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  Toll/interleukin-1 receptor adapter protein (tirap)Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Recombinant Dna Technique Included In Method Of Making A Protein Or PolypeptideThe Patent Description & Claims data below is from USPTO Patent Application 20080096250. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. application Ser. No. 10/101,398, filed Mar. 19, 2002, which in turn claims priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Application No. 60/289,738, filed May 9, 2001, U.S. Provisional Application No. 60/289,815, also filed May 9, 2001, and U.S. Provisional Application No. 60/289,866, filed Aug. 29, 2001. This application also claims priority to the Patent Cooperation Treaty filing having the Serial No. WO US 02/14915, which was filed on May 9, 2002. BACKGROUND OF THE INVENTION [0003] The drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biology. This approach is rapidly superceding earlier approaches based on positional cloning. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position. [0004] Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterize further genes and their related polypeptides/proteins, as targets for drug discovery with the potential for affecting immune response. [0005] The strategy of innate immune recognition is based on the detection of constitutive and conserved products of microbial metabolism. Many metabolic pathways and individual gene products are unique to microorganisms and absent from host cells. Although these targets of recognition are not absolutely identical between different species of microbes, the gene products may be found in the context of a common molecular pattern, which is typically highly conserved and invariant among microbes of a given class. Because the targets of innate immune recognition are conserved molecular patterns, they are called pathogen associated molecular patterns (PAMPs). [0006] The recent discovery and characterization of the Toll-like receptor (TLR) family have incited new interest in the field of innate immunity. TLRs are pattern recognition receptors that have a unique and critical function in animal immunity. TLRs typically are transmembrane receptors characterized by an extracellular leucine rich repeats domain and an intracellular TIR (Toll/Interleukin-1 Receptor) domain. The TIR domain is a conserved protein-protein interaction module and plays a role in host defense. In other words, TLRs play a critical role in microbial recognition and control of adaptive immune responses. [0007] In mammalian species, there are at least ten (10) TLRs, and each has a distinct function in innate immune recognition. The TLRs mainly differ from one another with regard to ligand specificity, the use of accessory molecules, expression profiles and differences in signal transduction pathways. [0008] Human TLR4 was the first identified and functionally characterized mammalian Toll. TLR4 functions as the signal transducing receptor for the PAMP lipopolysaccharide (LPS) as well as other PAMPs, which are apparent to one skilled in the art. [0009] Activation of signal transduction pathways by TLRs leads to the induction of a variety of genes that function in host defense including inflammatory cytokines, chemokines, MHC and co-stimulatory molecules. Mammalian TLRs also induce multiple effector molecules such as inducible nitric oxide synthetase and antimicrobial peptides that can directly destroy microbial pathogens. [0010] The signaling pathway, which appears to be shared by all members of the Toll and Interleukin-1 Receptor (IL-1R) families, includes four essential components: the adapters TRAF6, MyD88 and Tollip and a protein kinase, IRAK. MyD88 contains two protein interaction domains: an N-terminal death domain and a C-terminal TIR domain. The TIR domain of MyD88 associates with the TIR domain of TLR and IL-1R, while the death domain interacts with the death domain of IRAK. [0011] In cells wherein MyD88 expression has been suppressed (i.e. in MyD88 knockout mice), macrophages and dendritic cells do not produce cytokines IL-1.beta., TNF-.alpha., IL-6 and IL-12 when stimulated with LPS, MALP-2 or CpG, which signal through TLR4, TLR2 and TLR9, respectively. Consequently, MyD88 knockout mice are resistant to endotoxic shock. Furthermore, when normal bone marrow-derived dendritic cells (BMDCs) are stimulated with LPS or CpG, they produce large amounts of IL-12 and upregulate cell surface expression of MHC and co-stimulatory molecules. However, in MyD88 deficient BMDCs, stimulation with LPS or CpG does not produce IL-12 or IL-6. [0012] Additionally, RNA-dependent protein kinase (PKR)-deficient cells fail to activate c-Jun N-terminal Kinase (JNK) and p38 MAP Kinase (p38) in response to LPS stimulation. As TLR4 is required for signals downstream of LPS, this indicates that PKR is a component of the TLR4 signaling pathway. Although phosphorylated PKR can be detected in LPS stimulated wild-type macrophages, phosphorylated PKR has also been detected in LPS stimulated MyD88-deficient macrophages. Interestingly, PKR from the MyD88-deficient macrophages was activated with slower kinetics. [0013] Although some cellular responses are completely abolished in MyD88-deficient cells, TLR4, but not TLR9 or TLR2, can still activate NF-.kappa.B and MAP kinases. This difference indicates that another adapter protein exists that can mediate MyD88-independent signaling in response to TLR4 ligation. [0014] Thus, there is a need to determine the structure and function of the adapter protein involved in the MyD88 independent signaling downstream of TLR4 for various purposes including to develop compounds to treat diseases related to TLR4 function. SUMMARY OF THE INVENTION [0015] The present invention relates to Toll Interleukin-1 Receptor Adapter Proteins (TIRAPs), and in particular TIRAP polypeptides and TIRAP polynucleotides, recombinant materials and methods for their production. In yet a further aspect, the present invention relates to TIRAP inhibitors. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of inflammation and inducing or affecting immune response. In a further aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with TIRAP imbalance with the identified compounds. In yet a further aspect, the invention relates to transgenic mammals comprising TIRAP polynucleotides. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1 is a sequence alignment of human and mouse TIRAP. [0017] FIG. 2, comprising FIGS. 2A through 2D, is a series of graphs which demonstrate that TIRAP is a component of the TLR4 signaling pathway, but not of the IL-1R signaling pathway. On the y axis, luciferase activity is expressed as fold induction or relative light units (RLU). The x axis shows the transfected constructs: pcDNA3 (control vector); TIRAP (encodes full length TIRAP); TIRAP N (encodes N-terminal domain of TIRAP); TIRAP C (encodes C-terminal domain of TIRAP); TIRAP P125H (encodes TIRAP containing mutation at amino acid 125); CD4/TLR4 (encodes constitutively active form of CD4); MyD88 DN (encodes dominant negative mutant of MyD88); IL-1R/AcP (encodes ILI-1 receptor and receptor accessory protein); TLR9 (encodes full length TLR9). Shaded triangles below the constructs indicate that increasing amounts of that construct was added. [0018] FIG. 3, comprising FIGS. 3A and 3B, is a pair of images of immunoblots demonstrating co-immunoprecipitation of TIRAP with TLR4 and MyD88. Cells were transfected in the presence (+) or absence (-) of constructs encoding a wild-type portion of TLR4 (TLR4 wt), a mutant derivative thereof (TLR4 mut), wild-type TIRAP (TIRAP), a mutant TIRAP containing a P125H mutation (TIRAP PH), or wild-type MyD88 (MyD88), wherein each construct further encodes a Myc, Flag, or hemaglutianin (HA) tag, as indicated. Antibodies used for immunoprecipitation (IP) or immunoblot (IB) are indicated to the left of each blot. [0019] FIG. 4, comprising FIGS. 4A through 4C, is a series of images of immunoblots demonstrating that PKR co-immunoprecipitates with TIRAP and is a component of LPS and CpG-signaling pathways. (A) Bone marrow-derived macrophages from either wild-type (WT) or MyD88-deficient (MyD88.sup.-/-) mice were stimulated for the indicated time periods (minutes) with either LPS or CpG, and immunoblots were performed using an antibody that specifically recognizes phosphorylated PKR (PKR-P). B and C Cells were transfected with constructs encoding Flag-tagged TIRAP and either a construct encoding an HA-tagged kinase dead mutant of PKR (HA PKR KD) or p58, as indicated. Antibodies used for immunoprecipitation (IP) or immunoblot (IB) are indicated to the left of each blot, and arrows on the right show the location of the indicated proteins. [0020] FIG. 5, comprising FIGS. 5A through 5D, is a series of blots depicting inhibition of LPS- but not CpG-induced NF-.kappa.B activation, PKR phosphorylation, and JNK phosphorylation by TIRAP. (A) TIRAP peptide, but not a reverse sequence peptide, inhibited LPS-induced NF-.kappa.B activation. RAW .kappa.B cells pretreated for 1 h with either the TIRAP or control peptide were stimulated with LPS (10 ng/ml) for 5 h. Samples were stimulated in duplicate. (B) Pretreatment of RAW cells with TIRAP peptide blocks LPS-induced I.kappa.B-.alpha. degradation. RAW cells pretreated with the TIRAP peptide as indicated were either left unstimulated or stimulated with LPS (10 ng/ml) for the indicated time periods. Lysates (30 .mu.g/sample) were resolved by SDS-PAGE followed by immunoblotting with an anti-I.kappa.B-.alpha. antibody to assess I.kappa.B-.alpha. degradation. (C) TIRAP peptide inhibited LPS- but not CpG-induced NF-.kappa.B activity. RAW .kappa.B cells pretreated with the TIRAP peptide as indicated were stimulated with either LPS or CpG and harvested for reporter assay. (D) TIRAP peptide inhibited PKR and JNK phosphorylation induced by LPS but not CpG. RAW cells either untreated or pretreated with 40 .mu.M of the TIRAP peptide were stimulated with either LPS or CpG as indicated. Lysates (30 .mu.g/sample) were analyzed by SDS-PAGE followed by immunoblotting with antibodies that specifically recognize phosphorylated PKR or phosphorylated JNK. [0021] FIG. 6, comprising FIG. 6A through 6C, is a series of graphs indicating that TIRAP induces dendritic cell maturation. (A) Wild-type and MyD88.sup.-/- dendritic cells (DCs) were induced with LPS or CpG and analyzed for expression of the costimulatory molecules B7.1 and B7.1 using flow cytometry. (B) Production of the cytokines, IL-12 and IL-6, was measured in cells that were untreated (none) or treated with LPS or CpG in the presence or absence of TIRAP peptide. (C) T cell proliferation was measured in wild-type DCs that were untreated (none), treated with LPS alone (LPS), or treated with LPS in the presence of increasing amounts of TIRAP peptide (LPS+TIRAP peptide). T cell proliferation was measured by incorporation of .sup.3H-thymidine into cells, expressed as cpm.times.10.sup.3. Continue reading... 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