, on the pronuclei. Transgenesis can be accomplished by microinjection of an expression cassette comprising the genes modified in the two fertilized pronuclei. Thus, an animal according to the invention may be obtained by injection of an expression cassette comprising a nucleic acid. Preferably, this nucleic acid is a DNA which may be a genomic DNA (ADNg) or a complementary DNA (cDNA). The construction of transgenic animals according to the invention can be carried out according to the usual techniques well known to the person skilled in the art. The person skilled in the art will be able to refer in particular to the production of transgenic animals and, particularly, to the production of transgenic mice, as described in patents US 4873191, US 5464764 and US 5789215, the contents of these documents being incorporated herein by reference.

Briefly, a polynucleotide construct comprising a nucleic acid according to the invention is inserted into a line of ES-like stem cells. The insertion of the polynucleotide construct is preferably performed by electroporation, as described by Thomas et al. (1987, Cell, Vol. 51: 503-512). 16

Cells that undergo the electroporation step are then screened for the presence of the polynucleotide construct (for example, by selection using labels or further by PCR or Southern blotting) to select positive cells having integrated the exogenous polynucleotide construct into its genome, if necessary, following a homologous recombination event. This technique is, for example, described by MANSOUR et al. (Nature (1988) 336: 348-352).

Then, the positively selected cells are isolated, cloned and injected into 3.5-day-old mouse blastocysts, as described by BRADLEY (1987, Production and Analysis of Chimaeric Mice, in EJ ROBERTSON (ed., Teratocarcinomas and embryonic stem The blastocysts are then introduced into a female host animal and the development of the embryo continues through to the end.

According to one alternative, positively selected ES-type cells are contacted with 2.5 day embryos at a stage of 8-16 cells (morulae)

as described by WOOD et al. (1993, Proc Natl Acad Sci USA, vol 90: 4582-4585) or by NAGY et al. (1993, Proc Natl Acad Sci USA, vol 90: 8424-8428), the ES cells being internalized to colonize extensively the blastocyst, including the cells that give rise to the germ line.

The offspring are then tested to determine the ones that integrated the polynucleotide construct (the transgene). 17

The nucleic acids according to the invention may also be used for the preparation of genetic antisense or antisense oligonucleotides usable as pharmaceutical agents. The antisense sequences are oligonucleotides of small size, complementary to the coding strand for a given gene and therefore capable of specifically hybridizing to the transcribed mRNA, inhibiting their translation into protein. The aim of the invention is therefore to provide antisense sequences capable of at least partially inhibiting the interaction of PAP1 proteins with Parkin. Such sequences may comprise all or part of the nucleic sequences defined hereinbefore. These are generally sequences or sequence fragments complementary to coding sequences for peptides which interact 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 in vivo transfer and expression of antisense or peptide sequences capable of modulating the interaction of PAP1 protein with Parkin. In this context, the sequences may be incorporated into viral or non-viral vectors, allowing their administration in vivo (Kahn et al., 1991). By way of viral vectors according to the invention may be mentioned, in particular, adenovirus, retrovirus, adenovirus (AAV) -related (AAV) virus or herpes virus vectors. The subject of the present application is also deficient recombinant viruses comprising a nucleic acid encoding a polypeptide according to the invention, in particular a polypeptide or a peptide comprising all or part of the sequence SEQ H 2 or a derivative thereof, for example, all or part of the sequence SEQ ID NO: 12, 14, 42 or 44 or derivatives thereof. The invention also allows the preparation of nucleotide probes, synthetic or non-synthetic, capable of hybridizing to the nucleotide sequences defined hereinbefore or to the complementary strand thereof. These probes may be used in vitro as a diagnostic tool for detecting expression or over-expression of PAP1 or for the description of genetic abnormalities (incorrect processing, polymorphism, point modifications, etc.). These probes may also be used for the detection and isolation of homologous nucleic acid sequences encoding peptides, as defined above, from other cellular sources and preferably from cells of human origin. The probes of the invention generally comprise at least 10 bases and these may, for example, comprise up to all of the aforementioned sequences or their complementary strand. Preferably, these probes are labeled prior to use. Accordingly, various techniques known to the person skilled in the art (radioactive labeling, fluorescent, enzymatic, chemical, etc.) may be used. The invention also relates to primers or primer pairs that allow amplification of all or part of a nucleic acid encoding a PAP1, for example, a primer having a sequence selected from SEQ ID NOs: 16-41. The invention further aims at any pharmaceutical composition comprising as the active ingredient at least one compound as defined above, namely a peptide compound.

The subject of the invention is 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 at least one nucleic acid or vector as defined above.

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

In addition, it is also directed 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 Parkine protein and for this reason maintain the survival of the dopaminergic neurons. More particularly, these pharmaceutical compositions are intended to modulate the interaction between PAP1 protein and Parkin. It is a more preferred mode of pharmaceutical compositions for the treatment of diseases of the central nervous system, such as, for example, Parkinson's disease. The invention furthermore relates to the use of molecules described hereinbefore for modulating Parkin's activity or typing diseases of the central nervous system. In particular the invention relates to the use of these molecules to modulate, at least partially, the activity of Parkine. The invention also relates to a method for the screening or characterization of active molecules on the role of parkin, comprising the selection of molecules capable of binding to the sequence of SEQ ID NO: 2 or to the sequence of SEQ ID NO: 4 or a fragment thereof (or derivative thereof). The method advantageously comprises contacting in vitro the test molecule (s) with a polypeptide comprising the sequence of SEQ ID NO: 2 or the sequence SEQ ID NO: 4 or a fragment (or derivative thereof) thereof and selection of molecules capable of binding to the sequence SEQ ID NO: 2 (namely, the region comprised between residues 1 and 344) or sequence SEQ ID NO: 4. The molecules tested may be of a varied nature (peptide, nucleic , lipid, sugar, etc., or mixtures of such molecules, for example combinatorial libraries, etc.). As indicated hereinbefore, the molecules identified in this way can be used to modulate Parkine protein activity and represent potential therapeutic agents for the treatment of neurodegenerative disorders.

Other advantages of the present invention will emerge from reading the following examples and figures which are to be considered as illustrative and not limiting. FIGURE LEGENDS:

Figure 1: Representation of the vector pLex9-Parkin (135-290) 21

Figure 2: Results from the first 5'-RACE experiment. Eight clones were obtained. The initial electronic sequence is indicated at the end of the figure.

Figure 3: Results of the second 5'-RACE experiment. Only two of the 8 clones obtained in the first experiment (clones A12 and D5) were validated. The initial electronic sequence is indicated at the end of the figure. The complete DNA and protein sequence is provided in Sequences 12-15.

Figure 4: Detail of the organization of clones C5 and D4 from the second 5'-RACE experiment. The resulting consensus sequence is indicated at the top of the figure.

Figure 5: Structure of transcripts isolated from human brain.

Figure 6: Nuclear and protein sequence of LY111 (complete) human brain. Double underscore: conserved cysteines from the zinc finger domain. Bold: Domain C2I, Italic: domain C22.

Figure 7: LY111 (short version) nucleic and protein sequence of the human brain. Double underscore: conserved cysteines from the zinc finger domain. Bold: Domain C2I, Italic: domain C22.

Figure 8: Location of short (8b) or long (8a) LY111 protein after expression in Cos-7 cells.

Figure 9: Nucleic and protein sequence of LY111 (long version) of human lung. 22

Figure 10: Nuclear and protein sequence of LY 111 (short version) of human brain.

MATERIALS AND TECHNIQUES PERFORMED 1) Yeast strains:

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

This was cultured in the following culture medium:

Full YPD medium:

Yeast extract (10 g / L) (Difco)

Bactopeptone (20 g / L) (Difco)

Glucose (20 g / L) (Merck)

This medium was made solid by addition of 20 g / L agar (Difco). 23

Minimum YNB Medium:

Yeast Nitrogen Base (without amino acids) (6.7 g / L) (Difco)

Glucose (20 g / L) (Merck)

This medium can be rendered solid by the addition of 20 g / L agar (Difco). It may 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 / L), CSM-Trp (740 mg / L ) or CSM-Leu, -Trp (640 mg / L) (BiolO1) and / or 2.5 mM 3-amino-1,2,4-triazole. 2) Bacterial Strains:

Escherichia coli TG1 strain, supE genotype, hsdA5, thi, Δ (lac-proAB), F '[tra D36 pro A + B + laclq lacZAM15] was used in the construction of plasmids as a means of amplification and isolation of plasmids used. This was grown in the following medium:

LB medium:

NaCl (5 g / L) (Prolabo)

Bacto-tryptone (10 g / L) (Difco) Yeast extract (5 g / L) (Difco) 24

This medium is rendered solid by agar addition (Difco) 15 g / L.

Ampicillin at 100 pg / ml was used, this antibiotic is used to select the bacteria that received the plasmids containing the antibiotic resistance gene as marker.

Escherichia coli strain HB101 of the genotype supE44, aral4, galK2, lacY1, Δ (gpt-proA) 62, rpsL20 (Strr), xyl-5, mtl-1, recA13, A (mcrC-mrr), HsdS- (rm-) as amplification and isolation of plasmids from the human lymphocyte cDNA library. This was grown in

M1 Medium: Na2HP04 (7 g / L) (Prolabo) KH2P04 (3 g / L) (Prolabo) NH4 Cl (1 g / L) (Prolabo)

NaCl (0.5 g / L) (Prolabo)

Glucose (20 g / L) (Sigma)

MgSO4 (1 mM) (Prolabo)

Thiamine (0.001%) (Sigma)

This medium is rendered solid by agar addition (Difco) 15 g / L. 25

Leucine (50 mg / L) (Sigma) and proline (50 mg / L) (Sigma) should be added to the M9 medium to allow growth of the HB101 strain.

During selection of plasmids from the two hybrid lymphocyte cDNA library, no leucine was added to the medium as the plasmids contain a Leu2 selection marker. 3) plasmids: The 5 kb pLex9 (ρΒΤΜΙΙβ) (Bartel et al., 1993) vector, which is a pGBTIO vector containing multiple cloning site located downstream of the coding sequence of bacterial repressor LexA and upstream of a terminator to form a protein of fusion. pLex-HaRasVall2, plasmid pLex9, as described in application WO98 / 21327, which contains the coding sequence of the HaRas protein mutated at the Vall2 position known to interact with mammalian Raf protein (Vojtek et al., 1993). This plasmid was used to test the specificity of interaction of the PAP1 protein in the L40 strain. pLex9-cAPP, plasmid pLex9 which contains the coding sequence for the cytoplasmic domain of APP protein known to interact with the PT65 domain of FE65. This plasmid was used to test the specificity of interaction of the PAP1 protein in the L40 strain. 4) Synthetic oligonucleotides: TTAAGAATTC GGAAGTCCAG CAGGTAG (SEQ ID NO: 5) ATTAGGATCC CTACACACAA GGCAGGGAG (SEQ ID NO: 6)

Oligonucleotides that allowed to obtain the PCR fragment corresponding to the central region of Parkina flanked by the EcoRI and BamHI sites. GCGTTTGGAA TCACTACAG (SEQ ID NO: 7) GGTCTCGGTG TGGCATC (SEQ ID NO: 8) CCGCTTGCTT GGAGGAAC (SEQ ID NO: 9) CGTATTTCTC CGCCTTGG (SEQ ID NO: 10) AATAGCTCGA GTCAGTGCAG GACAAGAG (SEQ ID NO: 11)

Oligonucleotides that were used to sequence the PAP1 gene insert.

The oligonucleotides are synthesized in Applied System ABI 394-08. These are liberated from the ammonia synthesis matrix and are precipitated twice with 10 volumes of n-butanol then taken up in water. Quantification is performed by measuring the optical density (1 D026 corresponds to 30 pg / ml). 5) Preparation of plasmid DNAs.

Plasmid DNA preparations were performed in small quantities and in large quantities according to the protocols recommended by Quiagen manufacturer of the DNA purification kits: Quiaprep Spin Miniprep kit, ref: 27106 Quiaprep Plasmid Maxiprep kit, ref: 12163. 6) Enzyme amplification of DNA by PCR (Polymerase Chain Reaction):

PCR reactions are performed in a final volume of 100 pL in the presence of the DNA template, dNTP (0.2 mM), PCR buffer (10 mM Tris-HCl pH 8.5, 1 mM MgCl 2, 5 mM KCl, 0.01%), 10-20 pmol of each oligonucleotide and 2.5 IU Ampli Taq DNA polymerase (Perkin Elmer). The mixture is again covered with 2 drops of paraffin oil to limit evaporation of the sample. The device used is " Crocodile II " of Appligene. Applicant used a mold denaturation temperature of 94øC, a hybridization temperature of 52øC and an enzyme elongation temperature of 72øC. 7) Connections:

All binding reactions are performed at 37 ° C for one hour in a final volume of 20 μl in the presence of 100 to 200 μg of vector, 0.1 to 0.5 μg of insertion, 40 IU of DNA ligase enzyme of T4 (Biolabs) and a binding buffer (Tris-HCl pH 7.8 mM, 10 mM MgCl2, 10 mM DTT, 1 mM ATP). The negative control is comprised of vector binding in the absence of insertion. 8) Transformation of bacteria: Transformation of the bacteria by a plasmid is carried out according to the following protocol: 10 Âμl binding volume is used to transform TG1 bacteria according to the Chung method (Chung et al., 1989). After transformation the bacteria are spread on LB + ampicillin medium and are incubated for 16 h at 37 ° C. 9) DNA Separation and Extraction: DNA separation is performed according to its size by agarose gel electrophoresis according to Maniatis (Maniatis et al., 1989): 1% agarose gel (Gibco BRL) on a 10) Fluorescent Sequencing of Plasmid DNAs: The sequencing technique used is derived from the method of Sanger (Sanger et al., 1977) and is adapted for the sequencing by Tanger et al. fluorescence developed by Applied Biosystems. The protocol used is described by the creators of the system (Perkin Elmer, 1997). 29 11) Transformation of yeast:

Plasmids are introduced into the yeast by a classical yeast transformation technique, developed by Gietz (Gietz et al., 1992) and modified as follows:

In the particular case of yeast transformation by the lymphocyte cDNA library, the yeast used contains the plasmid pLex9-Parkin (135-290) encoding the central part of Parkina fused to the LexA protein. This is grown in 200 ml of minimal YNB medium supplemented with CSM-Trp amino acids at 30øC under agitation to a density of 107 cells / ml. To perform yeast transformation according to the above protocol, the cell suspension was separated into 10 50 μl tubes to which were added 5 μg of the library. Thermal shock was performed for 20 minutes, then the cells were collected by centrifugation and resuspended in 100 ml of YPD medium for 1 h at 30øC and in 100 ml of YNB medium supplemented with CSM-Leu, -Trp for 3.5 h at 30 ° C. The efficiency of the transformation is determined by spreading several dilutions of transformed cells onto solid YNB medium supplemented with CSM-Trp, -Leu. The colonies obtained were counted after culturing at 30øC for 3 days and the transformation rate per pg of lymphocyte library DNA was determined. Isolation of plasmids extracted from yeast: 5 ml of a yeast culture are incubated for 16 h at 30 ° C and taken up in 200 μl of a lysis buffer (Sorbitol 1 Μ, 0.1 M KH 2 PO 4 / K 2 HPO 4 pH 7.4, zymolyase 30 12.5 mg / ml) and incubated for 1 h 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) β-galactosidase activity test: A nitrocellulose sheet is previously deposited on a plate containing the individual yeast clones. This sheet is then submerged in liquid nitrogen for 30 seconds to cause yeasts to burst and thus release β-galactosidase activity. After thawing, the nitrocellulose sheet is deposited, colonized upwards, in another Petri dish containing a Whatman paper pre-soaked with 1.5 ml of PBS solution (60 mM Na2HPO4, 40 mM NathPCg, 10 mM KCl, MgSO4 1 mM, pH 7) containing 15 æl X-Gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside) with 40 mg / ml N, N-dimethylformamide. The plate is then placed in an oven at 37 ° C. The test is considered positive when the colonies become blue on the membrane at the end of 12 hours. EXAMPLE 1: CONSTRUCTION OF A VECTOR ALLOWING THE EXPRESSION OF A FUSION PROTEIN BETWEEN THE PARKINE CENTRAL PART AND THE BACTERIAL LEXA REPRESSOR. Screening a library using the double hybrid system requires that the central region of Parkina be fused to a DNA binding protein such as the bacterial repressor LexA. Expression of this fusion protein is performed using the pLex9 vector (see materials and methods), where the sequence corresponding to the LexA protein has been introduced into the same reading frame as the coding sequence of the central region of Parkina shown in sequence shown in SEQ ID No. 3 or 4.

The 468 bp DNA fragment corresponding to the 156 amino acids of the central region of Parkin starting at amino acid 135 was obtained by PCR from oligonucleotides (SEQ ID NO: 5 and No. 6) which also allowed to introduce the EcoRI site at the 5 '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 multiple cloning site of plasmid pLex9 downstream of the LexA protein coding sequence to give the vector pLex9-Parkin (135-290) (Fig. 1). The construct was checked by DNA sequencing. This check showed that this fragment did not show mutations produced during the PCR reaction and that it was fused in the same open reading frame as that of the corresponding LexA fragment.

EXAMPLE 2: SCREENING OF A LYMPHOCYTE FUSION LIBRARY. The applicant used the Double-Hybrid method (Fields and Song, 1989). Screening of a fusion library allows the identification of clones producing fused proteins with the GAL4 transactivator domain, which can interact with the protein of interest described in example 1 (central region of the parkin). 32

This interaction allows reconstituting a transactivator which will then be able to induce the form of the His3 and LacZ reporter genes in the L40 strain.

To carry out this screening the inventor selected a fusion library prepared from cDNA derived from peripheral human lymphocytes provided by Richard Benarous (Peytavi et al., 1999). Yeasts were transformed by the lymphocyte library 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 is present in at least one yeast simultaneously with the plasmid pLex9-Parkin (135-290). To preserve this probability it is important to have a good yeast transformation efficiency. As a result the inventors selected a yeast transformation protocol providing an efficacy of 2.6 105 transformed cells per pg of DNA. In addition, as cotransformation of yeast by two different plasmids reduces this efficacy, the inventor preferred to use a yeast previously transformed by the plasmid pLex9-Parkin (135-290). This strain L40 pLex9-Parkine (135-290) His-, Lys-, Leu-, Ade- phenotype was transformed with 50 pg of plasmid DNA from the fusion library. This amount of DNA allowed the applicants to obtain after estimation 1,3107 transformed cells, which corresponds to a number slightly higher than the number of independent plasmids constituting the library. According to this result the inventor believed that almost all of the plasmids from the library were used to transform the yeasts. Selection of the transformed cells capable of reconstituting a functional transactivator was performed on YNB medium supplemented with 2.5 mM 3-amino-1,2,4-triazole and CSM (BiolO1) 620 mg / L containing no histidine, leucine and tryptophan.

Numerous clones with His + phenotype were obtained as a result of this selection. A β-galactosidase activity test of these transformants was performed to validate this number of clones obtained by the expression of the other reporter gene, LacZ. 115 clones exhibiting the His +, β-Galr double phenotype may correspond to a protein-protein interaction. EXAMPLE 3: LIBRARY INSULATION OF PLASMIDES IN THE SELECTED CLONES.

Fusion library plasmids contained in selected yeasts were screened during double hybrid screening to identify proteins capable of interacting with the central region of Parkina. In order to be able to obtain them in large quantities, this isolation requires a transformation of E. coli into a DNA extract of the yeast positive strains. As the plasmid of the library contained in this extract is a yeast transporter plasmid / E. This coli can be easily replicated in the bacterium. The library plasmid was selected for complementation of the auxotrophic HB101 bacteria for leucine in leucine-depleted medium.

Plasmid DNAs from bacterial colonies obtained after transformation with yeast DNA extracts were digested by restriction enzyme digestion and separation of the DNA fragments on agarose gel. Of the 115 clones analyzed, a clone containing a plasmid from the library was obtained showing a profile different from the others. This plasmid, designated pGAD-Ly1111b, has been studied in more detail. EXAMPLE 4: DETERMINATION OF THE SEQUENCE OF THE INSERTION CONTAINED IN THE IDENTIFIED PLASMID. Sequencing of the insert contained in the identified plasmid was performed initially from the oligonucleotide SEQ ID NO: 7 complementary to the GAL4TA sequence in the vicinity of the EcoRI site of the lymphocyte DNA library insert. Then, in a second step, second from the oligonucleotides SEQ ID NO: 8 to SEQ ID NO: 11, corresponding to the insertion sequence obtained during the sequencing progression. The sequence obtained is shown in the sequence SEQ ID NO: 1. The protein thus identified was designated PAP1 (Parkin-Associated Protein 1). Comparison between the sequence of this insert and the sequences contained in the EMBL and GENBank (European Molecular Biology Lab) databases showed a 25% homology at protein level with several members of the Sinaptotagminas family. Sinaptotagminas belong to a family of membrane proteins encoded by at least eleven different genes expressed in the brain and other tissues. These contain a single transmembrane domain and two calcium-regulated domains designated C2. It is in this domain that the homology between Sinaptotagminas and the PAP1 protein is found. No other significant homology was observed. EXAMPLE 5: ANALYSIS OF THE SPECIFICITY OF INTERACTION BETWEEN THE PARKINE CENTRAL REGION AND PAP1 PROTEIN.

A two-hybrid specific interaction test was performed with other non-relevant proteins to determine the specificity of the interaction between the fragment corresponding to the PAP1 protein and the central region of Parkina. To carry out this test the applicants transformed the L40 strain with the control plasmids plex9-APP or pLex9-HaRasVall2 instead of plasmid pLex9-Parkin (135-290) encoding respectively for the APP cytoplasmic domain or the HaRasVall2 protein fused to the domain binding to LexA DNA and to the isolated plasmid during screening of the two hybrid library. A β-Gal activity test was performed on cells transformed by the different plasmids to determine a protein-protein interaction. According to the test result, only the yeast transformed by the plasmid isolated during screening of the two-hybrid library and the plasmid pLex9-Parkin (135-290) showed β-Galr activity, thus demonstrating an interaction between central region of Parkina and the PAP1 protein. This interaction therefore reveals to be specific since this fragment of PAP1 does not appear to interact with APP or HaRasVall2 proteins.

These results therefore show the existence of a new protein, designated PAP1, capable of interacting in a specific way with Parkin. This protein, related to synaptotagmines, has no significant homology to 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. 36

EXAMPLE 6: CLONING OF PAP1 GENE FROM A HUMAN LUNG DNA LIBRARY

In order to identify the complete sequence of the human PAP1 gene and to characterize the existence of variant forms, two electronic elongation approaches were performed from the sequence SEQ ID NO: 1. Thus, two electron sequences were obtained, respectively 1644 bp and 1646 bp, comprising an elongation of 330 bp compared to the sequence of SEQ ID NO: 1. However, analysis of these sequences revealed differences in the consensus region, apparent after translation. In this way, an ORF of 420 aa in one case and an ORF of 230 aa in the other sequence is obtained. The protein sequence obtained was compared to the known sequences and revealed a 24% homology in the 293 amino acids overlapped with the human synaptogamin 1 (p65) (p21579). The function of synaptogamin I may consist of a regulatory role in membrane interactions during the traffic of synaptic vesicles in the synapse zone. This binds to acid phospholipids with some specificity. In addition, a calcium-dependent interaction between synaptogamin and activated protein C kinase receptors has been described. Synaptogamin can also bind to three other proteins which are neurexin, syntaxin and ap2. In view of the brutal and early disappearance of any homology between the identified sequences and the family of the synaptogamines, it is possible that the identified sequence exhibits a deletion relative to the natural sequence. An RT-PCR and sequencing experiment was performed using the 1644 bp sequence to verify this hypothesis and validate the sequences. The sequence to be obtained comprises a 420 aa ORF having a homology of the same order with the synaptogamines. 37

In order to try to obtain a larger sequence and verify if the sequence obtained may correspond to one form of processing, a 5'-RACE elongation experiment was started from the 3 'region of the validated sequence, using the oligonucleotides Ll and L2 in a preparation of Human lung cDNA.

The results obtained are shown in figure 2 and show the identification of 8 corresponding clones at 6 different terminal 5 'ends. Three of these contain a stop codon that disrupts the ORF (clones A12, F2, F12) and clone A3 does not contain any ORF. The presence of different transcripts was confirmed by RT-PCR and RT-PCR with internal primers (Table 1).

Table 1 Primary RT-PCR Secondary PCR U3-L3 Secondary PCR U1-L4 Secondary PCR CB U3-L3 170 A-L4 153 + A-L3 Smear + U1-L4 130 U1-L3 Smear + Ul-B 415 + U2-B 515 + Expected size 170 130 120

Primer pairs U3-L3 and CB are specific for the common fragment of the sequence, oligonucleotides A and UI are specific for the initial sequence and clone Cl1, oligonucleotide L4 is specific for the initial sequence and primer U2 is specific for clone A3. A second 5'-RACE was performed with oligonucleotides L3 and L7 located in the common region of the different clones (Figure 2). The results obtained are presented in Figures 3 and 4. The presence of different transcripts was confirmed by RT-PCR and RT-PCR with internal primers (Table 2).

Table 2 RT-PCR Secondary PCR result CB Secondary PCR U3-B Secondary PCR U5-L7 U4-F Smear + + + U5-F Smear + + + U3-F 1550 pb + + Expected size 120 385 530 The sequence of the primers e oligonucleotides is provided in Tables 3 and 4 (SEQ ID NO: 16-37).

Table 3 SEQ ID LY111. _U4 CCAGTT CTGCCT GTT C ATC 23 to 41 16 LY111. _U5 TTCAAAACACAGAGGAGGAG 319 to 338 17 LY111. _U3 GAATTT GGT CAGTTT AGAGG 759 to 778 18 LY111. _L7 TTCTGGGATTT GGAGAGCTTTTT CAC 851 to 825 19 LY111. TCLGTCTGTCCCACACACTGCC 914 to 892 20 LY111. CTL CT G 928 to 910 21 LY111. AAGCAACAGAATCTCCCAT CC 1029 to 1049 22 LY111. GCATTGTCAAAATTGCCCATC 1147 to 1127 23 LY111. _ AGGCGGAGAAATACGAAGAC 1543 to 1562 24 LY111. GCAGAGTGAGACAGCCCTTAAC 1767 to 1746 Lylll. CTLACTCAGGACTGGCGACTTCAG 1811 to 1782 26 39 (continued) SEQ ID LIIIlL C AAGCGGT CGTT C ATT CC AAAGAG 1934 to 1913 27 LY111_F AAGAGGAGATAACCCACCAGAG 2288 to 2269 28

Table 4 LY111. _ A CGT AGAGCAGCAGGT CCAAG 14 to 34 46 LY111. _U1 AGGGCTGCTGGCTATTTTTC 36 to 55 29 LY111. AAGAAAT GGGTT GT GAAC 148 to 166 30 LY111. AAGCAACAGAAT CT CCCAT CC 1029 to 1049 31 LY111. GCATT GT CAAAATT GCCCAT C 1147 to 1127 32 LY111. _ AGGCGGAGAAATACGAAGAC 1543 to 1562 33 LY111. GCAGAGTGAGACAGCCCTTAAC 1767 to 1746 Lylll. CTLACTCAGG ACT GGC G ACTT CAG 1811 to 1782 35 Lylll. _L1 C AÂG CGGT CGTT CATT CC AAAGAG 1934 to 1913 36 LY111. AAGAGGAGATAACCCACCAGAG 2288 to 2269 37 The set of these results allows to validate the consensus sequence corresponding to the long isoform (Figure 9) and SEQ ID NO: 12 and 13 and the short isoform (SEQ ID NO: 14 and 15) of The long isoform is encoded by an 1833 bp ORF located at residues 237-2069 of SEQ ID NO: 12 and comprises 610 amino acids Polyadenylation signal is located from nucleotide 2315. The short isoform is encoded by a 942 bp ORF located at residues 429-1370 of SEQ ID NO: 14 and comprises 313 amino acids. The polyadenylation signal is located from of nucleotide 1616. 40

Northern blot experiments were then performed on different human tissues with probes (amplimer CD and EF) and allowed to reveal a transcript of 6 kb in the muscle, transcribed in the heart (3 kb), as well as a transcript of 6 kb in the fetal liver. Example 7 further described the cloning of a transcript in the human fetal brain.

Different homology studies were carried out in the different protein databases, the results of which are shown in table 5 below.

Table 5

Homology: Genotype G5926736 (AB025258) granulophyline-a Identity: 31% (215/679), Homology (POS): 46% (322/679) G5926738 (AB025259) Granuophyllin-b Identity: 31% (150/479), Homology (POS): 47% (230/479) G1235722 (D70830) Doc2 beta (homo sapiens) Identity: 25% (74/292), Homology (POS): 43% (127/292) G289718 (L 15302) Synapto- I Identity: 26% (77/293), Homology (POS): 45% (133/293) 41 (continued)

Homology (POS): 45% (133/293) SP: SYT2_MOUSE Synaptogamina II Identity: 24% (72/293), Homology (POS): 44% (131/293)

EXAMPLE 7: CLONING OF TWO COMPLETE TRANSCRIPTS OF PAP1 (LY111B) FROM HUMAN FETAL BRAIN COMPLEMENTARY DNA

To confirm the presence of a full-length Lylllb transcript (" complete ") in the human brain, a PCR was performed from complementary DNA from fetal human brain (Marathon Ready cDNA, Clontech) using oligonucleotides LyF1 (AAT GGA AGG GCG TGA CGC, Figure 5, SEQ ID NO: 38) and HA71 (CCT CAC GCC TGC TGC AAC CTG, SEQ ID NO: 39). A poorly represented DNA fragment of approximately two kilobases was amplified. The product of this first PCR was used as template for an internal primer PCR, performed with LyEcoF oligonucleotides (GCACGAATTC TGA GCC CAA GAA ATA GAT CTG, SEQ ID NO: 40) and HA72 (CTG TCT TCG TAT TTC TCC GCC TTG, SEQ ID NO: 41). The amplified products were digested with the restriction enzymes EcoRI (integrated into the LyEcoF oligonucleotide) and BstEII (Figure 5) and inserted into the pcDNA3 expression vector, then their sequence was determined. Sequence analysis of the clones obtained revealed the presence of two full potential Lylllb transcripts in the human fetal brain (Figure 5). The first of these transcripts (LyIIlbFuiIA) corresponds to mRNA identified in 42

human lung (Example 6) and encodes a 609 amino acid protein (pLyl 1 lbfuuA; Figures 5, 6, SEQ ID NO: 42-43). The second (LylllbfUuB) probably represents an alternative processing product of a common primary mRNA. In this transcript, identical to Lyl1lbfuuA, the sequence between nucleotides 752 and 956 of the validated sequence in the human lung is absent (SEQ ID NO: 42). Thus, the coding for a protein with 541 amino acids (pLlllbfBiB) identical to pLlllfullA, in which however the domain included between amino acids 172 and 240 (Figure 5, 7, SEQ ID NO: 44-45) is absent. The two pLlllbfIIa / foramiB proteins integrate the domain of the interaction with the Parkin fragment comprising amino acids 135 to 290, identified in the yeast (initial sequence Lylllb, figure 5) and can thus theoretically maintain this interaction.

The pLIII / γBUnB proteins belong to the RIM / Rabophyll A family pLyl1 lbfuUA / Î ± γB has a homology to the RIM / rabophyllin family proteins (Wang Y, Sugita S and Siidhof TG. Biol Chem (2000) 275, 20033-20044) and in particular granulophilins (Wang Jie, Takeuchi T, Yokota H and Izumi T Novel Rabphilin-3-like protein associates with insulin-containing granules in pancreatic beta calls. (1999) 274, 28542-28548). These are characterized by the presence of a zinc finger domain in the N-terminal part and two C2 domains in the C-terminal part (Figures 6 and 7). The zinc finger domain of RIM / rabophyllin family proteins was involved in the interaction with Rab proteins. The latter, GTP-binding proteins, are essential components of the membrane trafficking machinery in eukaryotic cells. In addition, it has been described that the C2 domains of RIM / rabophyllin family proteins can bind to membranes by interaction with phospholipids.

Expression of plylllbfunA / heliB proteins in cos-7 line cells: co-localization with Parkina The coding sequence for the LylllbfI / A transcripts was inserted into the eukaryotic expression vector pcDNA3 in phase with the sequence encoding an N-terminal myc epitope (pcDNA3-mycLylllbFuiiA / B) Transfected cos-7 line cells using these vectors produce proteins with an apparent molecular weight of approximately 67 kDa (pcDNA3-mycLylllbfUnA) and 60 kDa (pcDNA3-mycLylllbfUnB), corresponding to the expected molecular weight. These proteins, detected by immunoblotting using an antibody directed against the N-terminal myc epitope, are distributed in a non-homogeneous, dispersed form in the cytoplasm, in the extensions and sometimes in the nucleus of cos-7 line cells (Figure 8a , b, column A). When these are overexpressed with Parkin, revealed using the anti-Parkin Asp5 antibody on cos-7 line cells (Figure 8a, b, column B), a similar distribution and co-localization of these proteins can be observed (figure 8a, b, column C).

BIBLIOGRAPHIC 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. 44

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 and storage of bacterial cells in the same solution. Proc. Natl. Acad. Know. 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) Thérapie génique: espoirs et limites. Médecine et 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. 45

Lewy, F.H. (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, N. I.

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. Know. USA. 74, 5463-5467.

Shimura, H. et al. (1999). Immunohistochemical and subcellular localization of Parkin protein: absence of autosomal recessive protein in juvenile parkinsonism patients. Ann Neurol. 45, 668-672. 46

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.

SEQUENCE LISTING < 110 > AVENTIS PHARMA

INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE M < 120 > COMPOUNDS CAPABLE OF MODULAR PARKINE ACTIVITY, NUCLEOTHIC SEQUENCES AND UTILIZATIONS < 130 > PRJ00004 < 140 > < 141 > < 160 > 46 < 170 > Patentln Ver. 2.1

< 210 > 1 < 211 > 1313 < 212 > DNA < 213 > Homo sapiens < 220 >

< 221 > CDS < 222 > (1) .. (1032) < 4 0 0 > 1 cag aat ctc cca tcc agt ccg gea ccc agt acc ata ttc tet gga ggt 48 Gin Asn Leu Pro Ser Pro Pro Ala Pro Ser Thr Ile Phe Ser Gly Gly 1 5 10 15 ttt aga cac gga agt tta att age att gac age acc tgt aca gag atg 96 Phe Arg His Gly Ser Leu Ile Ser Asp Ser Thr Cys Thr Glu Met 20 25 30 ggc aat ttt gac aat gct aat gt ga g ga ga t ga g ttt gee att 144 Gly Asn Phe Asp Asn Ala Asn Go Thr Gly Glu Ile Glu Phe Ala Ile 35 40 45 cat tat tgc ttc aaa acc cat tet tta gaa ata tgc t ag gee tgt 192 His Tyr Cys Phe Lys Thr His Ser Leu Glu Ile Cys Ile Lys Ala Cys 50 55 60 aag aac ctt gee tat gga gaa gaa aag aag aaa aag tgc aat ccg tat 240 Lys Asn Leu Ala Tyr Gly Glu Glu Lys Lys Lys Lys Cys Asn Pro Tyr 65 70 75 80 gtg aag acc tac ctg ttg ccc gac aga tcc tcc cag gga aag age aag 288 Go Lys Thr Tyr Leu Leu Pro Asp Arg Ser Ser Gin Gly Lys Arg Lys 85 90 95 act gga gtc caa agg aac acc gtg gc ccg acc ttt cag gg acc ttg 336 Thr Gly Go Gin Arg Asn Thr Go Asp Pro Thr Phe Gin Glu Thr Leu 100 105 110 aag tat cag gt cg gee cag ctg gtg acc cgg cag ctg cag gtc 384 Lys Tyr Gin Go Ala Pro Ala Gin Leu Vai Thr Arg Gin Leu Gin Val 115 120 125 tcg gtg tgg cat ctg ggc acg ctg gee cgg aga gtg ttt ctt gga gaa 432 Ser Vai Trp His Leu Gly Thr Leu Ala Arg Arg Val Phe Leu Gly Glu 130 135 140 gtg att att tet ctg gee acg tgg gac ttt gaa gac age aca aca cag 480 Go Ile Ile Ser Leu Ala Thr Trp Asp Phe Glu Asp Ser Thr Thr Gin 145 150 155 160 48 528 tcc ttc ege tgg cat ceg ctc cgg gee aag gcg gag aaa tac gaa gac Ser Phe Arg Trp His Pro Leu Arg Ala Lys Ala Glu Lys Tyr Glu Asp 165 170 175 age gtt cct agg aat gga gag ctc aca gtc cgg gct aag ctg gtt Be Pro Gin Gin Asn Gly Glu Leu Thr Go Arg Ala Lys Leu Go 180 185 190 cctc cct cc ccc aga aaa ctc caa gag gct caa gaa ggg aca gat Leu Pro Ser Arg Pro Arg Lys Leu Gin Glu Ala Gin Glu Gly Thr Asp 195 200 205 cag cca tea ctt cat ggt caa ctt tgt ttg gta gtg cta gga gee aag Gin Pro Ser Leu His Gly Gin Leu Cys Leu Go Go Leu Gly Ala Lys 210 215 220 aat tta cct gtg cgg cca gat ggc acc ttg aac tea ttt gtt aag ggc Asn Leu Pro Go Arg Arg Asp Gly Thr Leu Asn Ser Phe Go Lys Gly 225 230 235 240 tgt ctg cca cca gca caa aaa ctg aga ctg aag teg cca gtc Cys Leu Thr Leu Pro Asp Gin Lys Leu Arg Leu Lys Ser Pro Go 245 250 255 ctg agg aag cag gct tgc ccc cag tgg aaa cac tea ttt gtc ttc agt Leu Arg Lys Gin Ala Cys Pro Gin Trp Lys His Ser Phe Will Phe Ser 260 265 270 ggc gta ac cca gct cag ctg agg cag teg age ttg gag tta act gtc Gly Vai Thr Pro Ala Gin Leu Arg Gin Ser Ser Leu Glu Leu Thr Go 275 280 285 tgg gat cag gee ctc ttt gga atg aat gac ege ttg ctt. gga gga acc Trp Asp Gin Ala Leu Phe Gly Met Asn Asp Arg Leu Leu Gly Gly Thr 290 295 300 aga ctt ggt tea aag gga gac ga gct gtt gg g g g g gea tgc tea Arg Leu Gly Ser Lys Gly Asp Thr Ala Go Gly Gly Asp Ala Cys Ser 305 310 315 320 cta teg aag ct c cg tg cg aaa gtc ctt tcc ccc aat cta tgg Leu Ser Lys Leu Gin Trp Gin Lys Go Leu Ser Ser Pro Asn Leu Trp 325 330 335 576 624 672 720 768 816 864 912 960 49 1008 CA gac atg act ctt gtc ctg cac tgacatgaag gcctcaaggt tccaggttgc 1062 Thr Asp Met Thr Leu Val Leu His 340 agcaggcgtg aggcactgtg cgtctgcaga ggggctacga accaggtgca gggtcccagc 1122 tggagacccc tttgaccttg agcagtctcc atctgcggcc ctgtcccatg gcttaaccgc 1182 ctattggtat ctgtgtatat ttacgttaaa cacaattatg ttacctaagc ctctggtggg 1242 ttatctcctc tttgagatgt agaaaatggc cagattttaa taaacgttgt tacccatgaa 1302 aaaaaaaaaa at 1313 < 210 > 2 < 211 > 344 < 212 > PRT < 213 > Homo sapiens < 40 > 2

Gin Asn Leu Pro Ser Ser Pro Ala 15

Phe Arg His Gly Ser Leu Ile Ser 20

Gly Asn Phe Asp Asn Ala Asn Go 35 40

His Tyr Cys Phe Lys Thr His Ser 50 55

Lys Asn Leu Ala Tyr Gly Glu Glu 65 70

Go Lys Thr Tyr Leu Leu Pro Asp 85

Thr Gly Go Gin Arg Asn Thr Go 100

Pro Ser Thr Ile Phe Ser Gly Gly 10 15

Ile Asp Ser Thr Cys Thr Glu Met 25 30

Thr Gly Glu Ile Glu Phe Ala Ile 45

Leu Glu Ile Cys Ile Lys Ala Cys 60

Lys Lys Lys Lys Cys Asn Pro Tyr 75 80

Arg Ser Ser Gin Gly Lys Arg Lys 90 95

Asp Pro Thr Phe Gin Glu Thr Leu 105 110 50

Leu Vai Thr Arg Gin Leu Gin Vai 125 Ala Arg Arg V Phe Leu Gly Glu 140 Asp Phe Glu Asp Ser Thr Thr Gin 155 160 Ala Lys Ala Glu Lys Tyr Glu Asp 170 175 Leu Thr Vai Arg Ala Lys Leu Vai 185 190 Gin Glu Ala Gin Glu Gly Thr Asp 205 Cys Leu Go Go Leu Gly Ala Lys 220 Thr Leu Asn Ser Phe Go Lys Gly 235 240 Lys Leu Arg Leu Lys Ser Pro Go 250 255 Trp Lys His Ser Phe Go Phe Ser 265 270 Gin Ser Ser Leu Glu Leu Thr Go 285 Asn Asp Arg Leu Leu Gly Gly Thr 300 Ala Go Gly Gly Asp Ala Cys Ser 315 320 Go Leu Ser Ser Pro Asn Leu Trp 330 335

Lys Tyr Gin Goes Ala Pro Wing Gin 115 120

Ser Vai Trp His Leu Gly Thr Leu 130 135

Ile Ile Ile Ser Leu Ala Thr Trp 145 150

Ser Phe Arg Trp His Pro Leu Arg 165

Being Go Pro Gin Be Asn Gly Glu 180

Leu Pro Ser Arg Arg Arg Lys Leu 195 200

Gin Pro Ser Leu His Gly Gin Leu 210 215

Asn Leu Pro Go Arg Pro Asp Gly 225 230

Cys Leu Thr Leu Pro Asp Gin Gin 245

Leu Arg Lys Gin Ala Cys Pro Gin 260

Gly Go Pro Thr Ala Gin Leu Arg 275 280

Trp Asp Gin Ala Leu Phe Gly Met 290 295

Arg Leu Gly Ser Lys Gly Asp Thr 305 310

Leu Ser Lys Leu Gin Trp Gin Lys 325

Thr Asp Met Thr Leu Vai Leu His 340 51

< 210 > 3 < 211 > 471 < 212 > DNA < 213 > Homo sapiens < 220 >

< 221 > CDS < 222 > (1) . . (471) < 400 > 3 gga agt cca gca gg aga Gly Ser Pro Ala Gly Arg 15 aaa ggc ccc tgt caa aga Lys Gly Pro Cys Gin Arg 20 ag acc g agg cag gca Ser Thr Cys Arg Gin Ala 35 tg gat gat gtt tta att Trp Asp Asp Vu Leu Ile 50 cca cac tgc cct ggg act Pro His Cys Pro Gly Thr 65 70 cac ccc acc tet gac aag His Pro Thr Ser Asp Lys 85 aca agat agt cg aac to Thr Asn Ser Arg Asn Ile 100 ccc gtc ctg gtt ttc cag Pro Vai Leu Go Phe Gin 115 tea to t aac age ttt Ser Ile Tyr Asn Ser Phe 10 g cg cg gga aaa etc. Go Gin Pro Gly Lys Leu 25 acg etc acc ttg acc cag Thr Leu Thr Leu Thr Gin 40 cca aac cgg atg agt ggt Pro Asn Arg Met Ser Gly 55 60 agt gca gaa ttt ttc ttt Ser Ala Glu Phe Phe Phe 75 gaa gta gct gt Gt Thr Ser Va Ala Leu 90 act tgc att acg tgc aca Thr Cys Ile Thr Cys Thr 105 tgc aac tcc ege cac gtg Cys Asn Ser Arg His Goat 120 tat gtg tat tgc 48 Tyr Go Tyr Cys 15 agg gta cag tgc 96

Arg Go to Gin Cys 30 ggt cca tet tgc 144 Gly Pro Ser Cys 45 gaa tgc caa tcc 192

Glu Cys Gin Ser aaa tgt gga gca 240

Lys Cys Gly Ala 80 cc gt 288

His Leu Ile Ala 95 gac gtc agg age 336

Asp Go Arg Arg 110 att tgc tta gac 384

Ile Cys Leu Asp 125 52 tgt ttc cacta Cys Phe His Leu 130 cac gac cacca His Asp Pro Gin 145 tgt gtg aca aga Tyr Cys GonA Thr Arg 135 cc ggc tac tcc ctg Leu Gly Tyr Ser Leu 150 ctc aat gat cgg Leu Asn Asp Arg 140 cct tgt gtg tag Pro Cys Go 155 cg ttt gtt 432 Gin Phe Go 471

< 210 > 4 < 211 > 156 < 212 > PRT < 213 > Homo sapiens < 400 > 4

Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn Ser Phe Tyr Val Tyr Cys 1 5 10 15 Lys Gly Pro Cys Gin Arg Pro Gly Lys Leu Arg Val Gin Cys 20 25 30 Ser Thr Cys Arg Gin Ala Thr Leu Thr Leu Thr Gin Gly Pro Ser Cys 35 40 45 Trp Asp Asp Val Leu Ile Pro Asn Arg Met Ser Gly Glu Cys Gin Ser 50 55 60 Pro His Cys Pro Gly Thr Ser Ala Glu Phe Phe Phe Lys Cys Gly Ala 65 70 75 80 His Pro Thr Ser Asp Lys Glu Thr Ser Vai Leu His Leu Ile Ala 85 90 95 Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr Cys Thr Asp Vai Arg Ser 100 105 110 Pro Val Leu Val Phe Gin Cys Asn Ser Arg His Ile Ile Cys Leu Asp 115 120 125 Cys Phe His Leu Tyr Cys Go Thr Arg Leu Asn Asp Arg Gin Phe Go 130 135 140 His Asp Pro Gin Leu Gly Tyr Ser Leu Pro Cys Go 145 150 155 53 < 210 > 5

< 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: Oligonucleotide < 400 > 5 ttaagaattc ggaagtccag caggtag ??? 27

< 210 > 6 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: Oligonucleotide < 400 > 6 attaggatcc ctacacacaa ggcagggag 29

< 210 > 7 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Oligonucleotide < 4 > 7 gcgtttggaa tcactacag ??? 19 < 210 > 8 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: Oligonucleotide < 40 0 > 8 ggtctcggtg tggcatc ??? 17 < 210 > 9 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: Oligonucleotide < 40 0 > 9 ccgctígct ggaggaac ??? 18 < 210 > 10 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: Oligonucleotide < 400 > 10 cgtatttctc cgccttgg 18 < 210 > 11 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: Oligonucleotide < 40 0 > 11 aatagctcga gtcagtgcag gacaagag 28 < 210 > 12 < 211 > 2347 < 212 > DNA < 213 > Homo sapiens < 40 > 12 ggccttgggg cactgaggga tgccagttct gcctgttcat ctggaacctg gatctaagga 60 gggaagaggc gttgcccctg ctggcatagt caggtaccag cccagccagg tattgaacgg 120 gctgagcttt tcatgatggt tcctgctgac ctggaaacat cttaaatgga agggcgtgag 180 cgcttggtcc atgcagtgaa gctcttccaa cctgggtcaa cgaaaacgga gaagaaatgg 240 cccaagaaat agatctgagt gctctcaagg agttagaacg cgaggccatt ctccaggtcc 300 tgtaccgaga ccaggcggtt caaaacacag aggaggagag gacacggaaa ctgaaaacac 360 acctgcagca tctccggtgg aaaggagcga agaacacgga ctgggagcac aaagagaagt 420 56 gctgtgcgcg ctgccagcag gtgctggggt tcctgctgca ccggggcgcc gtgtgccggg 480 gctgcagcca ccgcgtgtgt gcccagtgcc gagtgttcct gagggggacc catgcctgga 540 agtgcacggt gtgcttcgag gacaggaatg tcaaaataaa aactggagaa tggttctatg 600 aggaacgagc caagaaattt ccaactggag gcaaacatga gacagttgga gggcagctct 660 tgcaatctta tcagaagctg agcaaaattt ctgtggttcc tcctactcca cctcctgtca 720 gcgagagcca gtgcagccgc agtcctggca ggttacagga atttggtcag tttagaggat 780 ttaataagtc cgtggaaaat ttgtttctgt ctcttgctac ccacgtgaaa aagctctcca 840 aatcccagaa tgat atgact tctgagaagc atcttctcgc cacgggcccc aggcagtgtg 900 tgggacagac agagagacgg agccagtctg acactgcggt caacgtcacc accaggaagg 960 tcagtgcacc agatattctg aaacctctca atcaagagga tcccaaatgc tctactaacc 1020 ctattttgaa gcaacagaat ctcccatcca gtccggcacc cagtaccata ttctctggag 1080 gttttagaca cggaagttta attagcattg acagcacctg tacagagatg ggcaattttg 1140 acaatgctaa tgtcactgga gaaatagaat ttgccattca ttattgcttc aaaacccatt 1200 ctttagaaat atgcatcaag gcctgtaaga accttgccta tggagaagaa aagaagaaaa 1260 agtgcaatcc gtatgtgaag acctacctgt tgcccgacag atcctcccag ggaaagcgca 1320 agactggagt ccaaaggaac accgtggacc cgacctttca ggagaccttg aagtatcagg 1380 tggcccctgc ccagctggtg acccggcagc tgcaggtctc ggtgtggcat ctgggcacgc 1440 tggcccggag agtgtttctt ggagaagtga tcattcctct ggccacgtgg gactttgaag 1500 acagcacaac acagtccttc cgctggcatc cgctccgggc caaggcggag aaatacgaag 1560 acagcgttcc tcagagtaat ggagagctca cagtccgggc taagctggtt ctcccttcac 1620 ggcccagaaa actccaagag gctcaagaag ggacagatca gccatcactt catggtcaac 1680 tttgtttggt agtgctagga g ccaagaatt tacctgtgcg gccagatggc accttgaact 1740 catttgttaa gggctgtctc actctgccag accaacaaaa actgagactg aagtcgccag 1800 tcctgaggaa gcaggcttgc ccccagtgga aacactcatt tgtcttcagt ggcgtaaccc 1860 cagctcagct gaggcagtcg agcttggagt taactgtctg ggatcaggcc ctctttggaa 1920 tgaacgaccg cttgcttgga ggaaccagac ttggttcaaa gggagacaca gctgttggcg 1980 gggatgcatg ctcacaatcg aagctccagt ggcagaaagt cctttccagc cccaatctat 2040 ggacagacat gactcttgtc ctgcactgac atgaaggcct caaggttcca ggttgcagca 2100 ggcgtgaggc actgtgcgtc tgcagagggg ctacgaacca ggtgcagggt cccagctgga 2160 gacccctttg accttgagca gtctccatct gcggccctgt cccatggctt aaccgcctat gtatatttac gttaaacaca tggtatctgt 2220 attatgttac ctaagcctct ggtgggttat 2280 ctcctctttg agatgtagaa aatggccaga ttttaataaa cgttgttacc catgaaaaaa aaaaaaa 2340 2347 < 210 > 13 < 211 > 610 < 212 > PRT < 213 > Homo sapiens < 400 > 13

Met Ala Gin Glu Ile Asp Leu Ser Ala Leu Lys Glu Leu Glu Arg Glu 1 5 10 15

Ala Ile Leu Gin Vai Leu Tyr Arg Asp Gin Ala Go Gin Asn Thr Glu 20 25 30

Glu Glu Arg Thr Arg Lys Leu Lys Thr His Leu Gin His Leu Arg Trp 35 40 45

Lys Gly Ala Lys Asn Thr Asp Trp 50 55

Arg Cys Gin Gin Go Leu Gly Phe 65 70

Arg Gly Cys Ser His Arg Val Cys 85

Gly Thr His Ala Trp Lys Cys Thr 100

Lys Ile Lys Thr Gly Glu Trp Phe 115 120

Pro Thr Gly Gly Lys His Glu Thr 130 135

Tyr Gin Lys Leu Ser Lys Ile Ser 145 150

Will Be Glu Ser Gin Cys Ser Arg 165

Gly Gin Phe Arg Gly Phe Asn Lys 180

Leu Wing Thr His Vai Lys Lys Leu 195 200

Ser Glu Lys His Leu Leu Ala Thr 210 215

Glu His Lys Glu Lys Cys Cys Ala 60

Leu Leu His Arg Gly Ala Vai Cys 75 80

Ala Gin Cys Arg Vai Phe Leu Arg 90 95

Go to Cys Phe Glu Asp Arg Asn Go 105 105

Tyr Glu Glu Arg Ala Lys Lys Phe 125

Go Gly Gly Gin Leu Leu Gin Ser 140

Go Go Pro Pro Thr Pro Pro Pro 155 160

Ser Pro Gly Arg Leu Gin Glu Phe 170 175

Ser Vai Glu Asn Leu Phe Leu Ser 185 190

Ser Lys Ser Gin Asn Asp Met Thr 205

Gly Pro Arg Gin Cys Go Gly Gin 220 58

Thr Glu Arg Arg Ser Gin Be Asp Thr Ala Go Asn Go Thr Thr Arg 225 230 235 240

Lys Will Be Wing Pro Asp Ile Leu Lys Pro Leu Asn Gin Glu Asp Pro 245 250 255

Lys Cys Ser Thr Asn Pro Ile'Leu Lys Gin Gin Asn Leu Pro Ser Ser 260 265 270

Pro-Al-Pro-Ser-Thr Ile-Phe-Ser-Gly-Gly-Phe-Arg-His-Gly-Ser-Leu 275 280 285

Ile Ser Ile Asp Ser Thr Cys Thr Glu Met Gly Asn Phe Asp Asn Ala 290 295 300

Asn Go Thr Gly Glu Ile Glu Phe Ala Ile His Tyr Cys Phe Lys Thr 305 310 315 320

His Ser Leu Glu Ile Cys Ile Lys Ala Cys Lys Asn Leu Ala Tyr Gly 325 330 335

Glu Glu Lys Lys Lys Cys Asn Pro Tyr Go Lys Thr Tyr Leu Leu 340 345 350

Pro Asp Arg Ser Ser Gin Gly Lys Arg Lys Thr Gly Vai Gin Arg Asn 355 360 365

Thr Go Asp Pro Thr Phe Gin Glu. Thr Leu Lys Tyr Gin Goes Ala Pro 370 375 380

Ala Gin Leu Go Thr Arg Gin Leu Gin Gonna Be Go Trp His Leu Gly 385 390 395 400

Thr Leu Ala Arg Arg Go Phe Leu Gly Glu Go Ile Ile Pro Leu Ala 405 410 415

Thr Trp Asp Phe Glu Asp Ser Thr Thr Gin Ser Phe Arg Trp His Pro 420 425 430

Leu Arg Wing Lys Ala Glu Lys Tyr Glu Asp Ser Go Pro Pro Gin Ser Asn 435 440 445 59

Gly Glu Leu Thr Go Arg Ala Lys Leu Go Leu Pro Ser Arg Pro Arg 450 455 460

Lys Leu Gin Glu Ala Gin Glu Gly Thr Asp Gin Pro Ser Leu His Gly 465 470 475 480

Gin Leu Cys Leu Vai Leu Gly Wing Lys Asn Leu Pro Go Pro Arg 485 490 495

Asp Gly Thr Leu Asn Ser Phe Go Lys Gly Cys Leu Thr Leu Pro Asp 500 505 510

Gin Gin Lys Leu Arg Leu Lys Ser Pro Go Leu Arg Lys Gin Ala Cys 515 520 525

Pro Gin Trp Lys His Ser Phe Will Phe Be Gly Will Thr Pro Ala Gin 530 535 540

Leu Arg Gin Ser Ser Leu Glu Leu Thr Go Trp Asp Gin Ala Leu Phe 545 550 555 560

Gly Met Asn Asp Arg Leu Leu Gly Gly Thr Arg Leu Gly Ser Lys Gly 565 570 575

Asp Thr Ala Go Gly Gly Asp Ala Cys Ser Gin Ser Lys Leu Gin Trp 580 585 590

Gin Lys Will Be Ser Pro Asn Leu Trp Thr Asp Met Thr Leu Go 595 600 605

Leu His 610 < 210 > 14 < 211 > 1648 < 212 > DNA < 213 > Homo sapiens < 400 > 14 gaaatcatgc ccctcgtaga gcagcaggtc caagcagggc tgctggctat ttttccaaaa 60 agtgaggcag tttaaaaaaa aggcggagaa ctagaattat agaataatgg cacattttgt 120 gtatttgtaa aactaacggc ttgcatggtt cacaacccat ttcttatgcc tgtgttttcc 180 ttggcagcaa aatttctgtg gttcctccta ctccacctcc tgtcagcgag agccagtgca 240 gccgcagtcc tggcaggaag gtcagtgcac cagatattct gaaacctctc aatcaagagg 300 atcccaaatg ctctactaac cctattttga agcaacagaa tctcccatcc agtccggcac 360 ccagtaccat attctctgga ggttttagac acggaagttt aattagcatt gacagcacct 420 gtacagagat gggcaatttt gacaatgcta atgtcactgg agaaatagaa tttgccattc 480 attattgctt caaaacccat tctttagaaa tatgcatcaa ggcctgtaag aaccttgcct 540 atggagaaga aaagaagaaa aagtgcaatc cgtatgtgaa gacctacctg ttgcccgaca 600 gatcctccca gggaaagcgc aagactggag tccaaaggaa caccgtggac ccgacctttc 660 aggagacctt gaagtatcag gtggcccctg cccagctggt gacccggcag ctgcaggtct 720 cggtgtggca tctgggcacg ctggcccgga gagtgtttct tggagaagtg atcattcctc 780 tggccacgtg ggactttgaa gacagcacaa cacagtcctt ccgctggcat ccgctccggg 840 ccaaggcgga gaaatac GAA gacagcgttc ctcagagtaa tggagagctc acagtccggg 900 ctaagctggt tctcccttca cggcccagaa aactccaaga ggctcaagaa gggacagatc 960 agccatcact tcatggtcaa ctttgtttgg tagtgctagg agccaagaat ttacctgtgc 1020 ggccagatgg caccttgaac tcatttgtta agggctgtct cactctgcca gaccaacaaa 1080 aactgagact gaagtcgcca gtcctgagga agcaggcttg cccccagtgg aaacactcat 1140 ttgtcttcag tggcgtaacc ccagctcagc tgaggcagtc gagcttggag ttaactgtct 1200 gggatcaggc cctctttgga atgaacgacc gcttgcttgg aggaaccaga cttggttcaa 1260 agggagacac agctgttggc ggggatgcat gctcacaatc gaagctccag tggcagaaag 1320 tcctttccag ccccaatcta tggacagaca tgactcttgt cctgcactga catgaaggcc 1380 tcaaggttcc aggttgcagc aggcgtgagg cactgtgcgt ctgcagaggg gctacgaacc 1440 aggtgcaggg tcccagctgg agaccccttt gaccttgagc agtctccatc tgcggccctg 1500 tcccatggct taaccgccta ttggtatctg tgtatattta cgttaaacac aattatgtta 1560 cctaagcctc tggtgggtta tctcctcttt gagatgtaga aaatggccag attttaataa aaaaaaaa 1648 1620 acgttgttac ccatgaaaaa

< 210 > 15 < 211 > 313 < 212 > PRT < 213 > Homo sapiens < 40 > 15

Met Gly Asn Phe Asp Asn Ala Asn Go Thr Gly Glu Ile Glu Phe Ala 15 10 15

Ile His Tyr Cys Phe Lys Thr His Ser Leu Glu Ile Cys Ile Lys Ala 20 25 30 61

Cys Lys Asn Leu Ala Tyr Gly Glu Glu Lys Lys Lys Lys Cys Asn Pro 35 40. 45

Tyr Go Lys Thr Tyr Leu Leu Pro Asp Arg Ser Ser Gin Gly Lys Arg 50 55 60

Lys Thr Gly Go Gin Arg Asn Thr Go Asp Pro Thr Phe Gin Glu Thr 65 70 75 80

Leu Lys Tyr Gin Goes Ala Pro Ala Gin Leu Goes Thr Arg Gin Leu Gin 85 90 95

It will be Trp His Leu Gly Thr Leu Ala Arg Arg Go Phe Leu Gly 100 105 110

Glu Ile Ile Pro Leu Ala Thr Trp Asp Phe Glu Asp Ser Thr Thr 115 120 125

Gin Ser Phe Arg Trp His Pro Leu Arg Ala Lys Ala Glu Lys Tyr Glu 130 135 140

Asp Ser Go Pro Gin Ser Asn Gly Glu Leu Thr Go Arg Ala Lys Leu 145 150 155 160

Go Leu Pro Ser Arg Pro Arg Lys Leu Gin Glu Ala Gin Glu Gly Thr 165 170 175

Asp Gin Pro Ser Leu His Gly Gin Leu Cys Leu Go Go Leu Gly Ala 180 185 190

Lys Asn Leu Pro Go Arg Pro Asp Gly Thr Leu Asn Ser Phe Go Lys 195 200 205

Gly Cys Leu Thr Leu Pro Asp Gin Lys Leu Arg Leu Lys Ser Pro 210 215 220 Vai Leu Arg Lys Gin Ala Cys Pro Gin Trp Lys His Ser Phe Go Phe 225 230 235 240 Ser Gly Go Pro Thr Ala Gin Leu Arg Gin Ser Ser Leu Glu Leu Thr 245 250 255 Will Trp Asp Gin Ala Leu Phe Gly Met Asn Asp Arg Leu Leu Gly Gly 260 265 270 62

Thr Arg Leu Gly Ser Lys Gly Asp Thr Ala Go Gly Gly Asp Ala Cys 275 280 285

Being Gin Lys Leu Gin Trp Gin Lys Going Leu Be Being Pro Asn Leu 290 295 · 300

Trp Thr Asp Met Thr Leu Vai Leu His 305 310

< 210 > 16 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of the artificial sequence: oligonucleotide < 40 0 > 16 ccagtíctgc ctgtícatc 19

< 210 > 17 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of the artificial sequence: oligonucleotide < 40 0 > 17 ttcaaaacac agaggaggag ??? 20 < 210 > 18

< 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 18 gaatttggtc agtttagagg ??? 21 < 210 > 19 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 19 ttctgggatt tggagagctt ttcac 26 < 210 > 20 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 20 tctgtctgtc ccacacactg < cc ??? 21 < 210 > 21 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the sequence < 400 > 21 gactggccccctctctct 19 < 210 > 22 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of the sequence < 40 > 22 aagcaacaga atctcccatc c 21 < 210 > 23 < 211 > 21 < 212 > DNA < 213 > Artificial artificial sequence oligonucleotide oligonucleotide < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 23 gcattgtcaa aattgcccat c 21

< 210 > 24 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 24 aggcggagaa atacgaagac 20

< 210 > 25 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 25 gcagagag acagccctta ac 22 66

< 210 > 26 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 26 ctcctcagg actggcgact tcag 24

< 210 > 27 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 27 caagcggtcg ttcattccaa agag 24

< 210 > 28 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 28 aagaggagat aacccaccag ag 22

< 210 > 29 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 29 agggctgctg gctatitttc ??? 20

< 210 > 30 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 30 taagaaatgg gltgtgaac 19 < 210 > 31

< 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 31 aagcaacaga atctcccatc c 21

< 210 > 32 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 32 gcaftgtcaa aattgcccat c 21

< 210 > 33 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 33 aggcggagaa atacgaagac 20

< 210 > ≪ tb > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 34 gcagagtgag acagccctta ac 22

< 210 > ≪ tb > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 35 cttcctcagg actggcgact tcag 24

< 210 > 36 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 36 caagcggtcg ttcattccaa agag 24

< 210 > 37 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 37 aagaggagat aacccaccag ag 22

< 210 > 38 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 22 0 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 38 aatggaaggg cgtgacgc ??? 18 < 210 > 39 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the sequence < 400 > 39 cctcacgcct gctgcaacci 21 < 210 > 40 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the sequence < 400 > 40 artifici artifici: oligonucleotide al: oligonucleotide 31 gcacgaaitc atggcccaag aaaíagatct g

< 210 > 41 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence 72 < 22 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 41 ctgtcttcgt atttctccgc cttg ??? 24

< 210 > 42 < 211 > 2347 < 212 > DNA < 213 > Homo sapiens < 400 > 42 ggccttgggg cactgaggga tgccagttct gcctgttcat ctggaacctg gatctaagga 60 gggaagaggc gttgcccctg ctggcatagt caggtaccag cccagccagg tattgaacgg 120 gctgagcttt tcatgatggt tcctgctgac ctggaaacat cttaaatgga agggcgtgag 180 cgcttggtcc atgcagtgaa gctcttccaa cctgggtcaa cgaaaacgga gaagaaatgg 240 cccaagaaat agatctgagt gctctcaagg agttagaacg cgaggccatt ctccaggtcc 300 tgtaccgaga ccaggcggtt caaaacacag aggaggagag gacacggaaa ctgaaaacac 360 acctgcagca tctccggtgg aaaggagcga agaacacgga ctgggagcac aaagagaagt 420 gctgtgcgcg ctgccagcag gtgctggggt tcctgctgca ccggggcgcc gtgtgccggg 480 gctgcagcca ccgcgtgtgt gcccagtgcc gagtgttcct gagggggacc catgcctgga 540 agtgcacggt gtgcttcgag gacaggaatg tcaaaataaa aactggagaa tggttctatg 600 aggaacgagc caagaaattt ccaactggag gcaaacatga gacagttgga gggcagctct 660 tgcaatctta tcagaagctg agcaaaattt ctgtggttcc tcctactcca cctcctgtca 720 gcgagagcca gtgcagccgc agtcctggca ggttacagga atttggtcag tttagaggat 780 ttaataagtc cgtggaaaat ttgtttctgt ctcttgctac ccacgtgaaa aagctctcca 840 aatcccagaa tgatatg act tctgagaagc atcttctcgc cacgggcccc aggcagtgtg 900 tgggacagac agagagacgg agccagtctg acactgcggt caacgtcacc accaggaagg 960 tcagtgcacc agatattctg aaacctctca atcaagagga tcccaaatgc tctactaacc 1020 ctattttgaa gcaacagaat ctcccatcca gtccggcacc cagtaccata ttctctggag 1080 73 gttttagaca acaatgctaa ctttagaaat agtgcaatcc agactggagt tggcccctgc tggcccggag acagcacaac acagcgttcc ggcccagaaa tttgtttggt catttgttaa tcctgaggaa cagctcagct tgaacgaccg gggatgcatg ggacagacat ggcgtgaggc gacccctttg tggtatctgt ctcctctttg aaaaaaa cggaagttta attagcattg acagcacctg tacagagatg ggcaattttg tgtcactgga gaaatagaat ttgccattca ttattgcttc aaaacccatt atgcatcaag gcctgtaaga accttgccta tggagaagaa aagaagaaaa gtatgtgaag acctacctgt tgcccgacag atcctcccag ggaaagcgca ccaaaggaac accgtggacc cgacctttca ggagaccttg aagtatcagg ccagctggtg acccggcagc tgcaggtctc ggtgtggcat ctgggcacgc agtgtttctt ggagaagtga tcattcctct ggccacgtgg gactttgaag acagtccttc cgctggcatc cgctccgggc caaggcggag aaatacgaag tcagagtaat ggagagctca cagtccgggc taagctggtt ctcccttcac actccaagag gctcaagaag ggacagatca gccatcactt catggtcaac agtgctagga gccaagaatt tacctgtgcg gccagatggc accttgaact gggctgtctc actctgccag accaacaaaa actgagactg aagtcgccag gcaggcttgc ccccagtgga aacacteatt tgtcttcagt ggcgtaaccc gaggcagtcg agcttggagt taactgtctg ggatcaggcc ctctttggaa cttgcttgga ggaaccagac ttggttcaaa gggagacaca gctgttggcg ctcacaatcg aagctccagt ggcagaaagt cctttccagc cccaatctat gactcttgtc ctgcactgac atgaaggcct caaggttcca ggttgcagca actgtgcgtc tgcagagggg ctacgaacca ggtgcagggt cccagctgga accttgagca gtctccatct gcggccctgt cccatggctt aaccgcctat gtatatttac gttaaacaca attatgttac ctaagcctct ggtgggttat agatgtagaa aatggccaga ttttaataaa cgttgttacc catgaaaaaa

< 210 > 43 < 211 > 610 < 212 > PRT < 213 > Homo sapiens < 400 > 43

Met Ala Gin Glu Ile Asp Leu Ser Ala Leu Lys Glu Leu Glu Arg Glu 15 10 15

Ala Ile Leu Gin Vai Leu Tyr Arg Asp Gin Ala Go Gin Asn Thr Glu 20 25 30

Glu Glu Arg Thr Arg Lys Leu Lys Thr His Leu Gin His Leu Arg Trp 35 40 45

Lys Gly Ala Lys Asn Thr Asp Trp Glu His Lys Glu Lys Cys Cys Ala 50 55 60 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2347 74

Arg Cys Gin Gin Go Leu Gly Phe Leu Leu His Arg Gly Ala Go Cys 65 70 75 80

Arg Gly Cys Ser His Arg Val Cys Ala Gin Cys Arg Val Phe Leu Arg 85

Gly Thr His Ala Trp Lys Cys Thr 100

Lys Ile Lys Thr Gly Glu Trp Phe 115 120

Pro Thr Gly Gly Lys His Glu Thr 130 135

Tyr Gin Lys Leu Ser Lys Ile Ser 145 150

Will Be Glu Ser Gin Cys Ser Arg 165

Gly Gin Phe Arg Gly Phe Asn Lys 180

Leu Wing Thr His Vai Lys Lys Leu 195 200

Ser Glu Lys His Leu Leu Ala Thr 210 215

Thr Glu Arg Arg Ser Gin Ser Asp 225 230

Lys Will Be Wing Pro Asp Ile Leu 245

Lys Cys Ser Thr Asn Pro Ile Leu 260

Pro Wing Pro Ser Thr Ile Phe Ser 275 280 90 95

Val Cys Phe Glu Asp Arg Asn Go 105 105

Tyr Glu Glu Arg Ala Lys Lys Phe 125

Val Gly Gly Gin Leu Leu Gin Ser 140

Val Val Pro Pro Thr Pro Pro Pro 155 160

Ser Pro Gly Arg Leu Gin Glu Phe 170 175

Ser Val Glu Asn Leu Phe Leu Ser 185 190

Ser Lys Ser Gin Asn Asp Met Thr 205

Gly Pro Arg Gin Cys Val Gly Gin 220

Thr Ala Val Asn Val Thr Thr Arg 235 240

Lys Pro Leu Asn Gin Glu Asp Pro 250 255

Lys Gin Gin Asn Leu Pro Ser Ser 265 270

Gly Gly Phe Arg His Gly Ser Leu 285 75

Ile Ser Ile Asp Ser Thr Cys Thr Glu Met Gly Asn Phe Asp Asn Ala 290 295 300 Asn Go Thr Gly Gly Ile Glu Phe Ala Ile His Tyr Cys Phe Lys Thr 305 310 315 320 His Ser Leu Glu Ile Cys Ile Lys Ala Cys Lys Asn Leu Ala Tyr Gly 325 330 335 Glu Glu Lys Lys Lys Lys Cys Asn Pro Tyr Go Lys Thr Tyr Leu Leu 340 345 350

Pro Asp Arg Ser Ser Gin Gly Lys Arg Lys Thr Gly Vai Gin Arg Asn 355 360 365.

Thr Go Asp Pro Thr Phe Gin Glu Thr Leu Lys Tyr Gin Go Pro Wing 370 375 380

Ala Gin Leu Go Thr Arg Gin Leu Gin Gonna Be Go Trp His Leu Gly 385 390 395 400

Thr Leu Ala Arg Arg Go Phe Leu Gly Glu Go Ile Ile Pro Leu Ala 405 410 415

Thr Trp Asp Phe Glu Asp Ser Thr Thr Gin Ser Phe Arg Trp His Pro 420 425 430.

Leu Arg Wing Lys Ala Glu Lys Tyr Glu Asp Ser Go Pro Gin Ser Asn 435 440 445

Gly Glu Leu Thr Go Arg Ala Lys Leu Go Leu Pro Ser Arg Pro Arg 450 455 460

Lys Leu Gin Glu Ala Gin Glu Gly Thr Asp Gin Pro Ser Leu His Gly 465 470 475 480

Gin Leu Cys Leu Vai Leu Gly Wing Lys Asn Leu Pro Go Pro Arg 485 490 495

Asp Gly Thr Leu Asn Ser Phe Go Lys Gly Cys Leu Thr Leu Pro Asp 500 505 510

Gin Gin Lys Leu Arg Leu Lys Ser Pro Val Leu Arg Lys Gin Ala Cys 515 520 525 76 caagcagggc tgctggctat ttttccaaaa 60 ctagaattat agaataatgg cacattttgt 120 cacaacccat ttcttatgcc tgtgttttcc 180 ctccacctcc tgtcagcgag agccagtgca 240 cagatattct gaaacctctc aatcaagagg 300 agcaacagaa tctcccatcc agtccggcac 360 acggaagttt aattagcatt gacagcacct 420 atgtcactgg agaaatagaa tttgccattc 480 tatgcatcaa ggcctgtaag aaccttgcct 540 600 cgtatgtgaa gacctacctg ttgcccgaca tccaaaggaa caccgtggac ccgacctttc 660 cccagctggt gacccggcag ctgcaggtct 720 gagtgtttct tggagaagtg atcattcctc 780 840 cacagtcctt ccgctggcat ccgctccggg ctcagagtaa tggagagctc acagtccggg 900 960 aactccaaga ggctcaagaa gggacagatc

Pro Gin Trp Lys His Ser Phe Go 530 535

Leu Arg Gin Ser Ser Leu Glu Leu 545 550

Gly Met Asn Asp Arg Leu Leu Gly 565

Asp Thr Ala Go Gly Gly Asp Ala 580

Gin Lys Goes Leu Ser Ser Pro Asn 595 600

Leu His 610

< 210 > 44 < 211 > 1648 < 212 > DNA < 213 > Homo sapiens < 400 > 44 gaaatcatgc ccctcgtaga gcagcaggtc agtgaggcag tttaaaaaaa aggcggagaa gtatttgtaa aactaacggc ttgcatggtt ttggcagcaa aatttctgtg gttcctccta gccgcagtcc tggcaggaag gtcagtgcac atcccaaatg ctctactaac cctattttga ccagtaccat attctctgga ggttttagac gtacagagat gggcaatttt gacaatgcta attattgctt caaaacccat tctttagaaa atggagaaga aaagaagaaa aagtgcaatc gatcctccca gggaaagcgc aagactggag aggagacctt gaagtatcag gtggcccctg cggtgtggca tctgggcacg ctggcccgga tggccacgtg ggactttgaa gacagcacaa ccaaggcgga gaaatacgaa gacagcgttc ctaagctggt tctcccttca cggcccagaa

Phe Ser Gly Go Pro Thr Ala Gin 540

Thr Vai Trp Asp Gin Ala Leu Phe 555 560

Gly Thr Arg Leu Gly Ser Lys Gly 570 575

Cys Ser Gin Ser Lys Leu Gin Trp 585 590

Leu Trp Thr Asp Met Thr Leu Val 605 77 agccatcact tcatggtcaa ctttgtttgg tagtgctagg agccaagaat ttacctgtgc ggccagatgg caccttgaac tcatttgtta agggctgtct cactctgcca gaccaacaaa aactgagact gaagtcgcca gtcctgagga agcaggcttg cccccagtgg aaacactcat ttgtcttcag tggcgtaacc ccagctcagc tgaggcagtc gagcttggag ttaactgtct gggatcaggc cctctttgga atgaacgacc gcttgcttgg aggaaccaga cttggttcaa agggagacac agctgttggc ggggatgcat gctcacaatc gaagctccag tggcagaaag tcctttccag ccccaatcta tggacagaca tgactcttgt cctgcactga catgaaggcc tcaaggttcc aggttgcagc aggcgtgagg cactgtgegt ctgcagaggg gctacgaacc àggtgcaggg tcccagctgg agaccccttt gaccttgagc agtctccatc tgcggccctg tcccatggct taaccgccta ttggtatctg tgtatattta cgttaaacac aattatgtta cctaagcctc tggtgggtta tctcctcttt gagatgtaga aaatggccag attttaataa acgttgttac ccatgaaaaa aaaaaaaa

< 210 > 45 < 211 > 313 < 212 > PRT < 213 > Homo sapiens < 400 > 45

Met Gly Asn Phe Asp Asn Ala Asn Go Thr Gly Glu Ile Glu Phe 15 10 15

Ile His Tyr Cys Phe Lys Thr His Ser Leu Glu Ile Cys Ile Lys 20 25 30

Cys Lys Asn Leu Ala Tyr Gly Glu Glu Lys Lys Lys Lys Cys Asn 35 40 45

Tyr Go Lys Thr Tyr Leu Leu Pro Asp Arg Ser Ser Gin Gly Lys 50 55 60

Lys Thr Gly Go Gin Arg Asn Thr Go Asp Pro Thr Phe Gin Glu 65 70 75

Leu Lys Tyr Gin Goes Ala Pro Ala Gin Leu Goes Thr Arg Gin Leu 85 90 95

It will be Trp His Leu Gly Thr Leu Ala Arg Arg Go Phe Leu 100 105 110

Glu Ile Ile Pro Leu Ala Thr Trp Asp Phe Glu Asp Ser Thr 115 120 125 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1648

Allah

Allah

Pro

Arg

Thr 80 Gin

Gly

Thr 78

Gin Ser Phe Arg Trp His Pro Leu 130 135

Asp Ser Go Pro Gin Ser Asn Gly 145 150

Go Leu Pro Ser Arg Arg Arg Lys 165

Asp Gin Pro Ser Leu His Gly Gin 180

Lys Asn Leu Pro Go Arg Pro Asp 195 200

Gly Cys Leu Thr Leu Pro Asp Gin 210 215

Go Leu Arg Lys Gin Ala Cys Pro 225 230

Arg Ala Lys Ala Glu Lys Tyr Glu 140

Glu Leu Thr Vai Arg Ala Lys Leu 155 160

Leu Gin Glu Ala Gin Glu Gly Thr 170 175

Leu Cys Leu Go Go Leu Gly Ala 185 190

Gly Thr Leu Asn Ser Phe Go Lys 205

Gin Lys Leu Arg Leu Lys Ser Pro 220

Gin Trp Lys His Ser Phe Go Phe 235 240

Ser Gly Goes Thr Pro Ala Gin Leu Arg Gin Ser Ser Leu Glu Leu Thr 245 250 255

Go Trp Asp Gin Ala Leu Phe Gly Met Asn Asp Arg Leu Leu Gly Gly _260_265_270_

Thr Arg Leu Gly Ser Lys Gly Asp Thr Ala Go Gly Gly Asp Ala Cys 275 280 285. Ser Gin Ser Lys Leu Gin Trp Gin Lys Go Leu Ser Ser Pro Asn Leu 290 295 300

Trp Thr Asp Met Thr Leu Vai Leu His 305 310

< 210 > 46 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of artificial sequence: oligonucleotide < 400 > 46 tcgtagagca gcaggíccaa g ??? 21

Lisbon, July 26, 2010 80

Claims (18)

A polypeptide interacting with Parkin, having throughout its length an identity of at least 95% with the sequence of SEQ ID NO: 2. A polypeptide having one of the sequences SEQ ID NO: 13 and SEQ ID NO: 43. A polypeptide having one of the sequences SEQ ID NO: 15 and SEQ ID NO: 45. A nucleic acid encoding a polypeptide according to any one of claims 1 to 3. Nucleic acid according to claim 4, characterized in that it has an identity of at least 95% with the sequence of SEQ ID NO: 1. A nucleic acid according to claim 4, characterized in that it has one of the sequences SEQ ID NO: 12 and SEQ ID NO: 42. A nucleic acid according to claim 4, having one of the sequences SEQ ID NO: 14 and SEQ ID NO: 44. A vector comprising a nucleic acid according to one of claims 4 to 7. A defective recombinant virus comprising a nucleic acid according to one of claims 4 to 7. Pharmaceutical composition comprising at least one polypeptide according to one of claims 1 to 3. A pharmaceutical composition comprising at least one nucleic acid according to one of claims 4 to 7 or a vector according to one of claims 8 or 9. Composition according to one of Claims 10 or 11 for the treatment of neurodegenerative disorders. A method for the screening or characterization of molecules for the treatment of neurodegenerative disorders, comprising a step of selecting molecules capable of binding to the sequence of SEQ ID NO: 2 or a fragment thereof. A method for the screening or characterization of molecules for the treatment of neurodegenerative pathology, comprising a step of selecting molecules capable of binding to a sequence selected from the sequences SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 43 and SEQ ID NO: 45 or a fragment thereof. A process for the production of a polypeptide according to any one of claims 1 to 3, comprising culturing a cell containing a nucleic acid according to one of claims 4 to 7 or a vector according to claim 8 or 9, in the conditions of expression of said nucleic acid and the recovery of the peptidic compound produced. 2 A cell containing a nucleic acid according to one of claims 4 to 7 or a vector according to claim 8 or 9. A non-human mammal comprising in its cells a nucleic acid according to one of claims 4 to 7. Antibodies, fragments or derivatives of the antibodies directed against a polypeptide according to one of claims 1 to 3.