AU2006235783A1 - Compositions useful for regulating parkin gene activity - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

(ORIGINAL)

Name of Applicant(s): Aventis Pharma S.A. AND INSERM, of 20, avenue Raymond Aron, F-92160 Antony, France Actual Inventor(s): Hana Koutnikova Alexis Brice Alain Fournier Laurent Pradier Catherine Prades Isabelle Amould-Reguigne Marie-Francoise Rosier-Montus Olga Corti Address for Service: DAVIES COLLISON CAVE, Patent Trademark Attorneys, of 1 Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM "Compositions useful for regulating parkin gene activity" Invention Title: The following statement is a full description of this invention, including the best method of performing it known to us:- WO 01/60857 1 PCT/FR01/00461 O COMPOSITIONS WHICH CAN BE USED FOR REGULATING THE ACTIVITY OF PARKIN The present invention relates to compositions O and methods which can be used for regulating the r-- 5 activity of parkin. It relates in particular to a novel protein, referred to as PAP1, which is a partner of Sparkin, as well as to the peptides or polypeptides which are derived from or are homologous to this protein. It also relates to compounds which are capable of modulating, at least partially, the activity of parkin, in particular of interfering with the interaction between parkin and PAP1. The present invention can be used in the therapeutic or diagnostic domains, or for forming pharmacological targets which enable the development of novel medicaments.

The parkin gene is mutated in certain familial forms (autosomal recessive juvenile) 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 old. Patients suffering from this disease have neurological disorders which are grouped together under the term "Parkinsonian syndrome", which is characterized by rigidity, bradykinesia, and shaking while resting. These symptoms are the consequence of a degeneration of the ID 2 dopaminergic neurons of the substantia nigra of the O brain.

Most cases with a Parkinson's disease do not have a familial history. However, familial cases do 0 5 exist, of which certain correspond to a monogenic form r-- V' of the disease. At the current time, only three NO different genes have been identified in certain rare D hereditary forms. The first form corresponds to an autosomal dominant form, in which the gene responsible encodes alpha Synuclein (Polymeropoulos et al., 1997).

This protein is an abundant constituent of the intracytoplasmic inclusions, termed Lewy bodies, which are used as a marker for Parkinson's disease (Lewy, 1912). The second form, also autosomal dominant, is linked to a mutation in a gene which encodes a hydrolase termed ubiquitin carboxy-terminal hydrolase L1 (Leroy et al., 1998). This enzyme is assumed to hydrolyze ubiquitin polymers or conjugates into ubiquitin monomers. The third form differs from the previous forms in that it has an autosomal recessive transmission and a commencement often before 40 years of age, as well as an absence of Lewy bodies. These diseases respond more favorably to levodopa, a dopamine precursor which is used as treatment for Parkinson's disease. The gene involved in this form encodes a novel protein which is termed parkin (Kitada et al., 1998).

The parkin gene consists of 12 exons which cover a genomic region of more than 500,000 base pairs O3 on chromosome 6 (6q25.2-q27). At the current time, two o major types of mutation of this gene, which are at the 9 origin of the disease, are known; either deletions of variable size in the region which covers exons 2 to 9, 00 5 or point mutations which produce the premature C appearance of a stop codon or the change of an amino acid (Kitada et al., 1998; Abbas et al., 1999; Lucking et al., 1998; Hattori et al., 1998). The nature of these mutations and the autosomal recessive method of transmission suggest a loss of function of the parkin, which leads to Parkinson's disease.

This gene is expressed in a large number of tissues and in particular in the substantia nigra.

Several transcripts corresponding to this gene exist, which originate from different alternative splicing (Kitada et al., 1998; Sunada et al., 1998). In the brain, two types of messenger RNA are found, of which one lacks the portion corresponding to exon 5. In leukocytes, parkin messenger RNAs which do not contain the region encoding exons 3, 4 and 5 have been identified. The longest of the parkin messenger RNAs, which is present in the brain, contains 2960 bases and encodes a protein of 465 amino acids.

This protein has a weak homology, in its N-termimal portion, with ubiquitin. Its C-terminal half contains two ring finger motifs, separated by an IBR (In Between Ring) domain, which correspond to a cysteine-rich region and which are able to bind metals, ID 4 like the zing finger domains (Morett, 1999). It has o been shown, by immunocytochemistry, that parkin is located in the cytoplasm and the Golgi apparatus of neurons of the substantia nigra which contain melanin 00 5 (Shimura et al., 1999). In addition, this protein is present in certain Lewy bodies of Parkinsonians. The \0 cellular function of parkin has not yet been SD demonstrated, but it might play a transporter role in synaptic vesicles, in the maturation or degradation of proteins, and in the control of cellular growth, differentiation or development. In the autosomal recessive juvenile forms, parkin is absent, which thus confirms that the loss of this function is responsible for the disease.

The elucidation of the exact role of the parkin protein in the process of degeneration of the dopaminergic neurons thus constitutes a major asset for the understanding of and the therapeutical approach to Parkinson's disease, and more generally diseases of the central nervous system.

The present invention lies in the identification of a partner of parkin, which interacts with this protein under physiological conditions. This partner represents a novel pharmacological target for manufacturing or investigating compounds which are capable of modulating the activity of parkin, in particular its activity on the degeneration of dopaminergic neurons and/or the development of nervous IO pathologies. This protein, the antibodies, the o corresponding nucleic acids, as well as the specific probes or primers, can also be used for detecting or assaying the proteins in biological samples, in 00 5 particular nervous tissue samples. These proteins or nucleic acids can also be used in therapeutic O approaches, for modulating the activity of parkin and any compound according to the invention which is capable of modulating the interaction between parkin and the polypeptides of the invention.

The present invention results more particularly from the demonstration by the applicant of a novel human protein, referred to as PAP1 (Parkin Associated Protein 1) or LY111, which interacts with parkin. The PAP1 protein (sequence SEQ ID NO. 1 or 2) shows a certain homology with synaptotagmins and is capable of interacting more particularly with the central region of parkin (represented on the sequence SEQ ID NO. 3 or The PAP1 protein has also been cloned, sequenced and characterized using various tissues of human origin, in particular lung (SEQ ID NOS. 12 and 13) and brain (SEQ ID NOS. 42 and 43) as have short forms, corresponding to splicing variants (SEQ ID NOS. 14, 15, 44 and The present invention also results from the identification and from the characterization of specific regions of the PAP1 protein, which are implicated in the modulation of the function of parkin.

ND 6 The demonstration of the existence of this protein and O of regions which are implicated in its function makes it possible in particular to prepare novel compounds and/or compositions which can be used as pharmaceutical 00 5 agents, and to develop industrial methods of screening such compounds.

(cN A first subject of the invention thus relates

IND

O to compounds which are capable of modulating, at least partially, the interaction between the PAP1 protein (or homologs thereof) and parkin (in particular human parkin), or of interfering with the interaction between these proteins.

Another subject of the invention lies in the PAP1 protein and fragments, derivatives and homologs thereof.

Another aspect of the invention lies in a nucleic acid which encodes the PAP1 protein or fragments, derivatives or homologs thereof, as well as any vector which comprises such a nucleic acid, any recombinant cell which contains such a nucleic acid or vector and any nonhuman mammal comprising in its cells such a nucleic acid.

The invention also relates to antibodies which are capable of binding the PAP1 protein and fragments, derivatives and homologs thereof, in particular polyclonal or monoclonal antibodies, more preferably antibodies which are capable of binding the IND 7 SPAP1 protein and of inhibiting, at least partially, its O interaction with parkin.

Another aspect of the invention relates to nucleotide probes or primers, which are specific for 00 5 PAP1 and which can be used for detecting or amplifying the papl gene, or a region of this gene, in any IND biological sample.

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

As indicated above, a first aspect of the invention lies in a compound which is capable of interfering, at least partially, with the interaction between the PAP1 protein (or homologs thereof) and parkin.

For the purposes of the present invention, the name PAP1 protein refers to the protein per se, as well as to all homologous forms thereof. "Homologous form" is intended to refer to any protein which is equivalent to the protein under consideration, of varied cellular origin and in particular derived from cells of human origin, or from other organisms, and which possesses an activity of the same type. Such homologs also comprise natural variants of the PAP1 protein of sequence SEQ ID NO 2, in particular polymorphic or splicing variants. Such homologs can be IND 8 obtained by the experiments of hybridization between O the coding nucleic acids (in particular the nucleic acid of sequence SEQ ID NO. For the purposes of the invention, a sequence of this type only has to have a 00 5 significant percentage of identity to lead to a physiological behavior which is comparable to that of O the PAP1 protein as claimed. The expression

IND

O "significant percentage of identity" is intended to mean a percentage of at least 60%, preferentially of 80%, more preferentially of 90%, and even more preferentially of 95%. In this regard, variants and/or homologues of the sequence SEQ ID NO. 2 are described in the sequences SEQ ID NOS. 13, 15, 43 and identified using tissues of human origin. The name PAP1 therefore also encompasses these polypeptides.

For the purpose of the present invention, the "percentage of identity" between two nucleotide or amino acid sequences can be determined by comparing two optimally aligned sequences through a window of comparison.

The part of the nucleotide or polypeptide sequence within the window of comparison may thus comprise additions or deletions (for example gaps) compared with the reference sequence (which does not comprise these additions or these deletions) so as to obtain an optimal alignment of the two sequences.

The percentage is calculated by determining the number of positions at which an identical nucleic IND 9 c acid base or amino acid residue is observed for the two O sequences (nucleic acid or peptide sequences) compared, then dividing the number of positions at which there is identity between the two bases or amino acid residues 00 5 by the total number of positions in the window of ;I comparison, and then multiplying the result by 100 in order to obtain the percentage of sequence identity.

S The optimal alignment of the sequences for the comparison may be produced on a computer, using known algorithms contained in the WISCONSIN

GENETICS

SOFTARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN.

By way of illustration, the percentage of sequence identity may be produced using the BLAST program (versions BLAST 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 Res. (1997) 3389-3402). Blast searches for sequences which are similar/homologous to a "request" reference sequence, using the algorithm of Altschul et al. (above). The request sequence and the databases used may be peptide or nucleic acid based, any combination being possible.

The interference of a compound according to the invention can reveal itself in various ways. Thus, the compound can slow, inhibit or stimulate, at least partially, the interaction between the PAP1 protein, or INO c_ a homologous form thereof, and parkin. Preferably, they O are compounds which are capable of modulating this interaction in vitro, for example in a double-hybrid type system or in any acellular system for detecting an 00 5 interaction between two polypeptides. The compounds t'according to the invention are preferably compounds IND which are capable of modulating, at least partially, this interaction, preferably of increasing or inhibiting this reaction by at least 20%, more preferably by at least 50%, with respect to a control in the absence of the compound.

In a particular embodiment, they are compounds which are capable of interfering with the interaction between the region of parkin which is represented on the sequence SEQ ID NO. 4 and the region of the PAP1 protein which is represented on the sequence SEQ ID NOS. 2, 13, 15, 43 or According to a particular embodiment of the invention, the compounds are capable of binding at the level of the domain of interaction between the PAP1 protein, or a homologous form thereof, and parkin.

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

IND

CI According to a first variant, the compounds C.) o of the invention are peptide in nature. The term "peptide" refers to any molecule comprising a string of amino acids, such as for example a peptide, a polypeptide, a protein or an antibody (or antibody t fragment or derivative), which if necessary is modified O or combined with other compounds or chemical groups. In

IND

O this respect, the term "peptide" refers more specifically to a molecule comprising a string of at most 50 amino acids, more preferably of at most amino acids. A polypeptide (or a protein) preferably comprises from 50 to 500 amino acids, or more.

According to a first preferred embodiment, the compounds of the invention are peptide compounds comprising all or part of the peptide sequence SEQ ID NO. 2 or a derivative thereof, in particular all or part of the peptide sequence SEQ ID NOS. 13, 15, 43 or or derivatives of these sequences, more particularly of the PAP1 protein, which comprises the sequence

SEQ

ID NOS. 2, 13, 15, 43 or For the purposes of the present invention, the term "derivative" refers to any sequence which differs from the sequence under consideration because of a degeneracy of the genetic code, which is obtained by one or more modifications of genetic and/or chemical nature, as well as any peptide which is encoded by a sequence which hybridizes with the nucleic acid sequence SEQ ID NO. 1 or a fragment of this sequence, ND 12 u_ for example with the nucleic acid sequence SEQ ID NOS.

O 12, 14, 42 or 44 or a fragment of these sequences, and which is capable of interfering with the interaction between the PAP1 protein, or a homolog thereof, and 00 5 parkin. "Modification of genetic and/or chemical tnature" can be intended to mean any mutation, rn (N substitution, deletion, addition and/or modification of one or more residues. The term "derivative" also comprises the sequences which are homologous to the sequence under consideration, which are derived from other cellular sources and in particular cells of human origin, or from other organisms, and which possess an activity of the same type. Such homologous sequences can be obtained by hybridization experiments. The hybridizations can be carried out with nucleic acid libraries, using the native sequence or a fragment of this sequence as probe, under varied conditions of hybridization (Maniatis et al., 1989). Moreover, the term "fragment" or "part" refers to any portion of the molecule under consideration, which comprises at least consecutive residues, preferably at least 9 consecutive residues, even more preferably at least consecutive residues. Typical fragments can comprise at least 25 consecutive residues.

Such derivatives or fragments can be generated with different aims, such as in particular that of increasing their therapeutic effectiveness or of reducing their side effects, or that of conferring ID 13 novel pharmacokinetic and/or biological properties O thereon.

C-i As a peptide which is derived from the PAP1 protein and from the homologous forms, mention may be 00 5 made in particular of any peptide which is capable of Sinteracting with parkin, but which bears an effector IND region which has been made nonfunctional. Such peptides can be obtained by deletion, mutation or disruption of this effector region on the PAP1 protein and homologous forms. Such modifications can be carried out for example by in vitro mutagenesis, by introducing additional elements or synthetic sequences, or by deletions or substitutions of the original elements.

When such a derivative as defined above is prepared, its activity as partial inhibitor of the binding of the PAP1 protein and of the homologous forms, on its binding site on parkin, can be demonstrated. Any technique known to persons skilled in the art can of course be used for this.

They can also be fragments of the sequences indicated above. Such fragments can be generated in various ways. In particular they can be synthesized chemically, on the basis of the sequences given in the present application, using the peptide synthesizers known to persons skilled in the art. They can also be synthesized genetically, by expression in a host cell of a nucleotide sequence which encodes the desired peptide. In this case, the nucleotide sequence can be \O 14 cI prepared chemically using an oligonucleotide O synthesizer, on the basis of the peptide sequence given in the present application and of the genetic code. The nucleotide sequence can also be prepared from sequences 00 5 given in the present application, by enzymatic cleavage, ligation, cloning, etc., according to the O techniques known to persons skilled in the art, or by screening DNA libraries with probes which are developed from these sequences.

Moreover, the peptides of the invention, i.e.

which are capable of modulating, at least partially, the interaction between the PAP1 protein, and homologous forms, and parkin, can also be peptides which have a sequence corresponding to the site of interaction of the PAP1 protein and of the homologous forms on parkin.

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

A specific subject of the present invention relates to the PAP1 protein. It is more particularly the PAP1 protein comprising the sequence SEQ ID NO. 2 or a fragment or derivative of this sequence, for ND c example the PAP1 protein with the sequences SEQ ID NOS.

O 13, 15, 43 or 45 or with fragments of these sequences.

Another subject of the invention lies in polyclonal or monoclonal antibodies or antibody 00 5 fragments or derivatives, which are directed against f the polypeptide as defined above. Such antibodies can (Ni be generated by methods known to persons skilled in the art. In particular, these antibodies can be prepared by immunizing an animal against a peptide compound of the invention (in particular a polypeptide or a peptide comprising all or part of the sequence SEQ ID NO. 2), sampling the blood and isolating the antibodies. These antibodies can also be generated by preparing hybridomas according to the techniques known to persons skilled in the art.

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

Moreover, these antibodies can also be used for detecting and/or assaying the expression of PAP1 in biological samples and, consequently, for providing information on its activation state.

The antibody fragments or derivatives are for example Fab or Fab'2 fragments, single-chain antibodies (ScFv), etc. They are in particular any fragment or derivative which conserves the antigenic specificity of the antibodies from which they are derived.

ND 16 SThe antibodies according to the invention are O more preferably capable of binding the PAP1 proteins which comprise the sequence SEQ ID NOS. 2, 13, 15, 43 or 45, in particular the region of this protein which 00 5 is involved in the interaction with parkin. These Santibodies (or fragments or derivatives) are more ND preferably capable of binding an epitope which is O present in the sequence between residues 1 and 344 of the sequence SEQ ID NO. 2.

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

A subject of the present invention is also any nucleic acid which encodes a peptide compound according to the invention. It can be in particular a nucleic acid comprising all or part of the sequence SEQ ID NOS. i, 12, 14, 42 or 44, or a derivative thereof.

"Derived sequence" is intended to mean, for the purposes of the present invention, any sequence which hybridizes with the sequence which is presented in SEQ ND 17 ID No. i, or with a fragment of this sequence, and O which encodes a peptide compound according to the invention, as well as the sequences which result from the latter by degeneracy of the genetic code. Nucleic 00 5 acids according to the invention comprise, for example, all or part of the nucleic acid sequence SEQ ID NOS.

O 12, 14, 42 or 44.

The present invention also relates to sequences which have a significant percentage of identity with the sequence presented in SEQ ID NO. 1 or with a fragment of this sequence, and which encode a peptide compound having a physiological behavior similar to that of the PAP1 protein. The expression "significant percentage of identity" is intended to mean a percentage of at least 60%, Preferentially of more preferentially of 90%, and even more preferentially of The various nucleotide sequences of the invention may or may not be of artificial origin. They can be genomic, cDNA or RNA sequences, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained either by screening

DNA

libraries (cDNA library, genomic DNA library), by chemical synthesis, by mixed methods which include the chemical or enzymatic modification of sequences which are obtained by screening of libraries, or by searching for homology in nucleic acid or protein databases. The abovementioned hybridization is preferably carried out 0D 18 _c under the conditions described by Sambrook et al.

O

O (1989, pages 9.52-9.55).

1-- It is advantageously carried out under high stringency hybridization conditions. For the purpose of 00 5 the present invention, the expression "high stringency hybridization conditions" will be intended to mean the 0D following conditions:

(N

1 Membrane competition and PREHYBRIDIZATION: Mix: 40 p1 of salmon sperm DNA (10 mg/ml) 40 p1 of human placental DNA (10 mg/ml) Denature for 5 min at 96 0 C, and then plunge the mixture into ice.

Remove the 2X SSC buffer and pour 4 ml of formamide mix into the hybridization tube containing the membranes.

Add the mixture of the two denatured DNAs.

Incubate at 42 0 C for 5 to 6 hours, with rotation.

2 Labeled probe competition: Add 10 to 50 il of Cot 1 DNA, depending on the amount of nonspecific hybridization, to the labeled and purified probe.

Denature for 7 to 10 min at 95 0

C.

Incubate at 65 0 C for 2 to 5 hours.

3 Hybridization: Remove the prehybridization mix.

NO 19 Mix 40 1 of salmon sperm DNA 40 gi of human O placental DNA; denature for 5 min at 96 0 C and then plunge into ice.

Add 4 ml of formamide mix, the mixture of the two 00 5 DNAs and the labeled probe/denatured Cot 1 DNA to the hybridization tube.

cN kD Incubate for 15 to 20 hours at 42 0 C, with rotation.

O 4 Washing: Wash once at ambient temperature in 2X SSC, to rinse.

Wash twice 5 minutes at ambient temperature in 2X SSC and 0.1% SDS, wash twice 15 minutes in 0.1x SSC and 0.1% SDS at 65 0

C.

Wrap the membranes in Saran and expose.

The hybridization conditions described above are suitable for hybridizing, under high stringency conditions, a molecule of nucleic acid with a variable length of 20 nucleotides to several hundreds of nucleotides.

It goes without saying that the hybridization conditions described above may be adjusted as a function of the length of the nucleic acid for which the hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art.

The suitable hybridization conditions may, for example, be adjusted according to the teaching contained in the work by HAMES and HIGGINS (1985) IND (Nucleic acid Hybridization: a practical approach, 0 O Hames and Higgins Ed., IRL Press, Oxford) or in the work by F. AUSUBEL et al (1999) (Currents Protocols in Molecular Biology, Green Publishing Associates and 00 5 Wiley Interscience, SFor the purposes of the invention, a O particular nucleic acid encodes a polypeptide 3 comprising the sequence SEQ ID NO. 2 or a fragment or derivative of this sequence, in particular the human PAP1 protein. It is advantageously a nucleic acid comprising the nucleic acid sequence SEQ ID NOS. 1, 12, 14, 42 or 44.

Such nucleic acids can be used for producing the peptide compounds of the invention. The present application thus relates to a method for preparing such peptide compounds, according to which a cell which contains a nucleic acid according to the invention is cultured under conditions for expressing said nucleic acid, and the peptide compound produced is recovered.

In this case, the portion which encodes said peptide compound is generally placed under the control of signals which allow its expression in a host cell. The choice of these signals (promoters, terminators, secretion leader sequence, etc.) can vary as a function of the host cell used. Moreover, the nucleic acids of the invention can form part of a vector which can replicate autonomously or can integrate. More particularly, autonomously-replicating vectors can be ND 21 prepared using sequences which replicate autonomously O in the chosen host. As regards the integrating vectors, they can be prepared for example using sequences which are homologous to certain regions of the genome of the 00 5 host, which allow, by homologous recombination, the pq integration of the vector. It can be a vector of

(N

D plasmid, episomal, chromosomal, viral etc., type.

SThe host cells which can be used for producing the peptide compounds of the invention via the recombinant pathway are both eukaryotic and prokaryotic hosts. Among the eukaryotic hosts which are suitable, mention may be made of animal cells, yeasts or fungi. In particular, as regards yeasts mention may be made of the yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127, PC12 etc., cells. Among the fungi, mention may be made more particularly of Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, use of the following bacteria is preferred: E. coli, Bacillus or Streptomyces.

A subject of the present invention is also nonhuman mammals comprising in their cells a nucleic acid or a vector according to the invention.

Such mammals (rodents, canines, rabbits, etc.) can in particular be used for studying the properties of PAP1 and identifying compounds for therapeutic purposes. The modification of the genome of NO 22 such a transgenic animal may result from an alteration 0 or a modification of one or more genes by "knock-in" or by "knock-out". This modification may be produced using conventional altering agents or mutagenic agents, or by 00 5 site-directed mutagenesis. The modification of the genome may also result from an insertion of gene(s)

(N

I\O or from replacement of gene(s) in its (their) wildtype or mutated form. The modifications of the genome are advantageously performed on reproductive stem cells and advantageously on the pronuclei. The transgenesis may be performed by microinjection of an expression cassette comprising the modified genes into the two fertilized pronuclei. Thus, an animal according to the present invention may be obtained by injection of an expression cassette comprising a nucleic acid.

Preferentially, this nucleic acid is a DNA which may be a genomic DNA (gDNA) or a complementary DNA (cDNA).

The construction of transgenic animals according to the invention may be carried out according to conventional techniques known to those skilled in the art. Those skilled in the art may in particular refer to the production of transgenic animals, and particularly to the production of transgenic mice, as described in US patents 4,873,191, 5,464,764 and 5,789,215, the content of these documents being incorporated herein by way of reference.

Briefly, a polynucleotide construct comprising a nucleic acid according to the invention is NO 23 inserted into a line of stem cells of the ES type. The O polynucleotide construct is preferably inserted by electroporation, as described by Thomas et al. (1987, Cell, Vol. 51: 503-512).

O 5 The cells which have undergone the r-.

V' electroporation step are then screened for the presence D of the polynucleotide construct (for example by CD selection using markers, or alternatively by PCR or DNA analysis on an electrophoresis gel such as Southern blotting) in order to select the positive cells which have integrated the exogenous polynucleotide construct into their genome, where appropriate, following a homologous recombination event. Such a technique is, for example, described by MANSOUR et al. (Nature (1988) 336: 348-352).

Next, the positively selected cells are isolated, cloned and injected into 3.5-day mouse blastocysts, as described by BRADLEY (1987, Production and Analysis of Chimaeric mice. In: E.J. ROBERTSON teratocarcinomas and embryonic stem cells: A practical approach. IRL Press, Oxford, page 113).

Blastocysts are then introduced into a female host animal and the embryo is allowed to develop to term.

According to an alternative, positively selected cells of the ES type are brought into contact with 2.5-day embryos at an 8-16 cell stage (morulae) as described by WOOD et al. (1993, Proc. Natl. Acad. Sci.

USA, vol. 90: 4582-4585) or by NAGY et al. (1993, Proc.

ID 24 Natl. Acad. Sci. USA, vol. 90: 8424-8428), the ES cells O being internalized so as to extensively colonize the blastocyst, including the cells which give rise to the germ line.

00 5 The descendants are then tested in order to VB determine those which have integrated the 0D polynucleotide construct (the transgene).

D The nucleic acids according to the invention can also be used to prepare genetic antisense or antisense oligonucleotides which can be used as pharmaceutical agents. Antisense sequences are oligonucleotides of short length, which are complementary to the coding strand of a given gene, and consequently are capable of specifically hybridizing with the mRNA transcript, which inhibits its translation into a protein. A subject of the invention is thus antisense sequences which are capable of inhibiting, at least partially, the interaction of the PAP1 proteins on parkin. Such sequences can consist of all or part of the nucleic acid sequences defined above. They are generally sequences, or fragments of sequences, which are complementary to sequences encoding 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 transferring and expressing, in vivo, antisense sequences or peptides ND c, which are capable of modulating the interaction of the O PAP1 protein with parkin. In this respect, the sequences can be incorporated in viral or nonviral vectors, which allows their administration in vivo 00 5 (Kahn et al., 1991). As viral vectors in accordance with the invention, mention may be made most \D particularly of adenovirus, retrovirus, adeno- D associated virus (AAV) or herpesvirus type vectors.

A

subject of the present application is also defective recombinant viruses comprising a nucleic acid which encodes a polypeptide according to the invention, in particular a polypeptide or peptide comprising all or part of the sequence SEQ ID NO. 2 or of a derivative of this sequence, for example all or part of the sequence SEQ ID NOS. 12, 14, 42 or 44 or derivatives of these sequences.

The invention also enables the preparation of nucleotide probes, which may or may not be synthetic, and which are capable of hybridizing with the nucleotide sequences defined above or their complementary strand. Such probes can be used in vitro as a diagnostic tool for detecting the expression or overexpression of PAP1, or alternatively for revealing genetic abnormalities (incorrect splicing, polymorphism, point mutations, etc.). These probes can also be used for detecting and isolating homologous nucleic acid sequences which encode peptides as defined above, from other cellular sources and preferably from ND 26 cI cells of human origin. The probes of the invention o generally comprise at least 10 bases, and they can for example comprise up to the whole of one of the abovementioned sequences or of their complementary 00 5 strand. Preferably, these probes are labeled prior to t their use. For this, various techniques known to persons skilled in the art can be employed (radioactive, fluorescent, enzymatic, chemical labeling, etc.).

The invention also relates to primers or pairs of primers for amplifying all or part of a nucleic acid encoding a PAP1, for example a primer of sequence chosen from SEQ ID NOS. 16-41.

A subject of the invention is also any pharmaceutical composition which comprises, as an active principle, at least one compound as defined above, in particular a peptide compound.

A subject of the invention is in particular any pharmaceutical composition which comprises, as an active principle, at least one antibody and/or one antibody fragment as defined above, as well as any pharmaceutical composition which comprises, as an active principle at least one nucleic acid or one vector as defined above.

A subject of the invention is also any pharmaceutical composition which comprises, as an active principle, a chemical molecule which is capable IND 27 c of increasing or of decreasing the interaction between O the PAP1 protein and parkin.

Moreover, a subject of the invention is also pharmaceutical compositions in which the peptides, 00 5 antibodies, chemical molecules and nucleotide sequences Sdefined above are combined mutually or with other O active principles.

3 The pharmaceutical compositions according to the invention can be used for modulating the activity of the parkin protein, and consequently for maintaining the survival of the dopaminergic neurons. More particularly, these pharmaceutical compositions are intended for modulating the interaction between the PAP1 protein and parkin. They are, more preferably, pharmaceutical compositions which are intended for treating diseases of the central nervous system, such as for example Parkinson's disease.

A subject of the invention is also the use of the molecules described above for modulating the activity of parkin or for typing diseases of the central nervous system. In particular, the invention relates to the use of these molecules for modulating, at least partially, the activity of parkin.

The invention also relates to a method for screening or characterizing molecules which are active on the function of parkin, which comprises selecting molecules which are capable of binding the sequence SEQ ID NO. 2 or the sequence SEQ ID NO. 4, or a fragment IN 28 (or derivative) of these sequences. The method

O

O comprises, advantageously, bringing the molecule(s) to be tested into contact, in vitro, with a polypeptide which comprises the sequence SEQ ID NO. 2 or the 00 5 sequence SEQ ID NO. 4, or a fragment (or derivative) of t'these sequences, and selecting molecules which are INC capable of binding the sequence SEQ ID NO. 2 (in O particular the region between residues 1 and 344) or the sequence SEQ ID NO. 4. The molecules tested can be varied in nature (peptide, nucleic acid, lipid, sugar, etc., or mixtures of such molecules, for example combinatory libraries, etc.). As indicated above, the molecules thus identified can be used for modulating the activity of the parkin protein, and represent potential therapeutic agents for treating neurodegenerative pathologies.

Other advantages of the present invention will appear upon reading the following examples and figure, which should be considered as illustrative and nonlimiting.

LEGENDS TO THE FIGURE: Figure 1: Representation of the vector pLex9-parkin (135-290) Figure 2: Results of the first 5'-RACE experiment.

8 clones were obtained. The initial electronic sequence is indicated at the bottom of the figure.

ND 29 c_ Figure 3: Results of the second 5'-RACE experiment.

0 Only two of the 8 clones obtained in the first experiment were validated (clones A12 and D5). The initial electronic sequence is indicated at the bottom 00 5 of the figure. The complete sequence of the DNAs and Sproteins is given in the sequences 12-15.

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

Figure 5: Structure of the transcripts isolated from human brain.

Figure 6: Nucleic acid and protein sequence of LY111 (full length) from human brain.

Double underlined: conserved cysteines of the zinc finger domain. Bold: C 2 1 domain, italics: C 2 2 domain.

Figure 7: Nucleic acid and protein sequence of LY111 (short version) from human brain. Double underlined: conserved cysteines of the zinc finger domain. Bold: C 2 1 domain, italics: C 2 2 domain.

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

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

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

Figure 10: Nucleic acid and protein sequence of LY111 (short version) from human brain.

D MATERIALS AND TECHNIQUES USED Cp 1) Yeast strains: Strain L40 of the genus S. cerevisiae (Mata, 00 5 his3D200, trpl-901, leu2-3,112, ade2, LYS2:: (lexAop) 4 tn HIS3, URA3::(lexAop) 8 -LacZ, GAL4, GAL80) was used to \O verify the protein-protein interactions when one of the 0 protein partners is fused to the LexA protein. The latter is capable of recognizing the LexA response element which controls the expression of the reporter genes LacZ and His3.

It was cultured on the following culture media: Complete YPD medium: Yeast extract (10 g/l) (Difco) Bactopeptone (20 g/l) (Difco) Glucose (20 g/l) (Merck) This medium was solidified by addition of 20 g/l of agar (Difco).

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

2) Bacterial strains: ND 31 Strain TG1 of Escherichia coli, of genotype O supE, hsdA5, thi, A(lac-proAB), F'[tra D36 pro A'B* lacI q lacZAM15], was used for constructing plasmids, as a means of amplifying and of isolating recombinant 00 5 plasmids used. It was cultured on the following medium: Medium LB: NaCI (5g/l) (Prolabo) 0D Bactotryptone (10 g/l) (Difco) S- Yeast extract (5 g/l) (Difco) This medium is solidified by addition of 15 g/l of agar (Difco).

Ampicillin was used at 100 pg/ml; this antibiotic is used to select the bacteria which have received the plasmids bearing, as a marker, the gene for resistance to this antibiotic.

Strain HB101 of Escherichia coli of genotype supE44, aral4, galK2, lacY1, A(gpt-proA)62, xyl-5, mtl-l, recA13, A(mcrC-mrr), HsdS-(r-m-) was used as means for amplifying and for isolating plasmids which originate from the human lymphocyte cDNA library.

It was cultured on Medium M9: -Na 2 HP0 4 (7 g/l) (Prolabo)

-KH

2 P0 4 (3 g/l) (Prolabo)

-NH

4 C1 (1 g/l) (Prolabo) -NaC1 (0.5 g/l) (Prolabo) -Glucose (20 g/l) (Sigma) -MgS0 4 (1 mM) (Prolabo) -Thiamine (0.001%) (Sigma) NO ~32 This medium is solidified by addition of 15 g/l of agar O (Difco).

C--

Leucine (50 mg/1) (Sigma) and proline (50 mg/1) (Sigma) should be added to the M9 medium to enable the growth 00 5 of strain HB101.

c During the selection of plasmids which

(N

NO originate from the lymphocyte cDNA two-hybrid library, D leucine was not added to the medium because the plasmids bear a Leu2 selection marker.

3) Plasmids: The 5-kb vector pLex9 (pBTM116) (Bartel et al., 1993), which is homologous to pGBT10 and which contains a multiple cloning site located downstream of the sequence which encodes the LexA bacterial repressor, and upstream of a terminator, for forming a fusion protein.

pLex-HaRasVall2; plasmid pLex9, as described in application WO 98/21327, which contains the sequence encoding the HaRas protein mutated at position Vall2, which is known to interact with the mammalian Raf protein (Vojtek et al., 1993). This plasmid was used to test the specificity of interaction of the PAP1 protein in strain pLex9-cAPP; plasmid pLex9 which contains the sequence encoding the cytoplasmic domain of the APP protein, known to interact with the PTB2 domain of This plasmid was used to test the specificity of interaction of the PAP1 protein in strain NO 33 _c 4) Synthetic oligonucleotides: o TTAAGAATTC GGAAGTCCAG CAGGTAG (SEQ ID SATTAGGATCC CTACACACAA GGCAGGGAG (SEQ ID N"6) Oligonucleotides which made it possible to obtain the 00 PCR fragment which corresponds to the central region of rparkin, bordered with the sites EcoRI and BamHI.

GCGTTTGGAA TCACTACAG (SEQ ID N7) GGTCTCGGTG TGGCATC (SEQ ID N 0 8) CCGCTTGCTT GGAGGAAC (SEQ ID N°9) CGTATTTCTC CGCCTTGG (SEQ ID N AATAGCTCGA GTCAGTGCAG GACAAGAG (SEQ ID N 11) Oligonucleotides which were used to sequence the insert corresponding to the PAP1 gene.

The oligonucleotides are synthesized using an Applied System ABI 394-08 machine. They are removed from the synthesis matrix with ammonia and precipitated twice with 10 volumes of n-butanol, and then taken up in water. The quantification is carried out by measuring the optical density (10D 260 corresponds to pg/ml) Preparation of plasmid DNAs The preparations, in small amount and in large amount, of plasmid DNA were carried out according to the protocols recommended by Quiagen, the manufacturer of the DNA purification kits: Quiaprep Spin Miniprep kit, reference: 27106 Quiaprep Plasmid Maxiprep kit, reference: 12613.

NO 34 6) Enzymatic amplification of DNA by PCR (Polymerase O Chain Reaction): The PCR reactions are carried out in a final volume of 100 gl in the presence of the DNA matrix, of 00 5 dNTP (0.2 mM), of PCR buffer (10 mM Tris-HC1 pH 1 mM MgC1 2 5 mM KC1, 0.01% gelatin), of 10 to 20 pmol

(N

\D of each one of the oligonucleotides and of 2.5 IU of SAmpli Taq DNA polymerase (Perkin Elmer). The mixture is covered with 2 drops of liquid petroleum jelly to limit the evaporation of the sample. The machine used is the "Crocodile II" from Appligene.

We used a matrix denaturation temperature of 94 0 C, a hybridization temperature of 520C and a temperature for elongation by the enzyme at 72 0

C.

7) Ligations: All the ligation reactions are carried out at 370C for one hour in a final volume of 20 in the presence of 100 to 200 ng of vector, 0.1 to 0.5 |ig of insert, 40 IU of T4 DNA ligase enzyme (Biolabs) and a ligation buffer (50 mM Tris-HCl pH 7.8; 10 mM MgC1 2 mM DTT; 1 mM ATP). The negative control consists of ligating the vector in the absence of insert.

8) Transformation of bacteria: The transformation of bacteria with a plasmid is carried out according to the following protocol: gl of the ligation volume are used to transform the TG1 bacteria, according to the method of Chung (Chung et al., 1989). After transformation, the bacteria are ND plated on an LB medium ampicillin and incubated for O 16 h at 370C.

(C 9) Separation and extraction of DNAs: The separation of DNAs is carried out as a 00 5 function of their size, on agarose gel electrophoresis Saccording to Maniatis (Maniatis et al., 1989): 1%

(N

ND agarose gel (Gibco BRL) in a TBE buffer (90 mM Tris Sbase; 80 mM borate; 2 mM EDTA).

Fluorescent sequencing of plasmid DNAs: The sequencing technique used is derived from the method of Sanger (Sanger et al., 1977) and adapted for sequencing by fluorescence, which is developed by Applied Biosystems. The protocol used is that described by the designers of the system (Perkin Elmer, 1997).

11) Transformation of yeast: The plasmids are introduced into the yeast using a conventional technique for transforming yeast, which was developed by Gietz (Gietz et al., 1992) and which was modified in the following way: In the specific case of the transformation of yeast with the lymphocyte cDNA library, the yeast used contains the plasmid pLex9-parkin (135-290), which encodes the central portion of parkin fused to the LexA protein. It is cultured in 200 ml of YNB minimum medium, supplemented with amino acids CSM-Trp, at 30 0

C

with shaking until a density of 107 cells/ml is attained. To carry out the transformation of the yeasts, according to the above protocol, the cell INO 36 suspension was separated into 10 50-gl tubes, into

O

O which 5 gg of the library were added. Heat shock was eC carried out for 20 minutes, and the cells were collected by centrifugation and resuspended in 100 ml 00 5 of YPD medium for 1 h at 30 0 C, and in 100 ml of YNB medium, supplemented with CSM-Leu, -Trp, for 3 h 30 at

(N

OD 30 0 C. The efficiency of the transformation is O determined by placing various dilutions of transformed cells on solid YNB medium which is supplemented with CSM-Trp, -Leu. After 3 days of culture at 30 0 C, the colonies obtained were counted, and the degree of transformation per gg of lymphocyte library DNA was determined.

12) Isolation of plasmids extracted from yeast: 5 ml of a yeast culture, which is incubated for 16 h at 30 0 C, are centrifuged, and taken up in 200 l of a lysis buffer (1M Sorbitol, 0.1 M KH 2

PO

4

/K

2 HP04 pH 7.4, 12.5 mg/ml zymolyase) and incubated for 1 h at 37 0 C. The lysate is then treated according to the protocol recommended by Quiagen, the manufacturer of the DNA purification kit, Quiaprep Spin Miniprep kit, ref 27106.

13) P-galactosidase activity assay: A sheet of nitrocellulose is preplaced on the Petri dish containing the yeast clones, which are separated from each other. This sheet is then immersed in liquid nitrogen for 30 seconds, in order to rupture the yeasts and thus to release the B-galactosidase ID 37 c_ activity. After thawing, the sheet of nitrocellulose is O placed, colonies facing upwards, in another Petri dish 9 containing a Whatman paper which has been presoaked in ml of PBS solution (60 mM Na 2

HPO

4 40 mM NaH 2

PO

4 00 5 10 mM KC1, 1 mM MgS0 4 pH 7) containing 15 il of X-Gal (5-bromo-4-chloro-3-indoyl-P-D-galactoside) at 40 mg/ml ND of N,N-dimethylformamide. The dish is then placed in an CD incubator at 37 0 C. The assay is termed positive when the colonies on the membrane turn blue after 12 hours.

D 38 EXAMPLE 1: CONSTRUCTION OF A VECTOR WHICH ALLOWS THE o EXPRESSION OF A FUSION PROTEIN IN WHICH FUSION IS BETWEEN THE CENTRAL PORTION OF PARKIN AND THE LEXA BACTERIAL

REPRESSOR.

00 5 Screening a library using the double-hybrid on system requires the central region of parkin to be \D fused to a DNA binding protein, such as the LexA Sbacterial repressor. The expression of this fusion protein is carried out using the vector pLex9 (cf.

materials and methods), into which the sequence encoding the central region of parkin, which is in the sequence presented in sequence SEQ ID NO. 3 or 4, is introduced, in the same reading frame as the sequence corresponding to the LexA protein.

The 468 bp-fragment of DNA corresponding to the 156 amino acids of the central region of parkin which begins at amino acid 135, was obtained by PCR using the oligonucleotides (sequence SEQ ID NO. 5 and No. which also made it possible to introduce the site EcoRI at the 5' end and a stop codon and a site BamHI at the 3' end. The PCR fragment was introduced between the EcoRI and BamHI sites of the multiple cloning site of the plasmid pLex9, downstream of the sequence encoding the protein LexA, to give the vector pLex9-parkin (135-290) (Fig. 1).

The construct was verified by sequencing the DNA. This verification made it possible to show that this fragment does not have mutations generated during OD 39 c_ the PCR reaction, and that it was fused in the same O open reading frame as that of the fragment C corresponding to LexA.

00 5 EXAMPLE 2: SCREENING A LYMPHOCYTE FUSION LIBRARY lWe used the double-hybrid method (Fields and \0 Song, 1989).

0 Screening a fusion library makes it possible to identify clones producing proteins which are fused to the transactivating domain of GAL4, and which are able to interact with the protein of interest described in Example 1 (central region of parkin). This interaction makes it possible to reconstitute a transactivator which will then be capable of inducing the expression of the reporter genes His3 and LacZ in strain To carry out this screening we chose a fusion library which is prepared from cDNA originating from peripheral human lymphocytes, supplied by Richard Benarous (Peytavi et al., 1999). Yeasts were transformed with the lymphocyte library and positive clones were selected as described above.

During screening, it is necessary to maintain the probability that each separate plasmid from the fusion library is present in at least one yeast at the same time as the plasmid pLex9-parkin (135-290). To maintain this probability, it is important to have a good efficiency of transformation of the yeast. For D this, we chose a protocol for transforming yeast which O gives an efficiency of 2.6 x 105 transformed cells per jq .g of DNA. In addition, as cotransforming yeast with two different plasmids reduces this efficiency, we 00 5 preferred to use a yeast which is pretransformed with the plasmid pLex9-parkin (135-290). This strain

(N

pLex9-parkin (135-290), of phenotype His-, Lys-, Leu-, C Ade-, was transformed with 50 pg of plasmid DNA from the fusion library. This amount of DNA enabled us to obtain, after estimation, 1.3 x 107 transform cells, which corresponds to a number which is slightly higher than the number of separate plasmids which constitute the library. According to this result, it may be considered that virtually all of the plasmids of the library were used to transform the yeasts. The selection of the transform cells, which are capable of reconstituting a functional transactivator, was done on a YNB medium which was supplemented with 2.5 mM 3 -amino-l,2,4-triazole and 620 mg/l of CSM (BiolOl), and which contains no histidine, no leucine and no tryptophan.

Upon termination of this selection, many clones with a His+ phenotype were obtained. A B-galactosidase activity assay was carried out on these transformants, to validate, by the expression of the other reporter gene, LacZ, this number of clones obtained. 115 clones had the His+, P-Gal+ double ND 41 phenotype, which can correspond to a protein-protein O interaction.

EXAMPLE 3: ISOLATION OF THE LIBRARY PLASMIDS IN THE 00 5 CLONES SELECTED.

t- To identify the proteins which are able to 0D interact with the central region of parkin, the fusion D library plasmids contained in the yeasts which were selected during the double-hybrid screening were extracted. To be able to obtain a large amount thereof, this isolation requires a prior transformation of E. coli with an extract of DNA from the positive yeast strains. As the library plasmid which is contained in this extract is a yeast/E. coli shuttle plasmid, it can easily replicate in the bacterium. The library plasmid was selected by complementing the auxotrophic HB101 bacterium for leucine, on leucine-lacking medium.

The plasmid DNAs from the bacterial colonies which are obtained after transformation with extracts of DNA from yeasts were analyzed by digestion with restriction enzymes and separation of the DNA fragments on an agarose gel. Among the 115 clones analyzed, one clone containing a library plasmid, which showed a different profile from the others, was obtained. This plasmid, termed pGAD-Lylllb, was studied more precisely.

INO 42 (c EXAMPLE 4: DETERMINATION OF THE SEQUENCE OF THE INSERT O CONTAINED IN THE PLASMID IDENTIFIED.

[C Sequencing of the insert contained in the plasmid identified was carried out, firstly, using the 0 5 oligonucleotide SEQ ID No. 7, which is complementary to V the sequence GAL4TA, close to the EcoRI site of \0 insertion of the lymphocyte cDNA library; then, D secondly, using the oligonucleotides SEQ ID No. 8 to SEQ ID No. 11, which correspond to the sequence of the insert which is obtained during the course of the sequencing. The sequence obtained is presented on the sequence SEQ ID NO. 1. The protein thus identified was referred to as PAP1 (Parkin-Associated Protein 1).

Comparison of the sequence of this insert with the sequences which are contained in the GENBank and EMBL (European Molecular Biology Lab) databases showed a homology of 25%, at the protein level, with various members of the synaptotagmin family. The synaptotagmins are part of a family of membrane proteins which are encoded by at least eleven different genes, which are expressed in the brain and other tissues. They contain a single transmembrane domain and two calcium-regulated domains which are termed

C

2 It is in this domain that the homology between the synaptotagmins and the PAP1 protein is found. No other significant homology was observed.

MD 43 c_ EXAMPLE 5: ANALYSIS OF THE SPECIFICITY OF INTERACTION 0 BETWEEN THE CENTRAL REGION OF PARKIN AND THE PAP1

PROTEIN.

To determine the specificity of interaction 00 5 between the fragment corresponding to the PAP1 protein and the central region of parkin, a two-hybrid test for specific interaction with other nonrelevant proteins Swas carried out. To carry out this test, we transformed strain L40 with the control plasmids plex9-cAPP or pLex9-HaRasVall2, which respectively encode the cytoplasmic domain of the APP or the HaRasVall2 protein, which are fused to the domain the DNA binding of LexA, in place of the plasmid pLex9-parkin (135- 290), and with the plasmid which is isolated in the screening of the two-hybrid library. A 3-Gal activity assay was carried out on the cells which were transformed with the various plasmids, to determine a protein-protein interaction. According to the result of the assay, only the yeasts which were transformed with the plasmid which was isolated during the screening of the two-hybrid library, and with the plasmid pLex9-parkin (135-290), had a B-Gal+ activity, which thus shows an interaction between the central region of parkin and the PAP1 protein. This interaction thus turns out to be specific, since this fragment of PAP1 does not seem to interact with the cAPP or HaRasVall2 proteins.

ND 44 These results thus show the existence of a O novel protein, referred to as PAP1, which is capable of interacting specifically with parkin. This protein, which is related to the synaptotagmins, shows no 00 5 significant homology with known proteins, and can be used in therapeutic or diagnostic applications, for y\ producing antibodies, probes or peptides, or for O screening active molecules.

EXAMPLE 6: CLONING OF THE PAP1 GENE FROM A HUMAN LUNG DNA LIBRARY With the aim of identifying the complete sequence of the human PAP1 gene and of characterizing the existence of variant forms, two electronic extension approaches were carried out using the sequence SEQ ID NO. 1. Two electronic sequences were thus obtained, of 1644 bp and 1646 bp, respectively, comprising a 330 bp extension compared to the sequence SEQ ID NO. 1. However, analysis of these sequences showed differences in the consensus region, which were apparent after translation. Thus, a 420aa ORF is obtained in one case, and a 230aa ORF with the other sequence. The protein sequence obtained was compared with known sequences and revealed a 24% homology, over the 293 overlapping amino acids, with human synaptogamin I (p65)(p21579). The function of synaptogamin I may be a regulatory role in membrane interactions during the trafficking of synaptic IND vesicles in the area of the synapse. It binds acid O phospholipids with a certain specificity. In addition, tp a calcium-dependent interaction between synaptogamin and receptors for activated protein kinase C has been OO 5 reported. The synaptogamin may also bind three other proteins, namely neurexin, syntaxin and ap2. Given the (0 abrupt and early disappearance of any homology between C the sequences identified and the synaptogamin family, it is possible that the sequence identified has a deletion compared to the natural sequence. To verify this hypothesis and validate the sequences, an RT-PCR sequencing experiment was carried out using the 1644 bp sequence. The sequence to obtain comprises a 420aa ORF having a homology of the same order with synaptogamins.

To attempt to obtain a larger sequence, and to verify whether the sequence obtained could correspond to a splicing form, a 5'-RACE extension experiment was initiated from the 3' region of the validated sequence, using the oligos L1 and L2 on a preparation of human lung cDNA.

The results obtained are given in figure 2 and show the identification of 8 clones corresponding to 6 different terminal 5' ends. Three of them contain a STOP codon which interrupts the ORF (clones A12, F2, F12) and clone A3 contains no OPR. The presence of various transcripts was confirmed by RT-PCR and nested RT-PCR (table 1).

ND 46 STable 1

O

RT-PCR Primary Secondary PCR Secondary PCR Secondary

PCR

U3-L3 U1-L4

C-B

U3-L3 170 00 A-L4 153+ V' A-L3 Smear (CN Ul-L4 130 SU1-L3 Smear C(N Ul-B 415 U2-B 515 Expected size 170 130 120 The pairs of primers U3-L3 and C-B are specific for the common fragment of the sequence, the oligos A and U1 are specific for the initial sequence and for clone C11, the oligo L4 is specific for the initial sequence and the primer U2 is specific for clone A3. A second 5'-RACE was performed with the oligos L3 and L7 located in the region common to the various clones (figure The results obtained are given in figures 3 and 4. The presence of various transcripts was confirmed by RT-PCR and nested RT-PCR (table 2).

Table 2 RT-PCR Result Secondary PCR Secondary PCR Secondary PCR C-B U3-B U5-L7 U4-F Smear Smear U3-F 1550 bp Expected size (bp) 120 385 530 INO 47 The sequence of the primers and 0 oligonucleotides is given in tables 3 and 4 (SEQ ID NOS. 16-37) Table 3 SEQ ID 00 LY111 U4 CCAGTTCTGCCTGTTCATC 23 to 41 16 LY11_U5 TTCAAAACACAGAGGAGGAG 319 to 338 17 LY111_U3 GAATTGGTCAGTTTAGAGG 759 to 778 18 LY10_L7 1TCTGGGATTGGAGAGCT7TTCAC 851 to 825 19 LYi11_L6 TCrGTCTGTCCCACACACTGCC 914 to 892 LY11 -_L3 GACTGGCTCCGTCrCTCTG 928 to 910 21 LYl 11_C AAGCAACAGAATCTCCCATCC 1029 to 1049 22 LY111_B GCATTGTCAAAATTGCCCATC 1147 to 1127 23 LY11 AGGCGGAGAAATACGAAGAC 1543 to 1562 24 LY1 11_0 GCAGAGTGAGACAGCCCTTAAC 1767 to 1746 Ly11-L2 CTTCCCAGGACTGGCGACTTCAG 1811 to 1782 26 LylillL CAAGCGGTCG1TCATTCCAAAGAG 1934 to 1913 27 r *sA~Y-At f~ AC C.G17 9 to.7.269 78 Table 4 LYl 1 1A TCGTAGAGCAGCAGGTCCAAG 14 to 34 46 LYI 1Ul AGGGCTGCTGGCTATTTTTC 36 to 55 29 LY11-L4 TAAGAAATGGGTTGTGAAC 148 to 166 LY1 1C AAGCAACAGAATCTCCCATCC 1029 to 1049 31 LY1 1_B GCATTGTCAAAATTGCCCATC 1147 to 1127 32 LY1l I-E AGGCGGAGAAATACGAAGAC 1543 to 1562 33 LYl 1 1-D GCAGAGTGAGACAGCCCTTAAC 1767 to 1746 34 Ly1 1L.2 CTTCCTCAGGACTGGCGACTTCAG 1811 to 1782 Ly111L1 CAAGCGGTCGTTCATTCCAAAGAG 1934 to 1913 36 LYl 1 1j AAGAGGAGATAACCCACCAGAG 2288 to 2269 37 This set of results makes it possible to validate the consensus sequence corresponding to the long isoform (figure 9, SEQ ID NOS. 12 and 13) and to the short isoform (figure 10, SEQ ID NOS. 14 and 15) of the PAP1 protein identified from human lung. In the remainder of the examples, this protein is also referred to using the term LY111. The long isoform is encoded by a 1833 bp ORF located at residues 237-2069 of SEQ ID NO. 12, and comprises 610 amino acids. The polyadenylation signal is located starting from 0D 48 Cl nucleotide 2315. The short isoform is encoded by a O 942 bp ORF located at residues 429-1370 of SEQ ID NO.

14, and comprises 313 amino acids. The polyadenylation signal is located starting from nucleotide 1616.

00 5 Northern blotting experiments were then carried out on various human tissues with probes (amplimer CD and E-F) and made it possible to reveal a O 6 kb transcript in muscle, a transcript in heart (3 kb) and also a 6 kb transcript in fetal liver. Example 7 describes, moreover, the cloning of a transcript in human fetal brain.

Various homology studies were carried out in various protein databases, the results of which are given in table 5 below.

Table Databank Homology Genpeptll6 G5926736 (AB025258) granuphilin-a Identity: 31%(215/679), Homology (POS): 46%(322/679) G5926738 (AB025259) granuphilin-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 (L15302) Synaptogamin-1 Identity: 26%(77/293), Homology (POS): 45%(133/293) Swissprot SP:SYT1_CAEEL Synaptogamin 1 Identity: 26%(77/293), Homology (POS): 45%(133/293) SP:SYT2_MOUSE Synaptogamin II Identity: 24%(72/293), Homology (POS): 44% (131/293) EXAMPLE 7: CLONING OF TWO FULL-LENGTH TRANSCRIPTS OF PAP1 (LY111B) FROM HUMAN FETAL BRAIN COMPLEMENTARY

DNA

IND 49 SIn order to confirm the presence of a full- O length Lylllb transcript in human brain, a PCR was carried out using complementary DNA derived from human fetal brain (Marathon Ready cDNA, Clontech), using, as 00 5 primers, the oligonucleotides LyF1 (AAT GGA AGG GCG TGA SCTC, figure 5, SEQ ID NO. 38) and HA71 (CCT CAC GCC TGC

(N

TGC AAC CTG, SEQ ID NO. 39). A weakly represented DNA fragment of approximately two kilobases was amplified.

The product of this first PCR was used as matrix for a nested PCR, carried out with the oligonucleotides LyEcoF (GCACGAATTC ATG 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 oligonucleotide LyEcoF)and BstEII (figure 5) and inserted into the expression vector pcDNA3, and their sequence was then determined. Analysis of the sequence of clones obtained revealed the presence of two potential full-length Lylllb transcripts in human fetal brain (figure The first of these transcripts (LylllbfulA) corresponds to the mRNA identified in human lung (example 6) and encodes a 609 amino acid protein (pLylllbfuliA; figures 5 and 6, SEQ ID NOS. 42-43). The second (LylllbfuiiB) probably represents a product of alternative splicing of a common primary mRNA. In this transcript, which is identical to Lylllbfu 1 A, the sequence between nucleotides 752 and 956 of the sequence validated in human lung is absent (SEQ ID NO.

ND 51 In addition, it has been described that the C 2 domains o of the proteins of the RIM/rabphilin family may bind membranes through the interaction with phospholipids.

00 5 Expression of the pLylllbfu1A/fullB proteins in cells of Sthe cos-7 line: colocalization with Parkin

(N

I\D The coding sequence of the LylllbfulA/B C transcripts was inserted into the eukaryotic expression vector pcDNA3 in frame with the sequence encoding an N-terminal myc epitope (pcDNA 3 -mycLylllbfulA/B) Cells of the cos-7 line transfected using these vectors produce proteins with an apparent molecular weight of approximately 67 kDa (pcDNA3-mycLylllbfuiiA) and 60 kDa (pcDNA3-mycLylllbfu 1 corresponding to the expected molecular weight. These proteins, revealed by immunolabeling using an antibody directed against the N-terminal myc epitope, are distributed nonhomogeneously, in a punctate manner, in the cytoplasm, the extensions and sometimes the nucleus of the cells of the cos-7 line (figure 8a, b, column A).

When they are overexpressed with Parkin, revealed using the anti-Parkin antibody Asp5, in the cells of the cos- 7 line (figure 8a, b, column a similar distribution is a colocalization of these proteins may be observed (figure 8a, b, column C).

\O

IND

51a c O Throughout this specification and the claims c.i which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and M 5 "comprising", will be understood to imply the inclusion 00 of a stated integer or step or group of integers or steps M but not the exclusion of any other integer or step or O group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

0D 52 c_ BIBLIOGRAPHIC REFERENCES 0 0 eC Abbas, N. et al. (1999). A wide variety of mutations in the parkin gene are responsible for autosomal 00 5 recessive parkinsonism in Europe. Hum Mol Genet.

8, 567-574.

(c ID Bartel, P.L. et al. (1993). D.A. Hartley Ed, Oxford SUniversity Press, 153 Chung, C.T. et al. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution, Proc. Natl. Acad. Sci. USA. 86, 2172-2175.

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Claims (22)

1. 3. Compound according to either of claims 1 and 2, characterized in that it is capable of binding the domain of interaction between the PAP1 protein, or a homolog of this protein, and parkin.
2. 4. Compound according to one of claims 1 to 3, characterized in that it is a compound of peptide, nucleic acid, lipid or saccharide type, or an antibody. Compound according to claim 4, characterized in that it is a peptide compound comprising all or part of the peptide sequence SEQ ID NO. 2 or a derivative thereof.
3. 6. Compound according to claim 4, characterized in that it is a peptide compound comprising a region of which the sequence corresponds to all or a functional part of the site of interaction of the PAP1 protein with parkin.
4. 7. Compound according to claim 4, characterized in that it is a peptide compound which is ND 56 derived from the PAP1 protein (and/or from the O homologous forms), and which bears an effector region which has been made nonfunctional.
5. 8. Polypeptide comprising the sequence SEQ 00 5 ID NO. 2 or a derivative or fragment of this sequence.
6. 9. Polypeptide according to claim 8, IND comprising at least 5 consecutive residues of the Ssequence SEQ ID NO. 2, preferably at least 9, more preferably at least
7. 10. Polypeptide according to claim 8, comprising all or part of the sequence SEQ ID NOS. 13, 43 or 45 or a variant of these sequences, in particular at least 5 consecutive residues, preferably at least 9, more preferably at least 15 consecutive residues of the sequence SEQ ID NOS. 13, 15, 43 or
8. 11. Nucleic acid encoding a peptide compound according to one of claims 4 to
9. 12. Nucleic acid according to claim 11, characterized in that it comprises all or part of the sequence SEQ ID NOS. i, 12, 14, 42 or 44 or a sequence which is derived from this sequence.
10. 13. Nucleic acid encoding a polypeptide according to claim 8 or 11.
11. 14. Nucleic acid, in particular a nucleotide probe, which is capable of hybridizing with a nucleic acid according to one of claims 11 to 13, or with their complementary strand. ND 57
12. 15. Vector comprising a nucleic acid O O according to one of claims 11 to 14. C 16. Defective recombinant virus comprising a nucleic acid according to one of claims 11 to 14. 00 5 17. Nucleic acid chosen from the nucleic r- V' acids of sequence SEQ ID NOS. 16-41 and 46. (N ND 18. Antibody or antibody fragment or derivative, characterized in that it is directed against a peptide compound according to one of claims 4 to
13. 19. Antibody according to claim 18, characterized in that it recognizes a polypeptide according to claim 9 or Pharmaceutical composition comprising at least one compound according to one of claims 1 to or an antibody according to claim 18 or 19.
14. 21. Nonpeptide compound or a compound which is not of exclusively peptide nature, which is capable of modulating, at least partially, the interaction of the PAP1 protein, or a homolog of this protein, with parkin.
15. 22. Compound according to claim 21, characterized in that the active motifs of a peptide according to one of claims 5 to 7 have been duplicated with a structure which is not a peptide or which is not of exclusively peptide nature. ND 58
16. 23. Pharmaceutical composition comprising at O least one nucleic acid according to one of claims 11 to C- 14, or one vector according to claim 15 or 16.
17. 24. Pharmaceutical composition comprising a 00 5 peptide compound according to one of claims 4 to Composition according to claim 22, 23 or O 24, intended for treating neurodegenerative Spathologies.
18. 26. Method for screening or for characterizing active molecules, which comprises a step of selecting molecules which are capable of binding the sequence SEQ ID NO. 2 or the sequence SEQ ID NO. 4, or a fragment of these sequences.
19. 27. Method for screening or for characterizing active molecules, which comprises a step of selecting molecules which are capable of binding a sequence chosen from SEQ ID NOS. 13, 15, 43 and 45, or a fragment of these sequences.
20. 28. Method for producing a peptide compound according to one of claims 4 to 10, comprising the culture of a cell which contains a nucleic acid according to one of claims 11 to 14 or a vector according to claim 15 or 16, under conditions for expressing said nucleic acid, and the recovery of the peptide compound produced.
21. 29. Human PAP1 protein in isolated form. D 59 Cell containing a nucleic acid according O 0 to one of claims 11 to 14 or a vector according to 1-- C claim 15 or 16.
22. 31. Nonhuman mammal comprising in its cells 00 5 a nucleic acid according to one of claims 11 to 14. \O 0D 0D (N