IRAK-4: compositions and methods of use

Details IRAK-4: compositions and methods of use IRAK-4: compositions and methods of use

2001-01-11

2004-11-16

Eyler, Yvonne (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S006120, C435S320100, C435S325000, C530S350000, C536S023500

Reexamination Certificate

active

06818419

ABSTRACT:

BACKGROUND OF THE INVENTION
The proinflammatory cytokine interleukin-1 (IL-1) functions in the generation of systemic and local responses to infection, injury and immunological challenges. The importance of IL-1 in inflammation has been demonstrated by the ability of the highly specific IL-1 receptor antagonist protein (IL-1Ra, or IRAP) to relieve inflammatory conditions (for review, see, e.g., Dinarello,
Cytokine Growth Factor Rev.
8:253-265 (1997)). IL-1 is produced primarily by activated macrophages and monocytes, and is involved in lymphocyte activation, fever, leukocyte trafficking, the acute phase response, cartilage remodeling and other processes. IL-1 exerts its effects by binding to a receptor, IL-1RI, located on the plasma membrane of responsive cells. Among the results of IL-1 binding to the IL-1 RI receptor is the activation of the NF-&kgr;B transcription factor, ultimately leading to the expression of numerous genes involved in inflammation, such as cytokines, growth factors, immunoreceptors, and cell adhesion molecules (for review, see, e.g., Lee, et al.,
J. Clin. Pharmacol.
38(11):981-93 (1998)).
Several proteins have been discovered to mediate signal transduction following IL-1RI activation, ultimately leading to the activation of NF-KB. For example, the IL-1R accessory protein, IL-1RAcP, has been shown to associate with the IL-1RI receptor following binding to IL-1, thereby initiating the signal transduction cascade (Greenfeder, et al.,
J. Biol. Chem.
270(23):13757-65 (1995)). In addition, three IL-1 receptor-associated kinases (IRAKs) have been identified, IRAK (“IRAK-1;” Cao, et al.,
Science
271:1128-1131 (1996)), IRAK-2 (Muzio, et al.,
Science
278:1612-1615 (1997)), and the monomyeloic cell-specific IRAK-M (Wesche, et al.,
J. Biol. Chem.
274:19403-10 (1999)). IRAK has been shown to be phosphorylated and to associate with IL-1RI in an IL-1 dependent manner. In addition, the MyD88 protein has been shown to mediate the association of IRAK proteins to the activated IL-1 receptor (Wesche, et al., 7:837-47 (1997)). Also, TRAF6 transduces the IRAK signal to downstream effector molecules (Cao, et al.,
Nature
383:443-6 (1996)). The IRAK proteins, as well as MyD88, have been shown to play a role in transducing signals other than those originating from IL-1R receptors, including signals triggered by activation of IL-18 receptors (Kanakaraj, et al.
J. Exp. Med.
189(7):1129-38 (1999)) and LPS receptors (Yang, et al., J. Immunol. 163:639-643 (1999); Wesche, et al.,
J. Biol. Chem.
274:19403-10 (1999)). Overexpression of IRAK-2 and IRAK-M has been shown to be capable of reconstituting the response to IL-1 and LPS in an IRAK deficient cell line.
The IL-1 signal transduction cascade is analogous to a signaling cascade in
Drosophila melanogaster
that is involved in the establishment of dorsal ventral polarity during the early development of Drosophila embryos. Specifically, in Drosophila, the extracellular ligand Spaetzle binds to a receptor called Toll, which shares homology to IL-1R. In addition, a serine/threonine kinase acting downstream of Toll activation, Pelle is homologous to IRAK kinases (Cao, et al.,
Science
271:1128-1131 (1996); Muzio, et al.,
Science
278:1612-1615 (1997); Wesche, et al.,
J. Biol. Chem.
274:19403-19410 (1999)). Finally, activation of the Toll receptor results in the activation of the transcription factor Dorsal, which is homologous to NF-&kgr;B. Dorsal is inhibited in Drosophila cells by Cactus, which is itself homologous to the NF-&kgr;B inhibitor I&kgr;B.
The present invention is based on the identification of a novel member of the IRAK family, IRAK-4. Nucleic acid and protein sequences for IRAK-4 are provided, as are methods of making IRAK-4 nucleic acids and proteins. Also provided are methods of using IRAK-4 polynucleotides and polypeptides, including methods of using the herein-disclosed sequences to isolate compounds useful in the treatment or prevention of any of a number of inflammatory diseases and conditions.
SUMMARY OF THE INVENTION
The present invention provides novel nucleic acids and polypeptides for mammalian IRAK-4, a new member of the IRAK gene family. IRAK kinases associate with activated IL-1, IL-18 and other receptors and act to transduce signals originating from the activated receptors, ultimately leading to a variety of downstream effects such as NF-&kgr;B activation.
In one aspect, an isolated nucleic acid is provided encoding an IRAK-4 polypeptide, the polypeptide comprising at least about 98% amino acid sequence identity to SEQ ID NO:1 or to a subsequence thereof, wherein the amino acid sequence of the polypeptide comprises an alanine residue at an amino acid position corresponding to amino acid position 81 of SEQ ID NO:1, and wherein said nucleic acid comprises at least about 400 nucleotides.
In one embodiment, the polypeptide further comprises an amino acid selected from the group consisting of: (i) a valine residue at an amino acid position corresponding to amino acid position 432 of SEQ ID NO:1; (ii) a leucine residue at an amino acid position corresponding to amino acid position 437 of SEQ ID NO:1; (iii) an arginine residue at an amino acid position corresponding to amino acid position 444 of SEQ ID NO:1; and (iv) a glutamine residue at an amino acid position corresponding to amino acid position 451 of SEQ ID NO:1. In another embodiment, the polypeptide comprises each of the amino acids listed as (i) to (iv). In another embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide comprises at least about 100 amino acids. In another embodiment, the polypeptide comprises at least about 450 amino acids.
In another embodiment, the nucleic acid comprises a cytosine at a nucleotide position corresponding to nucleotide position 242 of SEQ ID NO:2. In another embodiment, the nucleic acid further comprises a nucleotide selected from the group consisting of: (i) a thymine at a nucleotide position corresponding to nucleotide position 1295 of SEQ ID NO:2; (ii) a thymine at a nucleotide position corresponding to nucleotide position 1302 of SEQ ID NO:2; (iii) a thymine at a nucleotide position corresponding to nucleotide position 1310 of SEQ ID NO:2; (iv) an adenine at a nucleotide position corresponding to nucleotide position 1332 of SEQ ID NO:2; and (v) an adenine at a nucleotide position corresponding to nucleotide position 1353 of SEQ ID NO:2. In another embodiment, the nucleic acid comprises each of the nucleotides listed as (i) to (v). In another embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:2. In another embodiment, the nucleic acid comprises at least about 350 nucleotides. In another embodiment, the polypeptide specifically binds to antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:1.
In another aspect, the present invention provides an isolated IRAK-4 polypeptide, the polypeptide having at least about 98% amino acid sequence identity to SEQ ID NO:1 or to a subsequence thereof, wherein the amino acid sequence of the polypeptide comprises an alanine residue at an amino acid position corresponding to amino acid position 81 of SEQ ID NO:1, and wherein the polypeptide comprises at least about 100 amino acids.
In one embodiment, the polypeptide further comprises an amino acid selected from the group consisting of: (i) a valine residue at an amino acid position corresponding to amino acid position 432 of SEQ ID NO:1; (ii) a leucine residue at an amino acid position corresponding to amino acid position 437 of SEQ ID NO:1; (iii) an arginine residue at an amino acid position corresponding to amino acid position 444 of SEQ ID NO:1; and (iv) a glutamine residue at an amino acid position corresponding to amino acid position 451 of SEQ ID NO:1. In another embodiment, the polypeptide comprises all of the amino acids listed as (i) to (iv). In another embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO:1. In another embodiment, the polypeptide is encoded by a nucleic aci

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