| Gamma subunit of cytokine responsive lkb-alpha kinase complex -> Monitor Keywords |
|
|
 
Gamma subunit of cytokine responsive lkb-alpha kinase complexRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Enzyme (e.g., Ligases (6. ), Etc.), Proenzyme; Compositions Thereof; Process For Preparing, Activating, Inhibiting, Separating, Or Purifying Enzymes, Transferase Other Than Ribonuclease (2.), Transferring Phosphorus Containing Group (e.g., Kineases, Etc.(2.7))Gamma subunit of cytokine responsive lkb-alpha kinase complex description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070166812, Gamma subunit of cytokine responsive lkb-alpha kinase complex. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 60/097,418, filed Aug. 20, 1998, and which is incorporated herein by reference. BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention relates generally to molecular biology and biochemistry and more specifically to a subunit of a protein kinase, the I.kappa.B kinase, which is activated in response to environmental stresses and proinflammatory signals to phosphorylate inhibitors of NF-.kappa.B. [0005] 2. Background Information [0006] In chronic inflammatory disease including asthma, rheumatoid arthritis, inflammatory bowel disease and psoriasis, cytokines recruit activated immune and inflammatory cells to the site of lesions, thereby amplifying and perpetuating the inflammatory states. Although the causes of chronic inflammatory disease are in large part unknown, both genetic and environmental factors contribute to pathology. Genes such as those for HLA antigens in rheumatoid arthritis and inflammatory bowel disease can determine a patient's susceptibility to the disease and disease severity, and environmental factors can determine its course. Once established, a chronic inflammatory process is virtually impossible to disrupt; there is no curative treatment for any chronic inflammatory disease. Although chronic disease can be suppressed by glucocorticoid or immunosuppressive therapy, side effects are associated with prolonged treatment. [0007] A ubiquitous transcription factor, nuclear factor-.kappa.B (NF-.kappa.B), has been identified as of particular importance in immune and inflammatory responses. NF-.kappa.B increases the expression of the genes for many cytokines, enzymes, and adhesion molecules in chronic inflammatory diseases. One gene regulated by NF-.kappa.B is the gene for inducible nitric oxide synthase, the expression of which is increased in airway epithelial cells and macrophages in patients with asthma; in colonic epithelial cells of patients with the inflammatory bowel disease, ulcerative colitis; and in synovial cells in inflamed joints. The increased expression is reflected by an increased amount of nitric oxide in the exhaled breath of patients with asthma and in the colons of patients with active ulcerative colitis, as well as by elevated urinary nitrite concentrations in patients with rheumatoid arthritis. Cyclooxygenase-2, another inducible enzyme regulated by NF-.kappa.B, is responsible for the increased production of prostaglandins and thromboxane in inflammatory diseases. [0008] NF-.kappa.B also is involved in expression of adhesion molecules, which recruit inflammatory cells such as neutrophils, eosinophils and T lymphocytes from the circulation to the site of inflammation in all chronic inflammatory diseases. NF-.kappa.B regulates the expression of several genes that encode adhesion molecules such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin. [0009] The production of pro-inflammatory cytokines also can be regulated by NF-.kappa.B. Interleukin-1.beta., TNF-.alpha., interleukin-6, granulocyte-macrophage colony-stimulating factor, and many chemotactic cytokines (chemokines) is increased in patients with asthma, rheumatoid arthritis, psoriasis and inflammatory bowel disease, and all have important roles in the inflammatory process. Interleukin-1.beta. and TNF-.alpha. appear to influence severity of disease, possibly by persistent activation of NF-.kappa.B. [0010] Thus, the transcription factor NF-.kappa.B plays a central role in inflammatory disease. This role is emphasized by the targeted disruption of an inhibitor of NF-.kappa.B in mice, which resulted in prolonged activation of NF-.kappa.B in response to inflammatory stimuli, and the death of these animals due to widespread inflammation. [0011] Unfortunately, the available therapies for chronic inflammatory diseases such as asthma, rheumatoid arthritis and inflammatory bowel disease are unsatisfactory. Identification of molecules that regulate NF-.kappa.B would provide new strategies for screening for anti-inflammatory therapeutics. The present invention satisfies this need by providing the novel I.kappa.B kinase subunit, IKK-.gamma., which is an essential regulatory subunit of the I.kappa.B kinase required for NF-.kappa.B activation. Related advantages also are provided. SUMMARY OF THE INVENTION [0012] The present invention provides a novel essential regulatory subunit of the I.kappa.B kinase (IKK) complex, IKK-.gamma.. The isolated IKK-.gamma. subunit of the invention has substantially the same amino acid sequence as SEQ ID NO: 2, for example, an amino acid sequence having at least 55% amino acid identity with SEQ ID NO: 2. [0013] Also provided by the invention is an IKK-.gamma.active fragment that has substantially the same amino acid sequence as a portion of the I.kappa.B kinase subunit, IKK-.gamma.. An IKK-.gamma. active fragment of the invention can contain, for example, ten, twenty, fifty or more contiguous amino acids of SEQ ID NO: 2 and can have, for example, IKK-.beta. binding activity. [0014] The invention also provides an isolated IKK-.gamma.nucleic acid molecule, which contains a nucleotide sequence encoding substantially the same amino acid sequence as SEQ ID NO: 2. Such an isolated IKK-.gamma. nucleic acid molecule of the invention can have, for example, a nucleotide sequence encoding an amino acid sequence having at least 55% amino acid identity with SEQ ID NO: 2. An isolated IKK-.gamma. nucleic acid molecule of the invention can have a nucleotide sequence encoding SEQ ID NO: 2, for example, nucleotides 149 to 1408 of SEQ ID NO: 1 or the entire sequence of SEQ ID NO: 1. [0015] IKK-.gamma. polynucleotides and antisense polynucleotides also are provided by the invention. The invention provides a polynucleotide containing at least nine contiguous nucleotides of SEQ ID NO: 1. The invention also provides an antisense polynucleotide containing a nucleotide sequence complementary to at least nine contiguous nucleotides of SEQ ID NO: 1. [0016] The present invention also provides a method of identifying an effective agent that modulates the specific association of an I.kappa.B kinase .gamma. (IKK-.gamma.) subunit and a second protein. Such agents can represent novel therapeutic agents, such as immunosuppressant, anti-inflammatory or anti-cancer therapeutics. The method includes the steps of contacting the IKK-.gamma. subunit and the second protein with an agent under conditions suitable for the specific association of the IKK-.gamma.subunit and the second protein, and detecting an altered association of the IKK-.gamma. subunit and the second protein in the presence of the agent, where the altered association identifies the agent as an effective agent that modulates the specific association of the IKK-.gamma. subunit and the second protein. In a method of the invention, the contacting can be, for example, in vitro with an isolated IKK-.gamma. subunit. Alternatively, the IKK-.gamma. subunit can be contacted, for example, in a cell such as a mammalian cell or yeast cell in culture. In a method of the invention, an altered association can be detected by a variety of methods, for example, by measuring the transcriptional activity of a reporter gene. In a preferred embodiment, the second protein is IKK-.beta. and an effective agent is identified that modulates the specific association of an IKK-.gamma. subunit and an IKK-.beta. subunit. [0017] Also provided herein is a method of modulating NF-.kappa.B activity in a cell by contacting the cell with an effective agent that modulates the specific association of an IKK-.gamma. subunit and a second protein. In a method of the invention, the second protein can be, for example, IKK-.beta.. Methods of modulating NF-.kappa.B activity in a cell by introducing into the cell an IKK-.gamma. antisense polynucleotide also are provided. The IKK-.gamma. antisense polynucleotide can be expressed in the cell, for example, in a vector. BRIEF DESCRIPTION OF THE DRAWINGS [0018] FIG. 1: Purification of the IKK complex and identification of the IKK subunits. a. Partially purified HeLa cell extracts were passed through an anti-IKK-.alpha. immunoaffinity column. The input, flowthrough (FT) and bound fractions were separated by SDS-PAGE and examined for IKK-.alpha. and IKK-.beta. content by immunoblotting. b. The purified IKK complex was separated by SDS-PAGE and stained with colloidal blue. The positions of the different subunits are indicated. The inset shows a portion of a gel run for a longer time in which better separation of IKK-.gamma.1 and IKK-.gamma.2 is seen. c. 293 cells were labeled with .sup.35S for 5 hrs using Pro-Mix (Amersham) followed by incubation without or with TNF. Lysates were immunoprecipitated (IP) with either anti-IKK-.alpha. or anti-HA antibody (used as a control). After extensive washing, the immunecomplexes were separated by SDS-PAGE and visualized by autoradiography. [0019] FIG. 2: Primary and secondary structure of IKK-.gamma.. a. The nucleotide sequence of the complete IKK-.gamma.cDNA (SEQ ID NO: 1). The initiator "ATG" is underlined. b. The amino acid sequence of the complete IKK-.gamma. ORF (SEQ ID NO: 2). Peptide sequences obtained by microsequencing are overlined; the leucines of the leucine zipper are indicated by solid dots. c. Secondary structure prediction of IKK-.gamma.. The boxes indicate .alpha.-helical regions. "Coil" designates coiled-coil regions, and "LZ" designates leucine zipper motif (which is a coiled-coil). The amino acid positions that mark the approximate boundaries of these motifs are indicated. [0020] FIG. 3: IKK-.gamma. physically interacts with IKK-.alpha./.beta.. a. HA-IKK-.gamma., Flag-IKK-.alpha., Flag-IKK-.beta. or empty expression vectors were transiently transfected into 293 cells as indicated. After 24 hrs the cells were lysed. Part of each lysate was precipitated with anti-HA antibody and another part with anti-Flag antibody (M2). The levels of Flag-IKK-.alpha., Flag-IKK-.beta. and HA-IKK-.gamma. were determined by immunoblotting. b. HA-IKK-.gamma. or "empty" vectors were transfected into HeLa cells. After 24 hrs the cells were left untreated or incubated with either TNF or IL-1, lysed, and immunoprecipitated with anti-HA antibody and immunoblotted with anti-IKK-.alpha. and anti-HA antibody. c. HA-IKK-.gamma. or "empty" vectors (VEC) were transfected into 293 cells that were treated and processed as in b. d. HA-IKK-.alpha. and Flag-IKK-.beta. were expressed in Sf9 cells using baculovirus vectors and purified as described in Zandi et al., Science 281:1360-1363 (1998). After incubation with or without purified recombinant Flag-IKK-.gamma., the proteins were immunoprecipitated with anti-IKK-.gamma. (NEMO) antibody (Yamaoka et al., Cell 93:1231-1240 (1998)) and immunoblotted with anti-HA and anti-Flag(M2) antibody. "IP" designates immunoprecipitation; "IB" designates immunoblot. [0021] FIG. 4: IKK-.gamma. is a component of the I.kappa.B kinase complex. a. HeLa cells were transiently transfected with HA-IKK-.gamma.. After 24 hrs, cells were treated or not with TNF or IL-1. Part of each lysate was immunoprecipitated (IP) with anti-HA antibody and another part with anti-IKK-.alpha.; I.kappa.B kinase activity was determined as described in DiDonato et al., Nature 388:548-554 (1997). Levels of endogenous IKK-.alpha. were determined by immunoblotting (IB) with anti-IKK-.alpha.. b. Extracts of unstimulated or TNF treated 293 cells that were transfected with an HA-IKK-.gamma. vector were fractionated on a Superose 6 column. Fractions were immunoprecipitated with anti-IKK-.alpha. antibody. Immunecomplex kinase assays (KA) and immunoblotting of IKK-.alpha. and HA-IKK-.gamma. were conducted with IKK-.alpha. and HA antibodies. [0022] FIG. 5: IKK-.gamma. is an essential component of the I.kappa.B kinase. a. HA-IKK-.alpha. and HA-IKK.beta. vectors were cotransfected into HeLa cells with either "empty" or antisense (AS)-IKK-.gamma. vectors. After 24 hrs, cells were treated or not with TNF and lysed. Lysates were precipitated with anti-HA antibody and IKK activity was determined by immunecomplex kinase assays (KA). Expression of HA-IKK-.alpha. and HA-IKK-.beta. was determined by immunoblotting (IB). Migration positions of IKK-.alpha. and IKK-.beta. and a nonspecific (ns) band are indicated. b. HA-IKK-.gamma., HA-IKK-.alpha. or HA-IKK-.beta. vectors were cotransfected into 293 cells with either "empty" or AS-IKK-.gamma. vectors as indicated. After 24 hrs the cells were lysed, immunoprecipitated with anti-HA and immunoblotted with anti-HA antibody. "ns" designates nonspecific bands. Continue reading about Gamma subunit of cytokine responsive lkb-alpha kinase complex... Full patent description for Gamma subunit of cytokine responsive lkb-alpha kinase complex Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Gamma subunit of cytokine responsive lkb-alpha kinase complex patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Gamma subunit of cytokine responsive lkb-alpha kinase complex or other areas of interest. ### Previous Patent Application: Methods and compositions for detecting enzymatic activity Next Patent Application: Gaucher disease drugs and methods of identifying same Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Gamma subunit of cytokine responsive lkb-alpha kinase complex patent info. IP-related news and info Results in 0.31381 seconds Other interesting Feshpatents.com categories: Electronics: Semiconductor , Audio , Illumination , Connectors , Crypto , 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
