Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-04-27
2004-03-09
Kemmerer, Elizabeth (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S351000, C530S324000, C536S023500, C435S325000, C435S252300, C435S254110, C435S254200, C435S069100
Reexamination Certificate
active
06703487
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to molecules that are members of a polypeptide family referred to as Pellino (also called Conserved Inflammatory Signal Target (CIST)). More particularly, the present invention includes Pellino polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, and fragments thereof, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, transgenic and knockout cells and animals, and uses thereof.
BACKGROUND OF THE INVENTION
The Interleukin-1 (IL-1) pathway is a cellular signaling pathway is that plays a crucial role in the mammalian inflammatory response. Several different receptors and ligands are involved in this pathway, including the ligands IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1ra), and two IL-1 receptors referred to as IL-1 receptor Type I (IL-1 RI) and IL-1 receptor Type II (IL-1 RII); a soluble form of the latter also exists. Of these, it appears that IL-1RI is the signaling receptor, whereas IL-1RII does not transduce signal to a cell, but instead may be involved in regulating an IL-1-mediated response (Colotta et al.,
Immunol. Today
15:562; 1994). Signaling via the IL-1 pathway is complex, requiring a number of accessory molecules in addition to IL-1 RI, including a receptor-associated kinase (IRAK). A serine/threonine kinase with homology to IRAK, referred to as Pelle, is found in Drosophila (for review, see Belvin and Armstrong,
Annu. Rev. Cell Dev. Biol.
12:393; 1996). Another Drosophila protein, Pellino, has been reported to interact with Pelle (Grosshans et al., Mech. Dev. 81:127; 1999).
Dorsal-ventral polarization in Drosophila embryos depends upon the establishment of a gradient of nuclear localization of the Rel-like transcription factor Dorsal. The transcriptional program mediated by Dorsal results from a signaling cascade triggered by binding of an extracellular ligand Spaetzle to its receptor Toll. Intermediates of this signaling cascade include the adaptor protein Tube, the serine/threonine kinase Pelle, and Cactus, a cytosolic binding partner of Dorsal. Signals transmitted by Toll result in the degradation of Cactus, and thereby permit the nuclear importation of Dorsal. The similarity between the cytosolic domains of Toll and the mammalian interleukin-1 receptor IL1-RI was first noted by Gay and Keith (Gay, N., and Keith, F., 1991
, Nature
351: 355-356), and the number of proteins which contain the homologous regions, called the Toll/IL-1 R (TIR) domain has subsequently been extended to include a larger family of receptors and intracellular signaling molecules from a variety of organisms. Those with leucine-rich repeats in their extracellular domains are broadly involved in innate immune responses and include at least ten mammalian toll-like receptors (TLRs) which initiate inflammatory responses to microbial pathogens such as peptidoglycan, bacterial lipopeptides, bacterial lipopolysaccharides, zymosan, CpG DNA, flagellin, lipoteichoic acids, and Respiratory Syncytial Virus proteins; and plant proteins such as the N resistance gene product which mediate disease resistance. Furthermore, it is now clear that an important function of Toll signaling in adult Drosophila is in controlling responses to fungal infections.
Downstream components of the Toll signaling pathway have also been evolutionarily conserved in mammalian TLR and interleukin-1 receptor signaling pathways which culminate in nuclear translocation of the transcription factor Nuclear Factor kappa B (NF-kB). Protein kinases IRAK-1 and IRAK2, close homologues of Pelle, are recruited to the activated IL-1 R or TLR receptor complexes through the adaptor protein MyD88 and undergo autophosphorylation reactions. Although MyD88 is not a strict analog of Tube, both proteins contain a so-called death domain, and Tube likely serves to mediate signal transmission between Toll and Pelle, to which it binds. IRAK subsequently interacts with another adaptor molecule TRAF-6, which is homologous to the recently described D-TRAF. Signals downstream of TRAF appear to be divergent, and not all of them are fully understood, but one consequence, in mammalian cells, is the activation of the IkB kinase (IKK) complex which directly phosphorylates the inhibitory Cactus homolog IkB at two N-terminal serine residues causing its ubiquitination and degradation. Released from a cytoplasmic association with IkB, NF-kB migrates into the nucleus. Recently, a candidate for an additional intermediate in Tube-Pelle interactions was found by yeast two-hybrid screening with Pelle as a bait sequence. This protein, called Pellino, was shown to interact with catalytically-competent Pelle, but not with a mutant form of Pelle that lacked kinase activity. Although a function for Pellino was not addressed in this study, it was suggested that it could either stabilize the activated form of Pelle, or mediate an interaction with downstream Pelle substrates.
IL-1 and other pro-inflammatory cytokines have been implicated in a variety of diseases and conditions, including rheumatoid arthritis, multiple myeloma, osteoporosis, endotoxemia and sepsis, osteoarthritis, inflammatory bowel disease, and allergy. Inhibition of the signaling of IL-1 using soluble forms of IL-1 Rs, and the IL-1ra, have been shown to be useful in treating or ameliorating disease characterized by excess levels of IL-1 (Rosenwasser, J. Allergy Clin. Immunol. 102:344; 1998). Other parts of the IL-1 signaling pathway and other pro-inflammatory MAP kinase-activated pathways have also been the target of attempts to identify additional molecules that can be used therapeutically to intervene in conditions related to IL-1 and pro-inflammatory cytokines generally. Thus, there is a need in the art to identify novel molecules involved in the IL-1 and MAP kinase-activated pro-inflammatory signaling pathways, both as tools with which to investigate cell signaling and for use in identifying inhibitors of pro-inflammatory signaling. Of particular interest are novel polypeptides that are involved in stimulation of multiple pro-inflammatory signaling pathways, as inhibition of such polypeptides would more effectively inhibit inflammatory effects than inhibition of a pathway-specific polypeptide.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery of new murine and human Pellino polypeptides, murine Pellino-1 and -2, and human Pellino-1, -2, and -3.
The invention provides an isolated polypeptide capable of stimulating MAP kinase-activated signaling pathways consisting of, consisting essentially of, or more preferably, comprising an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, and SEQ ID NO:12;
(b) an amino acid sequence selected from the group consisting of: amino acids x1 to x2 of SEQ ID NO:4, wherein x1 is any of amino acids 130 through 134 of SEQ ID NO:4, and x2 is any of amino acids 187 through 191 of SEQ ID NO:4; amino acids x1 to x2 of SEQ ID NO:8, wherein x1 is any of the amino acids 132 through 136 of SEQ ID NO:8, and x2 is any of amino acids 189 through 193 of SEQ ID NO:8; and amino acids x1 to x2 of SEQ ID NO:12, wherein x1 is any of the amino acids 155 through 160 of SEQ ID NO:12, and x2 is any of amino acids 212 through 217 of SEQ ID NO:12;
(c) an amino acid sequence selected from the group consisting of: amino acids x1 to x2 of SEQ ID NO:4, wherein x1 is any of amino acids 1 through 10 of SEQ ID NO:4, and x2 is any of amino acids 409 through 418 of SEQ ID NO:4; amino acids x1 to x2 of SEQ ID NO:8, wherein x1 is any of amino acids 1 through 10 of SEQ ID NO:8, and x2 is any of amino acids 410 through 419 of SEQ ID NO:8; and amino acids x1 to x2 of SEQ ID NO:12, wherein x1 is any of amino acids 1 through 10 of SEQ ID NO:12, and x2 is any of amino acids 435 through 445 of SEQ ID NO:12;
(d) an allelic variant of any of (a)-(c) above;
(e) a fragment of the amino acid sequences of any of (a)-(d) comprising at least 2
Bird Timothy A.
Cosman David J.
Immunex Corporation
Kemmerer Elizabeth
Li Ruixiang
Sprunger Suzanne A.
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