DE10059595A1 - Proteins and the DNA sequences underlying the proteins with function in inflammatory events - Google Patents

Abstract

The present invention relates to DNA sequences which code for at least one PYD domain, expression vectors which contain such DNA sequences, host cells which are transformed with such expression vectors, purified gene products of the aforementioned DNA sequences, antibodies against the aforementioned gene products, and Process for the isolation and / or expression of the aforementioned gene products. Furthermore, the present invention relates to the use of the aforementioned DNA sequences or of their gene products for the treatment of inflammatory events.

Description

The present invention relates to DNA sequences for encode at least one PYD domain, expression vectors, which contain such DNA sequences, host cells which are transformed with such expression vectors, purified gene products of the aforementioned DNA sequences, Antibodies against the aforementioned gene products, methods for isolation and / or expression of the aforementioned Gene products and the use of the DNA sequences or the Gene products for the treatment of inflammatory events.

Proteins have a modular structure, the individual sections of proteins being structurally and possibly functionally independent. These sections are called domains. Proteins with a modular structure occur, for example, in proteins of the apoptotic signal transduction chain (Aravind et al. ( 1999 ) TIBS, 24, 47-53; Hofmann ( 1999 ) Cell. Mol. Life. Sci., 55, 1113-28). For the class of apoptotic signal transduction proteins, three families of domains should be mentioned, namely the family of the death domains (DD), the family of the death defect domains (DED) and the family of the caspase recruitment domain (CARD), all of which are distantly related and also all belong to a superfamily, which typically forms a bundle of six helices ("six helix bundle") in its 3-dimensional structure. Proteins which have a death domain, death effector domain and / or a CARD domain include, for example, the proteins FLIP, CARDIAK-RIP2, ARC, Bcl10, DEDD, as described in the publications by Irmler et al., 1997, Nature, 388, 190-195; Koseki et al. 1998, Proct. Natl. Acad. Sci. USA, 95, 5156-60; McCarthy et al. 1998, J. Biol. Chem. 273, 16968-16975; Stegh, et al., 1998, EM-BO J., 17, 5974-86; Thome et al., 1999, J. Biol. Chem., 274, 9962-8. Thus, while numerous proteins that have domains of the structural superfamily (bundle of six helices) are involved in intracellular signal transduction processes, in particular due to their ability to associate with proteins that are connected upstream or downstream, the proteins involved in the inflammatory reaction are proteins , as well as the form of signal transmission in inflammatory reactions largely unknown.

The object of the present invention is on the one hand such proteins (with their amino acid sequences) and possibly identify the underlying DNA sequences, involved in the inflammatory response, or from other function known proteins have a function in assigned to the inflammatory response cascade and others on the one hand, by determining the signal transduction mechanism men, substances for the treatment of inflammatory reactions to provide.

To solve this problem, the inventors initially have found that PYD domains play a crucial role when an inflammatori is passed on intracellularly playing signal. According to the invention was festge represents that the domain PYD also the structure of the Has a bundle of 6 helices and that they in their In interaction potential with the structurally related domains DED, DD or CARD is comparable. In order to  according to the invention, a fourth family of "six Helix bundle "domains (here referred to as domain" PYD "), which occurs as a component of native proteins for Provided.

The present invention therefore relates to DNA sequences which code for a protein with at least one PYD domain, including all functionally homologous derivatives, fragments or alleles. In particular, all DNA sequences are included which hybridize with the DNA sequences according to the invention, including the sequences which are complementary in each case in the double strand (claim 1). In a preferred embodiment, such DNA sequences according to the invention are disclosed for which a significance level of p <10 ^-2 results if the PYD domain of a target DNA sequence (ie a DNA sequence potentially according to the invention) with a search profile is compared to Fig. 3 (claim 2). With regard to the relevant experimental procedure, reference is made to the publication by Bucher et al. (1996, Computer Chem., 20, 3-24) and to the corresponding explanations in the published German patent application DE 197 13 393.2-41, both of which are part of the disclosure of the present application.

In a further preferred embodiment, DNA sequences are disclosed whose gene product contains one of the amino acid sequences (for a PYD domain), as shown in FIG. 6, including all functionally homologous derivatives, alleles or fragments. DNA sequences hybridizing with these DNA sequences according to the invention (including the sequences of the complementary DNA strand) are also disclosed (claim 3).

DNA sequences which contain one of the (c) DNA sequences indicated in FIG. 1 are also preferred (claim 4). In the course of the findings according to the invention, it was found that DNA sequences according to the invention code for numerous proteins (amino acid sequences) with a PYD domain, which are involved in particular in the pathophysiological inflammation event, if appropriate. These DNA sequences (including the corresponding amino acid sequences) are shown in Fig. 1 Darge. As shown in FIG. 1 with coding number, this includes the proteins pyrine (see corresponding protein or cDNA sequences according to FIG. 1, coding number 1.2 ), pycard (protein and cDNA sequence, coding number 1.3 ), Pyc (protein sequence and cDNA sequence, coding number 1.1 ), NALP1 (protein sequence and cDNA sequence, coding number 1.4 ), NALP2 ((old name Py7) with protein and a DNA sequence, coding number 1.5 ), NALP3 ((old name PY5 ) with protein sequence and DNA sequence, coding number 1.6 ), NALP4 ((old name PY6) with protein sequence and DNA sequence, coding number 1.7 ), NALP5 ((old name Py8) with protein sequence and DNA sequence, coding number 1.8 ), NALP6 ( (old name PY9) with protein sequence and DNA sequence, coding number 1.9 ), PY10 (with protein and DNA sequence, coding number mer 1.10 ), NALP7 ((old name Py11) with protein sequence and cDNA sequence, coding number 1.11 ), NALP8 ((old name Py12) with protein sequence and DNA sequence , Coding number 1.12 ), NALP9 ((old name Py13) with protein sequence and cDNA sequence, coding number 1.13 ), NALP10 ((old name Py14) with protein and DNA sequence, coding number 1.14 ), NALP11 ((old name Py15) with protein and cDNA sequence, coding number 1.15 ), Py16 ((from the mouse)., With protein sequence and DNA sequence, coding number mer 1.16 ), NALP13 ((old name Py17), with protein and CDNA sequence , Coding number 1.17 ), NALP14 ((old name Py18), from the mouse, with protein and cDNA sequence, coding number 1.18 ), NALP15 ((old name Py19) with protein and partial DNA sequence, coding number 1.19 ) , NALP12 ((old name Py20), from the mouse, with protein sequence and cDNA sequence, coding number 1.20 ). The sequences mentioned above are all plotted in FIG. 1 and can be found there under the abovementioned designations or the coding numbers. The DNA and protein sequences that belong together, that is to say combined under a coding number, are separated from the next unit by dotted lines in bold type. Fig. 1 summarizes 22 numbered pages. Another preferred subject of the present invention is DNA sequences which code for one of the gene products shown in FIG. 1 (ie for one of the amino acid sequences contained in FIG. 1) or DNA sequences which comprise at least one section encode the overall sequence for one of the amino acid sequences indicated in FIG. 1 (claim 5).

Another object of the present invention are Expression vectors that contain a DNA Sequence, for example as previously disclosed or according to claims Claims 1 to 5 contain (claim 6). such expression vectors according to the invention (for example plasmids) contain in addition to at least one DNA Sequence typically also promoter regions and terms nator areas, possibly also marker genes (e.g. antibiotics Resistance genes) and / or signal sequences for transport of the translated protein.

The present invention further relates to host cells which have been transformed with an expression vector according to the invention (claim 7). Prokaryotic yeasts or higher eukaryotic cells are suitable as suitable host cells for cloning or expressing the DNA sequences according to the invention. In prokaryotes, gram-negative or gram-positive organisms are expressly included. E. coli or bacilli should be mentioned here. The strains E. coli 294, E. coli B and E. coli X1776 and E. coli W3110 are disclosed as preferred host cells for cloning the DNA sequences according to the invention. The bacilli are Bacillus subtilis, Salmonella typhimurium or the like. As already mentioned above, the expression vectors typically contain a signal sequence for transporting the protein into the culture medium, so per karyotic cells are used. In addition to prokaryotes, eukaryotic microbes can also be used as host cells that have been transfected with the expression vector. For example, filamentous fungi or yeasts can be used as suitable host cells for the vectors coding for the DNA sequences according to the invention. Particularly noteworthy is Saccharomycis cirevesiae or common baker's yeast (Stinchcomb et al., Nature, 282: 39, ( 1997 )).

In a preferred embodiment, however, cells from multicellular organisms are selected for expression of DNA sequences according to the invention. This also happens against the background of a possibly necessary glycosylation of the encoded proteins. This function can be carried out in a suitable manner in higher eukaryotic cells compared to prokaryotic cells. In principle, any higher eukaryotic cell culture is available as a host cell, although cells from mammals, for example monkeys, rats, hamsters or humans, are very particularly preferred. A large number of established cell lines are known to the person skilled in the art. In a list that is by no means exhaustive, the following cell lines are mentioned: 293T (embryonic kidney cell line), (Graham et al., J. Gen. Virol., 36: 59 ( 1997 )), BHK (baby hamster kidney cells), CHO (cells from the hamster ovaries) ), (Urlaub and Chasin, PNAS (USA) 77: 4216, ( 1980 )), HeLa (human cervical carcinoma cells) and other cell lines.

For the purposes of the present invention, expressions vectors which have the DNA sequences according to the invention sen, preferably cells of the mammalian immune system, above al lem of the human immune system, transfected (claim 8).

Another aspect of the present invention is that Gene products of the DNA sequences according to the invention 9). Gene products are understood in the sense of this invention both primary transcripts, ie RNA, preferably mRNA, as well as proteins or polypeptides, especially in cleaned form (claim 10). These proteins have been invented according to at least one PYD domain and regulate or in particular transport inflammatory signals.  

A purified gene product is preferred if it contains one of the amino acid sequences given in FIG. 7 (for a PYD domain), including all functionally homologous alleles, fragments or derivatives. However, the proteins according to the invention also include all those proteins which are derived from DNA derivatives, DNA fragments or DNA alleles according to the invention.

In addition, the proteins of the invention can be used be mixed modified. For example, a protecting group on N terminus are present. There can be glycosyl groups Hydroxyl or amino groups can be added, lipids can be covalently linked to the protein according to the invention, as well as phosphates or acetyl groups and the like. Also be any chemical substances, compounds or groups can in any way of synthesis to the invention appropriate protein bound. Also additional amino acids, z. B. in the form of individual amino acids or in the form of peptides or in the form of protein domains and the like be fused with the N and / or C terminus. Insbesonde re so-called signal or "leader" sequences here N-terminus of the amino acid sequence according to the invention gene that makes the peptide cotranslational or posttranslational in a certain cell organelle or in the extracellular Lead space (or the culture medium). At the N- or at the C- Terminus may also have amino acid sequences that appear as Antigen binding the amino acid sequence according to the invention allow for antibodies. This is particularly worth mentioning Flag peptide, the sequence of which in the one-letter code of the amino acids is: DYKDDDDK. This sequence has strong antigens Properties and thus allows a quick check and easy purification of the recombinant protein. Monoklo nale antibodies that bind the flag peptide are of the Eastman Kodak Co., Scientific Imaging Systems Divisi on, New Haven, Connecticut. The invention DNA sequences can also be found in numerous exons are separated from each other by introns, on the strand of the Genetic information molecule. With that also belong all conceivable SPLICE variants (at the mRNA level) as gene products  to the subject of the invention. Even that of these different SPLICE variants encoded proteins subject to this invention.

Another object of the present invention is an antibody that has an epitope on a ß gene product, especially a Pro according to the invention tein, recognizes (claim 12). The term "antibody" around summarizes i. S. of the present invention both polyclonal Antibodies as well as monoclonal antibodies (claim 13), chimeric antibodies, anti-idiotypic antibodies (directed against antibodies according to the invention), all are available in bound or soluble form and if necessary can be marked by "label", as well as fragments the aforementioned antibodies. In addition to the fragments of he antibodies according to the invention in isolation, he can Antibodies according to the invention also in recombinant form as Fusion proteins with other (protein) components to step. Fragments as such or fragments from inventions antibodies according to the invention as components of Fusionspro teins are typically enzymatic by the methods shear cleavage, protein synthesis or the subject known recombination methods.

The polyclonal antibodies are heterogeneous mixtures of antibody molecules which are produced from sera from animals which have been immunized with an antigen. A monoclonal antibody contains an essentially homogeneous population of antibodies which are specifically directed against antigens, the antibodies having essentially the same epitope binding sites. Monoclonal antibodies can be obtained by methods known in the art (e.g., Koehler and Milstein, Nature, 256, 495-397, ( 1975 ); U.S. Patent 4,376,110; Ausübel et al., Harlow and Lane "Antibodies ": Laboratory Manual, Cold Spring, Harbor Laboratory ( 1988 )). The description contained in the aforementioned litterature locations is included as part of the present invention in the disclosure of the vorlie invention. Antibodies according to the invention can belong to one of the following immunoglobulin classes: IgG, IgM, IgE, IgA, GILD and possibly a subclass of the aforementioned classes. A hybridoma cell clone, which he produces monoclonal antibodies according to the invention, can be cultivated in vitro, in situ or in vivo. The production of large titers on monoclonal antibodies is preferably carried out in vivo or in situ.

The chimeric antibodies according to the invention are molecules which contain various constituents, which are derived from different animal species (e.g. antibodies which have a variable region which is derived from a mouse monoclonal antibody and a constant one) Region of a human immunoglobulin). Chimeric antibodies are preferably used on the one hand to reduce the immunogenicity in the application and on the other hand to increase the yields in the production, for. B. murine monoclonal antibodies give higher yields from hybridoma cell lines, but also lead to a higher immunogenicity in humans, so that human / murine chimeric antibodies are preferably used before. Chimeric antibodies and methods for their preparation are known from the prior art (Cabilly et al., Proc. Natl. Sci. USA 81 : 3273-3277 ( 1984 ); Morrison et al. Proc. Natl. Acad. Sci USA 81 : 6851-6855 ( 1984 ); Boulianne et al. Nature 312 643-646 ( 1984 ); Cabilly et al., EP-A-125023; Neuberger et al., Nature 314 : 268-270 ( 1985 ); Taniguchi et al. , EP-A-171496; Morrion et al., EP-A-173494; Neuberger et al., WO 86/01533; Kudo et al., EP-A-184187; Sahagan et al., J. Immunol. 137: 1986 , 1066-1074; Robinson et al., WO 87/02671; Liu et al., Proc. Natl. Acad. Sci USA 84 : 3439-3443 ( 1987 ); Sun et al., Proc. Natl. Acad. Sci USA 84 : 214218 ( 1987 ), Better et al., Science 240: 1041-1043 ( 1988 ) and Harlow and Lane, Antibody: A Laboratory Manual, cited above, and these references are believed to be disclosed in the present invention included.

Such is particularly preferred according to the invention ß Antibody against a sequence section on the PYD Domain to be directed as an epitope (claim 14).

An anti-idiotypic antibody according to the invention is an antibody that is a determinant that in general with the antigen binding site of an invention is associated with the body. An anti-idiotypic Antibodies can be raised by immunizing an animal of the same type and the same genetic type (e.g. egg mouse strain) as the starting point for a monoclonal Antibodies against which an anti- idiotypic antibody is directed to who the. The immunized animal becomes the idiotypic deteter minants of the immunizing antibody by the Pro production of an antibody against the idiotypic Is directed determinants (namely a anti-idiotic antibody) (U.S. 4,699,880). An anti-idiotypic type according to the invention Antibody can also be used as an immunogen to to elicit an immune response in another animal and there to produce a so-called anti-anti to lead idiotypic antibody. The anti-anti idiotypic antibodies can, but need not, regarding its epitope construction is identical to the original monoclonal antibody that is the anti-idiotypic Response. That way, by the use of against idiotypic determinants monoclonal antibody directed antibody other Clones that have antibodies of identical specificity expri mate, be identified.

Monoclonal antibodies against Pro teine, analogs, fragments or derivatives of this invent Proteins according to the invention can be used  to the binding of anti-idiotypic antibodies in appropriate animals, such as. B. the BALB / c mouse induce. Immunize cells from the spleen of such ten mouse can be used to anti-idiotypic Hybridoma cell lines, the anti-idiotypic monoclonal An secretion, produce. Can continue anti-idiotypic monoclonal antibodies also to one Carriers are coupled (KLH, "keyhole limpet hemocya nin ") and then used to further BALB / c mice to immunize. The sera of these mice then contain ti-anti-idiotypic antibodies that bind the binding have original monoclonal antibodies and specific for an epitope of the protein according to the invention or a fragment or derivative thereof. The anti-idiotypic monoclonal antibodies have been raised this way their own idiotypic epitopes or "Idiotopes" that are structurally related to the subject under investigation Epitope are similar.

The term "antibody" is intended to include both intact molecules and fragments thereof, e.g. B. Fab and F (from ') ₂ . Fab and F (ab ') ₂ fragments lack an Fc fragment, such as in an intact antibody, so that they can be transported faster in the bloodstream and have comparatively less non-specific tissue binding than intact antibodies. It is emphasized here that Fab and F (ab _' ) ₂ fragments of antibodies according to the invention can be used in the detection and quantification of proteins according to the invention. Such fragments are typically produced by proteolytic cleavage by using enzymes such as e.g. B. papain (for the production of Fab fragments) or pepsin (for the production of F (ab _' ) ₂ , fragments) can be used.

Antibodies according to the invention, including the fragments of these antibodies, can be used for quantitative or qualitative detection of protein according to the invention in  a sample can be used or also for the detection of Cells expressing proteins according to the invention and secret if necessary. The detection can be done with the help of Im munofluorescence method can be achieved, the fluorescence zenz-labeled antibodies in combination with light micro scopy, flow cytometry or fluorometric detection be performed.

Antibodies according to the invention (or fragments of these anti body) are suitable for histological examinations, such as z. B. in the context of immunofluorescence or immunoelectro microscopy, for the in situ detection of an invention protein. He can perform in situ detection follow that up a histological sample from a patient is taken and labeled antibodies according to the invention to be added to such a sample. The antibody per (or a fragment of this antibody) is described in mar form on the biological sample. On this way it is not only possible for the presence of to determine protein according to the invention in the sample, son also the distribution of the protein according to the invention in the examined tissue. With the biological sample it can be a biological fluid, a tissue extract, harvested cells, such as B. immune cells or Cardiac muscle or liver cells, or generally around cells, that have been incubated in a tissue culture. The detection of the labeled antibody can, depending on the type the marking by Ver known in the prior art drive (e.g. by fluorescence method). The biological sample can also on a solid phase ger, such as B. nitrocellulose or another vehicle material, are applied so that the cells, cell parts or soluble proteins can be immobilized. The carrier can then be used one or more times with a suitable buffer be washed, followed by a detector bar labeled antibody according to the present invention is treated. The solid phase carrier can then with the Buffer washed a second time to free it  Eliminate antibodies. The amount of bun The marker on the solid phase support can then be used can be determined using a conventional method.

Glass, polystyrene and Po are particularly suitable as supports lypropylene, polyethylene, dextran, nylon amylases, natural Liche or modified celluloses, polyacrylamides and Magnetite. The carrier can either be partially soluble or insoluble in character to measure conditions object of the present invention to meet. The carrier material can take any shape, e.g. B. in the form of beads, or cylindrical or spherical be, with polystyrene beads preferred as a carrier are.

A detectable antibody label can be ver in different ways. For example, the anti body bound to an enzyme, the enzyme finally used in an immunoassay (EIA) can. The enzyme can then later with an appropriate React substrate so that a chemical compound ent stands in a manner familiar to the person skilled in the art can be detected and quantified if necessary, e.g. B. by spectrophotometry, fluorometry or other opti procedures. The enzyme can be malate Dehydrogenase, staphylococcal nuclease, delta-5 steroid Isomerase, yeast alcohol dehydrogenase, alpha Glycerophosphate dehydrogenase, triose phosphate isomerase, Horseradish peroxidase, alkaline phosphatase, Asparigi nose, glucose oxidase, beta-galactosidase, ribonuclease, Urease, catalase, glucose-6-phosphate dehydrogenase, Act glucoamylase or acetylcholinesterase. The De tection is then over a chromogenic substrate, the spe is specific for the enzyme used for the labeling, enables and can finally z. B. via visual comparison of the substrate converted by the enzyme reaction in Ver to control standards.  

Furthermore, the detection can be ensured by other immunoassays, e.g. B. by radioactive labeling of the antibodies or antibody fragments (ie by a radioimmunoassay (RIA; Laboratory Techniques and Biochemistry in Molecular Biology, Work, T. et al. North Holland Publishing Company, New York ( 1978 ). The radioactive isotope can be detected and quantified by using scintillation counters or by autoradigraphy.

Fluorescent compounds can also be used for labeling, for example compounds such as fluorescine isothiocyanate, rhodamine, phyoerythrin, phy cocyanin, allophycocyanin, o-phthaldehyde and fluoresca min. Also fluorescent-emitting metals, such as. B. ¹⁵² E or other metals from the lanthanide group can be used. These metals are attached to the antibody via chelate groups, such as. B. Diethylenetriaminepentaessig acid (ETPA) or EDTA coupled. Furthermore, the antibody according to the invention can be coupled via a compound which acts with the aid of chemiluminescence. The presence of the chemiluminescence-labeled antibody is then detected via the luminescence which arises in the course of a chemical reaction. Examples of the like compounds are luminol, isoluminol, acridiniu ester, imidazole, acridinium salt or oxalate ester. Likewise, bioluminescent compounds can also be used. Bioluminescence is a subspecies of chemiluminescence found in biological systems where a catalytic protein enhances the efficiency of the chemiluminescent reaction. The detection of the bioluminescent protein is in turn carried out via luminescence, with luciferin, luciferase or aequorin being suitable as the bioluminescent compound.

An antibody of the invention can be used in an immunometric assay, also known as a "twosite"  or "sandwich" assay. Ty Typical immunometric assay systems include so-called "Forward" assays, which are characterized in that Antibodies according to the invention to a solid phase system are bound and that the antibody with the sample, the un is sought, is brought into contact in this way. In this way the antigen is removed from the sample by formation of a binary solid phase antibody-antigen complex isolated from the sample. After a suitable incubation period the solid support is washed to remove the remaining Eliminate rest of the liquid sample, including the possibly unbound antigen, and then with egg contacted a solution that has an unknown amount contains on labeled detection antibody. The marked Antibody serves as a so-called reporter molecule. To a second incubation period, which corresponds to the marked An antibody allowed, with the An bound to the solid phase to associate, the solid phase carrier becomes again washed to labeled antibody that is unresponsive have to eliminate.

In an alternative form of an assay, a so-called. sandwich assay are used. Here, a the only incubation step is sufficient if the to the Solid phase bound antibody and the labeled antibody both applied simultaneously to the sample to be tested be brought. After the incubation is complete, the Solid phase carriers washed to remove liquid residues Sample and the unassociated labeled antibody remove. The presence of labeled antibody the solid phase carrier is determined in exactly the same way as for the conventional "forward" sandwich assay. With the so-called reverse assay is gradually a solution of the added labeled antibody to the liquid sample, ge follows from the addition of unlabeled antibody per, bound to a solid phase support, after expiry a suitable incubation period. After a second Inku The solid phase carrier is the conventional step  Way washed to remove it from sample remains and mar antibody that has not responded. Determination of the labeled antibody with the Solid phase carrier has reacted, then, as above wrote, performed.

Another aspect of the present invention is a ver drive to isolate gene products with at least one PYD domain, the host cells with a ß expression vector transformed and then under appropriate conditions that promote expression are cultivated the, so that the gene product eventually emerges from the culture can be cleaned (claim 15). The protein of the invent DNA sequence according to the invention can, depending on the Ex pression system, from a culture medium or from Cellx tracts are isolated. The expert can easily recognize that the respective insulation methods and Process for the purification of a fiction according to DNA encoded, recombinant protein strongly of the type the host cell or the fact whether the protein is in the medium is secreted depends. For example, you can Expression systems are used for the secretion of the recombinant protein. The culture medium must be in the case through commercially available protein concentrations tion filter, e.g. B. Amicon or Millipore Pelicon, aufkon be centered. After the concentration step, a Cleaning step take place, e.g. B. a gel filtration step. Alternatively, an anion exchanger can also be used that has a matrix with DEAE.

A1 s matrix are all used for protein purification known materials, e.g. B. acrylamide or agarose or Dextran or the like. But it can also be a cation exchangers are used, which then typically Contains carboxymethyl groups. For further cleaning a protein encoded by a DNA according to the invention then serve HPLC steps. It can be one or act several steps. In particular, the "reversed Phase "method is used. These steps are for preservation  an essentially homogeneous recombinant protein a DNA sequence according to the invention.

In addition to bacterial cell cultures for isolating the gene product, transformed yeast cells can also be used. In this case, the translated protein can be secreted so that the protein purification is simplified. Secreted recombinant protein from a yeast host cell can be obtained by methods as described in Urdal et al. (J. Chromato. 296: 171 ( 1994 )).

Another aspect of the present invention is a ver drive to the expression of gene products with at least one PYD domain, where host cells with an expression vector, which contains a DNA sequence according to the invention, transfor be lubricated (claim 17). This method of expression of gene products that are based on a DNA Sequence based, does not serve the corresponding gene to concentrate and purify product, but a lot more on this, the cell metabolism by introducing it DNA sequences according to the invention via the expression of the to influence the related gene product. Here is in particular re to the use of using expression vectors transformed host cells for the purpose of switching off the think inflammatory response. By using it a so-called constitutive promoter can in this Cells constant concentrations of fiction proteins based on sequences are expressed. In this way the triggering of the Inflamma becomes permanent tion prevented.

The corresponding cell lines are compared to egg Resistant to a wide range of inflammatory stimulants. These modified cells can also, if necessary, like which returns to the mammalian or human organism become. In this way, through in vitro with the inventions expression vectors of manipulated cells and the subsequent transfer (transplantation) into the Or ganismus a gene therapeutic use of the invention  DNA sequences possible. Expression vectors with the sequences according to the invention preferably in such Transfected cells that malfunction in the organism inflammation (e.g. hepatocytes in chro inflammation of the liver). In particular, in a sol Chen gene therapy approach sequences according to the invention used that block the inflammatory, for example. Fragments that do not carry the inflammatory signal gene and thus interrupt the signal transduction.

The present inventive idea also goes into it gene therapy process that is carried out in vivo that can, hand in hand. Vectors are inserted for this purpose sets (e.g. liposomes, adenoviruses, retroviruses or similar or also naked DNA), which the DNA according to the invention Sequences targeted to the desired target cells of the organization insertism. The target cells are ty typically around cells whose inflammation is regulated is particularly disruptive to cells that are pathologically a Show increased disposition to the inflammatory reaction (e.g. with chronic inflammation of the liver). In this together fragments of a DNA Sequence are used, the inhibitory effect ent fold, for example. DNA sequences that essentially only the PYD domain included and thus only an association can perform a function, but not more extensive biolo pass on signals (for inflammation) - that is have no further biological functionality.

Another object of the present invention is the use of a DNA sequence according to the invention (alleles, derivatives, fragments) or a gene product according to the invention for the treatment of diseases which are based on incorrectly controlled intracellular signal transduction (claim 17). The aforementioned use of DNA sequences according to the invention also includes the use of the expression vectors according to the invention described above which contain a nucleotide sequence according to the invention, for example a nucleotide sequence disclosed in FIG. 1 or a functional derivative or allele of such a sequence (or also an infunctional derivative of such a sequence), with the aim of correcting the misguided intra-cellular signal transduction. The use can be carried out by gene therapy methods via injection of naked DNA according to the invention or the protein or via gene transporters, in particular viral or liposomal vectors. The present invention therefore relates both to the use of such expression vectors according to the invention and to the use according to the invention of cells (ie the use for triggering (or blocking) the inflammation process) which are transfected with expression vectors according to the invention.

Preferred is the use of an inventive DNA sequence or a gene product according to the invention, if the disease is one with a excessive inflammatory response (claim 18). The use of a is very particularly preferred such a DNA sequence or such a gene product Treatment (for the manufacture of a medicinal product action) of psoriasis, atherosclerosis, bacterial or viral infectious diseases, especially bacteria rial or viral meningitis or bacterial pneumo never, multiple sclerosis, rheumatoid arthritis, asthma, Sarcoidosis, glomerular nephritis or osteoarthritis (Claim 19). This can be achieved, for example, using fragments that block signal transduction, for example Sequences only for the PYD domain (or parts here of) encode or contain only these.

According to another object of the present invention a connection is made available there characterized in that they function as inventions gene products according to the invention (proteins) as intracellular Signal molecule of an inflammatory signal cascade Triggering of inflammatory reactions inhibited. Insbeson others block compounds according to the invention the specific  Interaction of PYD domains with the intracellular Signal transmission (claim 20). It is preferred in a chemical compound according to the invention, which the PYD function according to the invention (for example. With Inflam mation or apoptosis) to an oligopeptide that chemically modified (e.g. to facilitate passage through the cell membrane, especially through terminal (especially N-terminal) sequence regions) can be or may not be modified. In particular, it can be a native Se involved in the PYD association act range of a protein according to the invention. For example, an inhibitory Mo lekül, preferably a tetra to dodecamer, one of the native substrate corresponding amino acid sequence ten or consist of a native substrate sequence, where typically with the preferably tetra to dodecamer also a sequence section from a PYD domain protein according to the invention. Possibly. can a derar oligopeptide also be chemically modified that the amide-like bond between the individual ami Resistant to proteolytic degradation te alternative chemical group (e.g. sulfur or Phosphorus bridges) is replaced.

A chemical compound according to the invention is preferably an organic chemical compound with a molecular weight of <5000, in particular <3000, especially <1500 and is typically physiologically well tolerated (claim 21). Possibly. it will be part of a composition with at least one further active ingredient, such as preferably auxiliaries and / or additives, and can be used as a medicament. The organic molecule will be particularly preferred if the binding constant for binding to a protein according to the invention, in particular to the PYD domain of a protein according to the invention, is at least 10 ⁷ mol ^-1 . The compound of the invention will preferably be such that it can pass through the cell membrane, be it by diffusion or via (intra) membrane transport proteins (claim 22).

In another preferred embodiment of the present The present invention is the compound of the invention Antibodies, preferably one against the PYD domain Protein directed antibody according to the invention, the ex vivo in retransplanted host cells or by genthe therapeutic in vivo procedures in host cells lock and is not secreted there as an "intrabody" but can exert its effect intracellularly. Through such "intrabodies" according to the invention Protect cells from an inflammatory response. Such an approach is typically used for Cells of those tissues that are considered in the patient a pathophysiologically exaggerated inflammatori show behavior, for example with hepatocytes (Inflammation of the liver), keratinocytes, connective tissue cells, Im mun cells or muscle cells. The same is true like with "intrabodies" gentherapeu according to the invention table modified cells part of the present Invention.

A chemical compound according to the invention with the function of blocking the, for example, inflammatory function of physiological proteins according to the invention (see FIG. 1) can be used as a medicament. In particular, a chemical compound according to the invention (for the manufacture of a medicament) for the treatment of diseases for which at least tw. a pathological hyperinflammatory response is causal or symptomatic. An inhibitor according to the invention of the cellular function of a protein according to the invention, such as the inflammatory reaction, in particular an inhibitor of the association of PYD domains, can be used for inhibiting inflammation, particularly in the treatment of the following diseases or for the manufacture of a medicament for treatment of the following diseases are used: autoimmune diseases, psoriasis, arthriosclerosis, bacterial or viral infectious diseases, in particular bacterial or viral meningitis or bacterial pneumonia, multiple sclerosis, rheumatoid arthritis, asthma, sarcoidosis, glomerular nephritis or osteoarthritis, Parkinson's disease (Parkinson's disease or disease) Claim 23).

The present invention further relates to methods (“screening methods”) for identifying compounds with inhibitory properties with a view to triggering or forwarding signals which are associated with inflammatory reactions, in particular compounds which interact with proteins block the inflammatory signal cascade, in particular of physiologically reacting PYD domains (same or different proteins). Methods according to the invention provide that (a) cells, especially hepatocytes, in particular T-lymphocytes, are modified so that they form a show inflammatory reaction, (b) these are provided in a cell culture according to (a) modified cells, (c) test substances are added to the cell culture, (d) the extent of the inflammatory reaction of the cells in the cell culture is determined. For this purpose, several parallel tests with increasing concentrations of the test substance are preferably carried out according to the method according to the invention, in order to be able to determine the ID ₅₀ value in the event of an inflammation-inhibiting effect of the test substance.

A "screening" method according to the invention can also be carried out with With the help of so-called "proteomics" techniques become. For this purpose, ty pische Differences in the expression pattern of cells with inflammatory response and control cell experiments tell raised. The method is used for such a procedure ren typically used 2D gel electrophoresis.  

The present invention is illustrated by the following Figures explained in more detail:

Fig. 1 represents DNA sequences (including the corre sponding amino acid sequences) which are involved in the inflammatory signal cascade.

Fig. 2 shows a summary of the aforementioned sequences in tabular form, arranged according to the names already used in Fig. 1, with any EST clones, the origin of the sequence, the location on a chromosome and a summary of the respective sequences contained domains (eg the PYD domain, SPRY domain, CARD domain, NIGHT domain, the LRR domain) are specified. The sequences of human origin are summarized under (A), (B) and (C), whereas (D) contains murine sequences.

FIG. 3 shows the generalized "PYD" search profile that was used to find further PYD proteins in, for example, EST databases. Here, the publication of Bucher et al. (1996, Computer Chem., 20, 3-24) and to the corresponding explanations in the published German patent application DE 197 13 393.2-41, both of which are part of the present application. As a result, two further PYD-containing proteins were identified, namely NALP1 and NALP2 (the name NALP is composed of the individual characteristic domains that occur in both proteins: domain NACHT, LRR and PYD). Both proteins play a crucial role as signal transduction proteins for inflammatory signals. Furthermore, their function and thus their use in connection with apoptotic signal protein transduction come into consideration.

As a result, FIG. 4 shows that Pycard homodimerizes using its PYD domain and interacts with the PYD domain of NALP1. In the middle representation, cell extracts lysed 24 hours after the transfection were used and the anti-flag immunoprecipitates were examined for the presence of VSV-Pycard. In the illustration below, the various flag-marked constructs (namely Pycard-PYD (i.e.PYD from Pycard)), RAIDD, Apaf1-CARD (CARD from Apaf1, NALP1-PYD (PYD from NALP1), NALP1-CARD (CARD from NALP1) and a dummy vector, with regard to their respective presence in the immunoprecipitate by anti-flag antibodies.

Fig. 5 shows in part A, an alignment of PYD domains from different proteins ( "alignment" from the N-terminus to C-terminus). The respective sequence positions with at least 50% identity or with at least similar amino acids are highlighted in black or with a gray background. The abbreviations stand for HS: homo sa piens and DR: Danio rerio (zebrafish). The respective accession numbers in the database "Gen Bank" (EMBL) are the following: AF310103 for human pycard, AF310104 for murine pycard, 015553 for pyrine, AF310105 for NALP1, AF310106 for NALP2, AAF66964 for the protein CASPY from zebrafish, AAF66956 for that Protein pycard from zebra fish.

FIG. 5B illustrates schematically the Domän structure of proteins with a PYD domain (pyrin domain). The naming of the individual homology domains is shown in the figure legend in Appendix A12 under Fig. 5.

FIG. 5C shows the amino acid sequence of NALP1. The different shades correspond to the domain-specific shades chosen for FIG. 5B: dark gray background: PYD domain, framed light background NACHT domain, light gray background: LRR domain and dark background: CARD -Domain.

FIG. 6 shows an alignment of representative pyrine domains (PYD domain), death effector domain (DED) and caspase recruitment domain (CARD) and death domain (DD). The similarity of the different domains is shown by corresponding amino acids which are shown shaded, clearly. There are also considerable similarities at the level of the secondary structure, as is evident from the secondary structure prediction (here for 6 α helices in each case).

FIG. 7 shows an alignment (from the N- to the C-terminus, "alignment") of amino acid sequences of the PYD domains of the following proteins: pyrine, from humans (hs), from the mouse (mm) and rn, pycard from humans and from mouse human Pyc, human NALP1, human NALP2, human NALP3, human NALP4, human NALP5, human NALP6, human NALP7, human NALP8, human NALP9, human NALP10, human NALP11, murine NALP13, human NALP12, human human NALP15, human PY10, murine PY16, CASPY1 from zebra fish, CASPY2 from zebra fish, pycard from zebra fish. In the last line, a highlighting of the sequence position occurring in a consensus sequence is marked.

Figure 8 represents a family tree of the identified PYD domain containing proteins, taking into account the proximity of the degree of kinship in this family tree. The family tree thus shows the supposed divergence of family trees obtained through genomic evolutionary duplications.

The attached Appendix A1 to A13 is part of the front lying revelation.

embodiment

To check whether the PYD domains - as well as the domain NEN DD, DED, CARD - have the property only with Mit structure within the own family of "6-Helix- Bundle proteins "to interact have been expression vectors gates for PYD proteins and with Coimmunopräzi pitation experiments their suitability to interact with other proteins checked. Pycard- Constructs by PCR techniques from the following IMAGE EST clones amplified: AA528254 (965955) and AI148558 (1,714,818). Pycard was primed with the following amplified: JT1509 5'-ATGGGGCGCGCGCGCGAC-3 'and JT1512 5'-TCAGCTCCGCTCCAGG-3 '. Pycard's PYD domain was amplified with JT1509 and JT1510 5'CGACTGAGGAGGGGCC-3 '.

NALP1 constructs were amplified by PCR methods using the KIAA0926 EST clones from the Kazusa DNA Research Institute as a "template". NALP1-PYD was amplified with JT1497 5'-ATGGCTGGCGGAGCCTGGGGCCGCCTGGCCTGTTACTTG-3 'and JT1525 5'-GATCCAGGGCATTAGCAC-3'. NALP1-CARD was amplified with JT1500 5'-GTTGATACTTCAGCTGCTGAGTGGCAGGAG- 3 'and JT1527 5'-GATGAGACTCTGGTGTGG-3'.

The amplified fragments were ligated into PCR zero blunt (from Invitrogen) and subcloned into the EcoR1 site of VSV or Flag containing the PCR-3 (from Invitrogen) derived vectors as described by Thome et al. (1999, J. Biol. Chem., 274, 9962-8). The other constructs used were previously described by Thome et al. (1999 J. Biol. Chem., 274, 9962-8) be described and are part of the present disclosure with regard to the representation of the experimental execution. The publication by Burns et al. (1998, J. Biol. Chem., 273, 12203-12209) describes the implementation of immunoprecipitation. It is also part of the present disclosure and can be summarized as follows:
293T cells, which were cultured in DMEM medium supplemented with 10% fetal calf serum glutamine, were set up with a density of 1-3 × 10 ⁶ cells per 10 cm plate and the next with 3 μg of the respective construct Day transfected by the calcium phosphate precipitation method. The cells were collected and lysed in lysis buffer 24 to 26 hours after transfection (the lysis buffer contains 0.2% NP40, 150 mM NaCl, 50 mM EDTA, 30 mM Tris, pH 7.4). The cell lysates were placed on Sepharose 6 B (from Pharmacia) for at least three hours before precipitation with an equal amount of proteins at 4 ° C. for 4 hours with 3 μl of a flag agarose (from Kodak International Biotechnology) of 3 µl of Sepharose 6 B beads pre-cleaned. The resin was washed six times in lysis buffer and after the last wash, bound protein was eluted by boiling in the sample buffer, separated by SDS-PAGE and transferred to nitrocellulose (from Hybond ECL, Pharmacia) to subsequently perform Western blotting. The anti-VSV and anti-flag antibodies are from Sigma. An HRP-conjugated antibody was used which specifically detected the heavy chains of murine IgG1 (from Southern Biotechnology Associates).

As shown in FIG. 4 as a result of the present embodiment, specific binding of Pycard to the PYD domain of Pycard and NALP could be detected when coexpression with VSV-labeled Pycard, flag-labeled constructs that either the PYD domain from Pycard or the PYD domain from NALP1 contained co-expressed. In contrast, no interaction of the PYD domain from Pycard with other PYD domains or with death domains, CARD domains or death effector domains (the latter is not shown in FIG. 4) was found. Therefore, a PYD domain specifically interacts with PYD domains through a protein-protein interaction. As a result, FIG. 4 shows that Pycard homodimerizes using its PYD domain and interacts with the PYD domain of NALP1.

German translation of the figures A1-A13 A1

In pro-apoptotic signal transduction cascades, the interaction between the various initiator units, such as. B. the death receptor Fas, the various Adapter proteins and the caspases primarily through three structurally related protein Protein domains, namely the death domain (DD), death effector domain (DED) and the Caspase Recruitment Domain (CARD). In the present case, the proof revealed that a fourth related domain called the pyrine domain (PYD) exists. The PYD is found in pyrine, a protein that mutates in patients with familial Mediterranean fever is in Pycard, a regulator of etoposide-mediated apoptosis, in a caspase from Zebrafish and in two new proteins (NALP1, NALP2) that are structurally related to apoptosis Regulatory protein Card4 / Nod1 are observed. For Pycard's PYD domain has been shown to homodimerize and interact with PYD from NALP1. The Identification of the PYD family members can be used for short-term characterization of pro-apoptotic and / or pro-inflammatory signal transduction pathways.

introduction

Apoptosis or programmed cell death is an essential process in animals and Plants, especially for the removal of unwanted cells in an orderly manner and way. There has been significant progress in identification over the past few years and characterization of the modular nature of molecules required for regulation and Execution of apoptosis are responsible (Aravind et al., 1999, Hofmann, 1999). Of particular importance here is that three families of homology domains that Death Domain (DD), Death Effect Domain (DED) and Caspase Recruitment Domain (CARD) exist that are distantly related, and a super family of Form "six-helix bundle" protein interaction domains. Is with these domains especially important that they have highly specific interaction with members of the same Exercise subfamily and in most cases also a role in Play signal transduction process leading to apoptosis and / or inflammation. in the It should be noted in particular that the "adapter level" of proteins, namely the Transport death receptor signals to the caspases, which is heavily loaded with proteins Combinations of domains from DD / DED / CARD. Because of the big The importance and predictability of their function are several systematic searches for new proteins containing these domains have been successfully carried out. hereby the discovery of proteins such as FLIP, CARDIAK / RIP2, ARC, Bcl10, DEDD (Irmler et al., 1997; Koseki et al., 1998; McCarthy et al., 1998; Stegh et al., 1998, Thome et al., 1999) possible. In the following we report the discovery of a new type of domain, which is hereinafter called PYD (pyrine domain). This can be the fourth subfamily within the "six-helix" bundle "interaction domains", namely based on sequence homologies, structure prediction and interaction properties.  

RESULTS AND DISCUSSION Identifying a New Domain: The Pyrine Domain

In the course of analyzing the sequence of the recently identified protein Pycard with a CARD domain (protein containing PYD and CARD), which is also known as ASC ("apoptosis-associated speckle-like protein"), it was realized that the second structural domain, that occurs at the N-terminus of Pycard (pyrine domain, PYD) has a weak but significant sequence homology to a variety of other proteins ( Fig. 1A). Pycard is a 22 kDa protein that forms aggregates as soon as apoptosis is induced by certain antitumor substances (Masumoto et al., 1999).

In addition, it can be seen that cells that have been forced to express reduced amounts of Pycard are also significantly suppressed by etoposide-mediated apoptosis. Using Pycard's PYD, a simple BLAST search revealed that two additional PYD-containing proteins were included in the sequence database, namely Pyrin and Caspy. Pyrine was initially identified as a product of the MEFV (Mediterranean Fever) gene, which is mutated in patients with familial Mediterranean fever (FrenchFMFConsortium, 1997; InternationalFMFConsortium, 1997), a hereditary periodic fever syndrome characterized by episodic fever and serous and synovial inflammation. The state of knowledge with regard to pyrine is essentially based on its domain structure, which, in addition to the PYD domain, also has a B-box zinc finger and a "Spry" domain ( FIG. 1B). It has therefore been suggested that Pyriri is a member of the RoRet gene family of nuclear transcription factors, which has given rise to speculation that the protein acts as a transcriptional inflammation regulator (Centola st al., 1998). However, recent results suggest that pyrine is localized in cytoplasm and has no detectable transcriptional activity (Chen et al., 2000; Tidow et al., 2000). Therefore, the exact function of pyrine in inflammatory diseases is still unclear.

A general PYD profile was created on the basis of the PYD sequence of these two proteins (Bucher et al., 1996) and used in the subsequent search steps in the EST database. Two additional PYD-containing proteins NALP1 and NALP2 (NIGHT; LRR and PYD-containing proteins) were identified. Sequence analysis revealed that the NALPs have a PYD, NIGHT and LRR modular organization ( Fig. 1A, B). The NIGHT and LRR domain architecture is found in proteins involved in infection or apoptosis, particularly CARD4 / Nod1 ( Fig. 1B), an NF-kB inducing molecule (Bertin et al., 1999; Inohara et al ., 1999), a neuronal apoptosis inhibitor protein, NAIP, and the MHC class II transcription activator, CIITA (Koonin and Aravind, 2000). Interestingly, the domain PYD of NALP2 is replaced by CARD at CARD4 / Nod1, while the overall structural organization is conserved, which suggests a similar functionality ( FIG. 1B). CASPY is a protein containing PYD and caspase domains that was initially identified with a database search for zebrafish homologues of mammalian apoptosis regulators (Inohara and Nunez, 2000). This caspase is most homologous to caspase-13, which contains CARD instead of PYD in humans. It is noteworthy that the same study identified a pycard-related protein in zebrafish, indicating a high level of evolutionary conservation of these proteins ( Fig. 1A).

The pyrine domain is related to the DD family

Based on sequence comparisons, it was suggested that the domains DD, DED and CARD represent structurally related protein-protein interaction modules (Hofmann et al., 1997). All three types of domains are of a similar size and the secondary structure analysis revealed a similar arrangement of the six a-helices. All three domains therefore share the property of being able to form homo- or heterodimers and are also highly conserved (Hofmann et al., 1997). The structure prediction was later confirmed by NMR analysis of the DDs, DEDs and CARD5 (Chou et al., 1998; Eberstadt et al. 1998; Huang et al., 1996; Zhou et al., 1999), since the structural topology of these domains proved to be very similar, especially with regard to the structural core formed by helices a2 to a5 ( Fig. 2). Including the PYD-containing sequences in a general "alignment" with DED, CARD and DD, the PYD domain was identified according to the invention as a potential fourth member of the "DD-folded" superfamily ( FIG. 2).

Despite the similarity of their folding, it is known that DD, DED and CARD interact exclusively with members of their own subfamily, so that no promiscuity between the domains could be determined. In order to check whether the PYDs have the same properties, expression vectors were generated for the PYD proteins and tested for their ability to interact with other proteins in co-immunoprecipitation experiments. As shown in Figure 3, specific binding of Pycard to the PYDs of Pycard and NALP1 was detected when coexpressed with a VSV-labeled Pycard containing flag-labeled constructs containing the PYD from Pycard or the PYD from NALP1. No interactions of the Pycard PYD with other PYDs or with DDs, CARDs or DEDs ( Fig. 3) were detected. Therefore, the PYD domain is a protein-protein interaction domain that specifically interacts with PYD domains.

Pycard with its PYD-CARD divided into two domains is reminiscent of the DD and DAD containing molecule FADD or the protein RAIDD containing DD and CARD. For Both proteins are known to have a protein containing a DD or a DED Adapt the protein containing the CARD domain. For example, Fas's DD requires FADD to bind to the Caspase-8 DED. Our results suggest that Pycard represents a new adapter molecule, the NALP1 to a still unknown and too defining protein containing CARD couples. In fact, initial results show that the Caspase-5 CARD is the target structure of Pycard-CARD. The physiological role this interaction is still under investigation.

To summarize, we see PYD as a new protein-protein Interaction module identified that all criteria of a member of the DD Folding family fulfilled. Comparable to the limited interaction ability available to others Members apply, PYDs only interact with PYDs and not with members of the other three Subfamilies. In addition, PYDs are related to proteins involved in the Apoptosis and inflammation are involved, what is best found through the caspase Caspy is busy. PYDs regularly occur together with CARDs, such as B. at NALP1 and Pycard, on. The identification of the new, PYD-containing proteins is therefore possible probably the characterization of new pro-apoptotic or pro inflammatory signaling pathways, as was the case after the Identification of the DD of Fas a few years ago (Itoh and Nagata, 1993). We think, that the characterization of NALP1 and the Pycard complex is the first step in this  Direction is what ultimately leads to a better understanding of the molecular causes of inflammatory diseases.

METHODS Sequence and structure analysis

The blast and profile algorithms were used (Bucher et al., 1996), which are available on the ISREC server (www.isrec.isb-sib.ch). The secondary structure was predicted using the algorithms of Rost and Sander ( 1993 ).

Cloning, Expression and Immunoprecipitation

Pycard constructs were amplified by PCR from the following IMAGE EST clones:
AA528254 (965955) and AI148558 (1714818). Pycard was amplified with the following primers: JT1509 5'-ATGGGGCGCGCGCGCGAC-3 'and JT1512 5'-TCAGCTCCGCTCCAGG-3'. Pycard's PYD domain was amplified with JT1509 and JT1510 5'-CGACTGAGGAGGGGCC-3 '.

NALP1 constructs were amplified by PCR using the KIAA0926 EST Clones from the Kazusa DNA Research Institute as a "template". NALP1-PYD was amplified with JT1497 S'- ATGGCTGGCGGAGCCTGGGGCCGCCTGGCCTGTTACTTG-3 'and JT1525 5'- GATCCAGGGCATTAGCAC-3 ', NALP1-CARD was amplified with JT1500 5'- GTTGATACTTCAGCTGCTGAGTGGCAGGAG-3 'and JT1527 5'- GATGAGACTCTGGTGTGG-3 '.

Amplified section fragments were ligated into PCR zero blunt (Invitrogen) and then into the EcoR1 interface of VSV or flag-containing PCR-3 (Invitrogen) derived vectors (Thome et al., 1999) subcloned. Other constructs used corresponded to those previously described (Thome et al., 1999) subcloned.

Immunoprecipitation was carried out as previously described (Burns et al., 1998). In brief, 293 T cells were cultured in DMEM medium enriched with 10% fetal calf serum glutamine, exposed to 1-3 × 10 ⁶ cells per 10 cm plate and exposed to 3 µg of the indicated constructs next day transfected by the calcium phosphate precipitation method. The cells were harvested and lysed in lysis buffer (0.2% NP40, 150mM NaCl, 50mM EDTA, 50mM Tris, pH 7.4) 24-26 hours after transfection. The cell lysates were pre-cleaned for at least 3 hours on Sepharose 6 B (Pharmacia), specifically before the precipitation of an equal amount of proteins at 4 ° C. for 4 hours with 3 μl of flag agarose (Kodak International Biotechnology) and 3 µl of Sepharose 6B beads. The resin was washed 6 × in lysis buffer and after the last washing step, the bound proteins were eluted by boiling in sample buffer, separated by SDS page and transferred to nitrocellulose (Hybond ECL, Pharmacia) for the subsequent Western blotting. Both anti-VSV and anti-flag antibodies were purchased from Sigma. An HRP-conjugated antibody, which was able to specifically detect the mouse IgG1 heavy chain (Southern Biotechnology Associates), was used.

Announcements

We thank Kim Burns for critically reviewing the manuscript.  

credentials

Aravind, L., Dixit, V. M. and Koonin, E. V. (

1999

) The domains of death: evolution of the apoptosis machinery. TIBS, 24, 47-53.
Bertin, J., Nir, WJ, Fischer, CM, Tayber, OV, Errada, PR, Grant, JR, Keilty, JJ, Gosselin, ML, Robison, KE, Wong, GH, Glucksmann, MA and DiStefano, PS (

1999

) Human CARD4 protein is a novel CED-4 / Apaf-1 cell death family member that activates NF-kappaB. J. Biol. Chem., 274, 12955-8.
Bucher, P., Karplus, K., Moeri, N. and Hofmann, K. (

1996

) A flexible search technique based on generalized profiles. Computer Chem., 20, 3-24.
Burns, K., Martinon, F., Esslinger, C., Pahl, H., Schneider, P., Bodmer, JL, Di Marco, F., French, L. and Tschopp, J. (

1998

) MyD88, an adapter protein involved in interleukin-1 signaling. J. Biol. Chent, 273, 12203-12209.
Centola, M., Aksentijevich, I. and Kastner, DL (

1998

) The hereditary periodic fever syndromes: molecular analysis of a new family of inflammatory diseases. Hum. Mol. Genet., 7, 1581-8.
Chen, X., Bykhovskaya, Y., Tidow, N., Hamon, M., Bercovitz, Z., Spirina, O. and Fischel-Ghodsian, N. (

2000

) The familial mediterranean fever protein interacts and colocalizes with a putative Golgi transporter. Proc. Soc. Exp. Biol. Med., 224, 32-40.
Chou, JJ, Matsuo, H., Duan, H. and Wagner, G. (

1998

) Solution structure of the RAIDD CARD and model for CARD / CARD interaction in caspase-2 and caspase-9 recruitment. Cell, 94, 171-80.
Eberstadt, M., Huang, B., Chen, Z., Meadows, RP, Ng, SC, Zheng, L., Lenardo, MJ and Fesik, SW (

1998

) NMR structure and mutagenesis of the FADD (Mort1) death-effector domain. Nature, 392, 941-5.
FrenchFMFConsortium. (

1997

) A candidate gene for familial Mediterranean fever. The French FMF Consortium. Nat. Genet., 17, 25-31.
Hofmann, K. (

1999

) The modular nature of apoptotic signaling proteins. Cell. Mol. Life. Sci., 55, 1113-28.
Hofmann, K., Bucher, P. and Tschopp, J. (

1997

) The CARD domain: a new apoptotic signaling motif. Trends biochem. Sci., 22, 155-156.
Huang, B., Eberstadt, M., Olejniczak, ET, Meadows, RP and Fesik, SW (

1996

) NMR swcture and mutagenesis of the Fas (APO-1 / CD95) death domain. Nature, 384, 638-41.
Inohara, N., Koseki, T., del Peso, L., Hu, Y., Yee, C., Chen, S., Carrio, R., Merino, J., Liu, D., Ni, J. and Nunez, G. (

1999

) Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J. Beol. Chent, 274, 14560-7.
Inohara, N. and Nunez, G. (

2000

) Genes with homology to mammalian apoptosis regulators identified in zebrafish. Cell Death Differ, 7, 509-10.
InternationalFMFConsortium. (

1997

) Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. The International FMF Consortium. Cell, 90, 797-807.
Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, JL, Schroter, M., Burns, K., Mattmann, C., Rimoldi , D., French, LE and Tschopp, J. (

1997

) Inhibition of death receptor signals by cellular FLIP. Nature, 388, 190-195.
Itoh, N. and Nagata, S. (

1993

) A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J. Biol. Chem., 268, 10932-7.
Koonin, EV and Aravind, L. (

2000

) The NACHT family - a new group of predicted NTPases implicated in apoptosis and MHC transcription activation. TIBS, 25, 223-4.
Koseki, T., Inohara, N., Chen, S. and Nunez, G. (

1998

) ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases. Proc. Natl. Acad. Sci. USA, 95, 5156-60.
Masumoto, J., Taniguchi, S., Ayukawa, K., Sarvotham, H., Kishino, T., Niikawa, N., Hidaka, E., Katsuyama, T., Higuchi, T. and Sagara, J. (

1999

) ASC, a novel 22- kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-

60

cells. J. Biol. Chent, 274, 33835-8.
McCarthy, JV, Ni, J. and Dixit, VM (

1998

) RIP2 is a novel NF-kappaB-activating and cell death-inducing kinase. J. Biol. Chent, 273, 16968-16975.
Rost, B. and Sander, C. (

1993

) Secondary structure prediction of all-helical proteins in two states. Protein Eng., 6, 831-6.
Stegh, AH, Schickling, O., Ehret, A., Scaffidi, C., Peterhansel, C., Hofmann, TG, Grummt, I., Krammer, PH and Peter, ME (

1998

) DEDD, a novel death effector domain-containing protein, targeted to the nucleolus. EMBO J., 17, 5974-86.
Thome, M., Martinon, F., Hofmann, K., Rubio, V., Steiner, V., Schneider, P., Mattmann, C. and Tschopp, J. (

1999

) Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c- Jun N-terminal kinase. J. Biol. Chem., 274, 9962-8.
Tidow, N., Chen, X., Muller, C., Kawano, S., Gombart, AF, Fischel-Ghodsian, N. and Koeffler, HP (

2000

) Hematopoietic-specific expression of MEFV, the gene mutated in familial Mediterranean fever, and subcellular localization of its corresponding protein, pyrin. Blood, 95, 1451-5.
Zhou, P., Chou, J., Olea, RS, Yuan, J. and Wagner, G. (

1999

) Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific adapter / caspase interaction. Proc. Natl. Acad. Sci. USA, 96, 11265-70.

FIGURE LEGENDS

Fig. 4 (A) Multiple alignment (alignment) of the pyrine domain. Positions with more than 50% identical or similar amino acids are shown on a black or gray background. The species abbreviation is as follows: HS, Homo sapiens and DR, Danio rerio. Genebank / EMBL accession numbers are: AF310103 for human pycard, AF310104 for murine pycard, 015553 for pyrine, AF310105 for NALP1; AF310106 for NALP2, AAF66964 for zebra fish CASPY, AAF66956 for zebra fish Pycard. (B) Domain structure of the proteins containing a pyrine domain. Homology domains are named as follows: PYD for pyrine domain, CARD for caspase recruitment domain; NIGHT for NAIP, CIITA, HET-E and TP1 domain; LRR for Leucine-Rich Repeats, SPRY for domain at the SPla and Ryanodine receptor. B for B box. (C) NALP 1 amino acid sequence. The different shades of the boxes correspond to the domains, such as in Fig. 1B (4 B).

Figure 5 Alignment of representative pyrine domains (PYD), death effector domains (DED), caspase recruitment domains (CARD) and death domains (DD); this shows the similarity of these interaction domains. α lines indicate the predicted α helices for PYD (Rost and Sander, 1993) and the indicated α helices for the DD, CARD, and DED solution structures, respectively (Eberstadt et al., 1998; Huang et al., 1996; Zhou et al., 1999)

Fig. 6 Pycard homodimerized using its PYD and interacts with PYD from NALP1. Flag-marked constructs contain: PYD from Pycard (Pycard-PYD), RAIDD, CARD from Apaf-1 (Apaf1-CARD), PYD from NALPI (NALP1-PYD), CARD from NALPI (NALP1-CARD) and an empty vector ( Mock vector) were co-transfected in 293 T cells with a VSV-labeled pycard construct. The cells were lysed 24 hours after transfection and the anti-flag immunoprecipitates were analyzed for the presence of a VSV pycard. The expression of the different constructs was analyzed in the cell lysates (lower illustration).

Claims (23)

1. DNA sequence, characterized in that it codes for a protein with at least one PYD domain, including all functionally homologous derivatives, fragments or alleles. 2. DNA sequence according to claim 1, characterized in that a level of significance of p <10 ^-2 results when the PYD domain of the DNA sequence is compared with a search profile according to FIG. 3. 3. DNA sequence according to claim 1 or 2, characterized in that its gene product contains one of the amino acid sequences (for a PYD domain) as shown in Fig. 6 as shown, including all functionally homologous derivatives, alleles or fragments. 4. DNA sequence according to one of the preceding claims, characterized in that it contains one of the (c) DNA sequences shown in Fig. 1. 5. DNA sequence according to one of the preceding claims, characterized in that its gene product contains one of the amino acid sequences shown in Fig. 1 ent. 6. Expression vector, characterized in that it a DNA sequence according to any one of claims 1 to 5 contains. 7. host cell, characterized in that it with egg transformed expression vector according to claim 6 is.   8. Host cell according to claim 7, characterized in that they are a mammalian cell, especially a huma ne cell, is. 9. Purified gene product, characterized in that it is by a DNA sequence according to one of the claims che 1 to 5 is encoded. 10. Purified gene product according to claim 9, characterized characterized as being a polypeptide. 11. Purified gene product according to claim 9 or 10, characterized in that it contains one of the amino acid sequences shown in Fig. 7 (for a PYD domain), including all functionally homologous alleles, fragments or derivatives. 12. Antibody, characterized in that it is an epi top on a gene product according to one of claims 9 to 11 recognizes. 13. Antibody according to claim 12, characterized in that it is monoclonal. 14. Antibody according to one of claims 12 or 13, because characterized in that it is against a sequence cut on the PYD domain as an epitope. 15. Process for the isolation of gene products with min at least one PYD domain, characterized in that Host cells according to claim 7 or 8 under suitable, cultivated the conditions promoting expression and the gene product is finally from culture is cleaned. 16. Process for the expression of gene products with min at least one PYD domain, characterized in that Host cells with an An expression vector to be transformed.   17. Use of a DNA sequence according to one of the claims che 1 to 5 or a gene product according to one of the Claims 9 to 11 for the treatment of diseases, based on miscontrolled intracellular signal trans production based. 18. Use of a DNA sequence or a gene product according to claim 17, characterized in that it the disease is one with a excessive inflammatory response. 19. Use of a DNA sequence or a gene product according to claim 17 or 18, characterized in that the disease is psoriasis, arthe riosclerosis, bacterial or viral infections diseases, especially bacterial or viral Meningitis or bacterial pneumonia, multiple Sclerosis, rheumatoid arthritis, asthma, sarcoidosis, glomerular nephritis or osteoarthritis. 20. Connection, characterized in that it the spe specific interaction of PYD domains to the intracellu blocked signal transmission. 21. A compound according to claim 20, characterized in that they have an organic chemical connection with egg its molecular weight is preferably <3000. 22. A compound according to claim 21, characterized in that they can diffuse or cross the cell membrane membrane transport proteins happen. 23. Use of a compound according to claim 20 or 21 for the treatment of (or for the production of an Ar neimittels for the treatment of) Psoriasis, Artherios clerosis, bacterial or viral infection crane kungen, especially bacterial or viral menin gitis or bacterial pneumonia, multiple sclerosis,  rheumatoid arthritis, asthma, sarcoidosis, glomeru laral nephritis or osteoarthritis.