U.S. patent application number 10/657146 was filed with the patent office on 2004-06-10 for human irak-2.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Dixit, Vishva M., Feng, Ping, Muzio, Marta, Ni, Jian.
Application Number | 20040110926 10/657146 |
Document ID | / |
Family ID | 25527328 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110926 |
Kind Code |
A1 |
Ni, Jian ; et al. |
June 10, 2004 |
Human IRAK-2
Abstract
The present invention relates to a novel IRAK-2 protein which is
a member of the IL-1 signaling pathway. In particular, isolated
nucleic acid molecules are provided encoding the human IRAK-2
protein. IRAK-2 polypeptides are also provided as are vectors, host
cells and recombinant methods for producing the same. Also provided
are diagnostic methods for detecting IRAK-2 related disorders and
therapeutic methods for treating IRAK-2 related disorders.
Inventors: |
Ni, Jian; (Germantown,
MD) ; Feng, Ping; (Germantown, MD) ; Muzio,
Marta; (Milan, IT) ; Dixit, Vishva M.; (Los
Altos Hills, CA) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
9410 Key West Avenue
Rockville
MD
20850
The Regents of the University of Michigan
3003 South State Street
Ann Arbor
MI
48109
|
Family ID: |
25527328 |
Appl. No.: |
10/657146 |
Filed: |
September 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10657146 |
Sep 9, 2003 |
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09773753 |
Feb 2, 2001 |
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6653452 |
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09773753 |
Feb 2, 2001 |
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09307185 |
May 7, 1999 |
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6222019 |
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09307185 |
May 7, 1999 |
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08980060 |
Nov 26, 1997 |
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5965421 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C12N 9/12 20130101; A61K
38/00 20130101; Y02A 50/30 20180101; Y02A 50/422 20180101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 014/705 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a polynucleotide at
least 95% identical to a nucleotide sequence encoding amino acids 1
to 96 of SEQ ID NO:2.
2. The isolated nucleic acid molecule of claim 1, comprising a
polynucleotide encoding amino acids 1 to 96 of SEQ ID NO:2.
3. The isolated nucleic acid molecule of claim 2, comprising
nucleotides 34 to 321 of SEQ ID NO:1.
4. The isolated nucleic acid molecule of claim 1, which is DNA.
5. The isolated nucleic acid molecule of claim 1, which is RNA.
6. The isolated nucleic acid molecule of claim 1, further
comprising a heterologous polynucleotide.
7. The isolated nucleic acid molecule of claim 6, wherein said
heterologous polynucleotide encodes a polypeptide.
8. A recombinant vector comprising the isolated nucleic acid
molecule of claim 1.
9. A genetically engineered host cell that comprises the isolated
nucleic acid molecule of claim 1.
10. A genetically engineered host cell that comprises the
polynucleotide of claim 1 operatively associated with a regulatory
sequence that controls gene expression.
11. A recombinant method for producing an IRAK-2 polypeptide,
comprising culturing the recombinant host cell of claim 10 under
conditions such that said polypeptide is expressed and recovering
said polypeptide.
12. A recombinant polypeptide produced by the method of claim
11.
13. An isolated polypeptide comprising an amino acid sequence at
least 95% identical to amino acids 1 to 96 of SEQ ID NO:2.
14. The isolated polypeptide of claim 13, comprising amino acids 1
to 96 of SEQ ID NO:2.
15. The isolated polypeptide of claim 13, further comprising a
heterologous polypeptide.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 09/773,753, filed Feb. 2, 2001, which is a continuation of U.S.
application Ser. No. 09/307,185, filed May 7, 1999 (now U.S. Pat.
No. 6,222,019, issued Apr. 24, 2001), which is a divisional of U.S.
application Ser. No. 08/980,060, filed Nov. 26, 1997 (now U.S. Pat.
No. 5,965,421, issued Oct. 12, 1999).
FIELD OF THE INVENTION
[0002] The present invention relates to a novel interleukin-1
receptor signaling protein. More specifically, isolated nucleic
acid molecules are provided encoding a human interleukin-1 receptor
associated kinase-2 (IRAK-2). IRAK-2 polypeptides are also
provided, as are vectors, host cells and recombinant methods for
producing the same.
BACKGROUND OF THE INVENTION
[0003] Interleukin-1 (IL-1). Interleukin-1 (IL-1.alpha. and
IL-1.beta.) is a "multifunctional" cytokine that affects nearly
every cell type, and often in concert with other cytokines or small
mediator molecules. (Dinarello, C. A., Blood 87:2095-2147 (Mar. 15,
1996).) There are three members of the IL-1 gene family:
IL-1.alpha., IL-1.beta., and IL-1 receptor antagonist (IL-1Ra).
IL-1.alpha. and IL-1.beta. are agonists and IL-1Ra is a specific
receptor antagonist. IL-1.alpha. and .beta. are synthesized as
precursors without leader sequences. The molecular weight of each
precursor is 31 kD. Processing of IL-1.alpha. or IL-1.beta. to
"mature" forms of 17 kD requires specific cellular proteases. In
contrast, IL-1Ra evolved with a signal peptide and is readily
transported out of the cells and termed secreted IL-1Ra
(sIL-1Ra).
[0004] IL-1 Receptor and Ligands. The receptors and ligands of the
IL-1 pathway have been well defined (for review, see Dinarello, C.
A., FASEB J. 8:1314-1325 (1994); Sims, J. E. et al., Interleukin-1
signal transduction: Advances in Cell and Molecular Biology of
Membranes and Organelles, Vol. 3, JAI Press, Inc., Greenwich, Conn.
(1994), pp. 197-222). Three ligands, IL-1.alpha., IL-1.beta., and
IL-1 receptor antagonist (IL-1Ra) bind three forms of IL-1
receptor, an 80-kDa type I IL-1 receptor (IL-1R1) (Sims, J. E. et
al., Science 241:585-589 (1988)), a 68-kDa type II IL-1 receptor
(IL-1RII) (McMahan, C. J. et al., EMBO J. 10:2821-2832 (1991)), and
a soluble form of the type II IL-1R (sIL-1RII) Colotta, F. et al.,
Science 261:472-475 (1993)).
[0005] IL-1 production in various disease states. Increased IL-1
production has been reported in patients with various viral,
bacterial, fungal, and parasitic infections; intravascular
coagulation; high-dose IL-2 therapy; solid tumors; leukemias;
Alzheimer's disease; HIV-1 infection; autoimmune disorders; trauma
(surgery); hemodialysis; ischemic diseases (myocardial infarction);
noninfectious hepatitis; asthma; UV radiation; closed head injury;
pancreatitis; periodontitis; graft-versus-host disease; transplant
rejection; and in healthy subjects after strenuous exercise. There
is an association of increased IL-1.beta. production in patients
with Alzheimer's disease and a possible role for IL-1 in the
release of the amyloid precursor protein (Vasilakos, J. P., et al.,
FEBS Lett. 354:289 (1994)). However, in most conditions, IL-1 is
not the only cytokine exhibiting increased production and hence the
specificity of the IL-1 findings as related to the pathogenesis of
any particular disease is lacking. In various disease states,
IL-1.beta., but not IL-1.alpha., is detected in the
circulation.
[0006] IL-1 in Therapy. Although IL-1 has been found to exhibit
many important biological activities, it is also found to be toxic
at doses that are close to therapeutic dosages (Dinarello, C. A.,
Blood 87:2095-2147 (Mar. 15, 1996)). In general, the acute
toxicities of either isoform of IL-1 were greater after intravenous
compared with subcutaneous injection. Subcutaneous injection was
associated with significant local pain, erythema, and swelling
(Kitamura, T., & Takaku, F., Exp. Med. 7:170 (1989); Laughlin,
M. J., Ann. Hematol. 67:267 (1993)). Patients receiving intravenous
IL-1 at doses of 100 ng/kg or greater experienced significant
hypotension. In patients receiving IL-1.beta. from 4 to 32 ng/kg
subcutaneously, there was only one episode of hypotension at the
highest dose level (Laughlin, M. J., Ann. Hematol. 67:267
(1993)).
[0007] Contrary to IL-1-associated myelostimulation in patients
with normal marrow reserves, patients with aplastic anemia treated
with 5 daily doses of IL-1.alpha. (30 to 100 ng/kg) had no
increases in peripheral blood counts or bone marrow cellularity
(Walsh, C. E., et al., Br. J. Haematol 80:106 (1992)). IL-1 has
been administered to patients undergoing various regiments of
chemotherapy to reduce the nadir of neutropenia and
thrombocytopenia.
[0008] Daily treatment with 40 ng/kg IL-1.alpha. from day 0 to day
13 of autologous bone marrow or stem cells resulted in an earlier
recovery of neutropenia (median, 12 days; P<0.001) (Weisdorf,
D., et al., Blood 84:2044 (1994)). After 14 days of treatment, the
bone marrow was significantly enriched with committed myeloid
progenitor cells. Similar results were reported in patients with
AML receiving 50 ng/kg/d of IL-1.beta. for 5 days starting at the
time of transplantation with purged or nonpurged bone marrow
(Nemunaitis, J., et al., Blood 83:3473 (1994)). Injecting humans
with low doses of either IL-1.alpha. or IL-1.beta. confirms the
impressive pyrogenic and hypotension-inducing properties of the
molecules.
[0009] IL-1 signaling mechanisms. After binding to interleukin-1
(IL-1), the IL-1 receptor type I (IL-1RI) associates with the IL-1R
Accessory Protein (IL-1RAcP) and initiates a signaling cascade that
results in the activation of NF-kB, (Greenfeder, S. A., et al., J.
Biol. Chem. 270:13757-65 (1995); Sims, J. E., et al., Science
241:585-9 (1988); Korherr, C.,et al., Eur. J. Immunol. 27:262-7
(1997); Wesche, H., et al., J. Biol. Chem. 272:7727-31 (1997);
Freshney, N. W., et al., Cell 78:1039-49 (1994); and Martin, M., et
al., Eur. J. Immunol. 24:1566 (1994)). Significant similarity
exists between the IL-1R signaling pathway in mammals and the Toll
signaling pathway in Drosophila. Toll, which shares sequence
homology with the cytoplasmic domain of the IL-1RAcP, induces
Dorsal activation (a homologue of NF-kB) via the adapter protein
Tube and the protein kinase Pelle, (Galindo, R. L., et al.,
Development 121:2209-18 (1995); Norris, J. L. & Manley, J. L.,
Genes Devel. 10:862-72 (1996); Letsou, A., et al., EMBO
12:3449-3458 (1993); and Grosshans, J., et al., Nature 372:563-566
(1994)); significantly the recently identified IRAK (IL-1R
Associated Kinase) is homologous to Pelle, (Cao, Z., et al.,
Science 271:1128-31 (1996)). However, in mammalian cells,
additional complexity is thought to exist based on the observation
that multiple protein kinase activities coprecipitate with the
IL1RI (Singh, R., et al., J. Clin. Invest. 100:419 (1997); and
Eriksson, A., et al., Cytokine 7:649 (1995)). Furthermore, given
that in Drosophila the adapter protein Tube interacts with and
regulates Pelle's activity, it is likely that analogous
adapter/regulatory molecules might participate in IL-1 signaling.
There is a need in the art to characterize molecules involved in
the IL-1 signaling pathway.
[0010] Nuclear factor kappa B (NF-kB). NF-kB is a member of a
family of dimeric transcription factors made from monomers that
have approximately 300 amino-acid Rel regions which bind to DNA,
interact with each other, and bind the IkB inhibitors (for review,
see Baeuerle and Baltimore, Cell 87:13-20 (1996)). Disregulation of
NF-kB has been implicated in malignant transformation and
hyperplasia (Gilmore et al., Oncogene 9:2391-2398 (1996)). NF-kB
plays an important role in the antiviral response as a
virus-inducible transcriptional regulator of .beta.-interferon, MHC
class I, and inflammatory cytokine genes. NF-kB has also been shown
to protect cells from pro-apoptotic stimuli (Beg et al., Nature
376:167-170 (1995)).
SUMMARY OF THE INVENTION
[0011] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding the IRAK-2
polypeptide having the amino acid sequence shown in SEQ ID NO:2 or
SEQ ID NO:4 or the amino acid sequence encoded by the cDNA clone
deposited in a bacterial host as ATCC Deposit Number 209340 on Oct.
7, 1997.
[0012] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of IRAK-2 polypeptides or peptides by
recombinant techniques.
[0013] The invention further provides an isolated IRAK-2
polypeptide having an amino acid sequence encoded by a
polynucleotide described herein.
[0014] The present invention also provides a screening method for
identifying compounds capable of enhancing or inhibiting a cellular
response induced by the IRAK-2, which involves contacting cells
which express the IRAK-2 with the candidate compound, assaying a
cellular response, and comparing the cellular response to a
standard cellular response, the standard being assayed when contact
is made in absence of the candidate compound; whereby, an increased
cellular response over the standard indicates that the compound is
an agonist and a decreased cellular response over the standard
indicates that the compound is an antagonist.
[0015] The invention provides a diagnostic method useful during
diagnosis of a IRAK-2 or IL-1 disorder.
[0016] An additional aspect of the invention is related to a method
for treating an individual in need of an increased level of IRAK-2
activity in the body comprising administering to such an individual
a composition comprising a therapeutically effective amount of an
isolated IRAK-2 polypeptide of the invention or an agonist
thereof.
[0017] A still further aspect of the invention is related to a
method for treating an individual in need of a decreased level of
IRAK-2 activity in the body comprising, administering to such an
individual a composition comprising a therapeutically effective
amount of an IRAK-2 antagonist.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1A-C show the nucleotide (SEQ ID NO: 1) and deduced
amino acid (SEQ ID NO:2) sequences of IRAK-2.alpha.. The protein
has a deduced molecular weight of about 65 kDa.
[0019] FIGS. 2A-D show the nucleotide (SEQ ID NO:3) and deduced
amino acid (SEQ ID NO:4) sequences of IRAK-2.beta..
[0020] FIGS. 3A-B show the regions of similarity between the amino
acid sequences of the IRAK-2.alpha. (SEQ ID NO:2) and IRAK-2.beta.
(SEQ ID NO:4) proteins and human IRAK (SEQ ID NO:5) and Pelle (SEQ
ID NO:6). Alignment was performed with Custall software.
[0021] FIG. 4. FIG. 4A shows that ectopic expression of IRAK-2 but
not the mutant version of IRAK-2 (1-96) activates NF-kB in 293
cells as measured by NF-kB reporter gene activity. FIG. 4B shows
that IRAK-2 (1-96) and IRAK-2 (97-590) inhibit IL-1Rs-induced NF-kB
activity. Transfection with TRAF-2 (87-501) and NIK (KK429-430AA)
expression vectors served as negative and positive controls,
respectively. 0.1 .mu.g of IL-1RI plus 0.1 .mu.g of IL-1RAcP and
0.6 .mu.g of putative inhibitory expression constructs were
transfected. Data are expressed as percentage of relative
IL-1Rs-induced NF-kB activity.
[0022] FIG. 5 shows that IRAK-2 induced NF-kB activity is
specifically abrogated by TRAF6 (289-522) but not TRAF2 (87-501).
293 cells were transfected with 0.2 .mu.g of IRAK-2 and increasing
amounts of TRAF constructs.
[0023] FIG. 6. FIG. 6A shows that ectopic expression of MyD88 in
293 cells results in the induction of NF-kB activity. A mutant
version of MyD88 encoding a N-terminal region, MyD88 (1-152), was
similarly capable of inducing NF-kB activity albeit to a lesser
extent; in contrast a mutant version of MyD88 coding for amino
acids 152 to the end, MyD88 (152-296) failed to induce any
luciferase activity (not evident in graph). FIG. 6B shows that
MyD88-induced NF-kB activity was selectively inhibited by a
dominant negative version of TRAF6, TRAF6 (298-522) but not TRAF2
(87-501). 0.1 .mu.g of MyD88 and increasing amount of TRAF
expression constructs were used. Data are expressed as percentage
of relative MyD88-induced NF-kB activity.
[0024] FIGS. 7A-B show that MyD88 (106-296) selectively inhibits
IL-1Rs--but not TNFR2-induced NF-kB activity. TRAF6 (298-522) and
the related TRAF2 (87-501) were used as controls. 0.5 .mu.g
receptors and increasing amounts of putative dominant negative
expression constructs were transfected. Data are expressed as
percentage of relative IL-1Rs or TNFR2-induced NF-kB activity.
[0025] FIGS. 8A-C show that MyD88 dominant negative version, MyD88
(152-296), abrogates IL-1Rs-induced but not IRAK-2-induced NF-kB
activity. Conversely IRAK-2 dominant negative versions, IRAK-2
(1-96) and IRAK-2 (97-590), significantly inhibit both IL-1Rs and
MyD88-induced NF-kB activity. 0.2 .mu.g of inducer and 0.6 .mu.g of
dominant negative expression constructs were used in each
transfection. Data are expressed as percentage of relative induced
NF-kB activity.
[0026] FIG. 9 is a schematic representation of the molecular order
of mediators of the IL-1Rs-induced NF-kB activation.
[0027] FIG. 10 shows an analysis of the IRAK-2.alpha. amino acid
sequence. Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic
Index--Jameson-Wolf" graph, amino acid residues about 96 to about
193, about 207 to about 254, about 293 to about 316, about 416 to
about 472, and about 487 to about 541 in FIG. 1 (SEQ ID NO:2)
correspond to the shown highly antigenic regions of the
IRAK-2.alpha. protein.
[0028] FIG. 11 shows an analysis of the IRAK-2.beta. amino acid
sequence. Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic
Index--Jameson-Wolf" graph, amino acid residues 96 to about 193,
about 207 to about 254, about 293 to about 316, about 416 to about
472, about 487 to about 541, and about 559 to about 619 in FIG. 2
(SEQ ID NO:4) correspond to the shown highly antigenic regions of
the IRAK-2.beta. protein.
DETAILED DESCRIPTION
[0029] The present inventors have identified a human IRAK-2,
IRAK-2.alpha., and a splice variant thereof, IRAK-2.beta.. Thus,
the present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding an IRAK-2 polypeptide having
the amino acid sequence shown in SEQ ID NO:2. The present invention
also provides isolated nucleic acid molecules comprising a
polynucleotide encoding an IRAK-2 polypeptide having the amino acid
sequence shown in SEQ ID NO:4, which was determined by sequencing a
cloned cDNA. The IRAK-2.alpha. and IRAK-2.beta. proteins of the
present invention shares sequence homology with IRAK (SEQ ID NO:5)
and Pelle (SEQ ID NO:6). The nucleotide sequence shown in SEQ ID
NO:3 was obtained by sequencing a cDNA clone, which was deposited
on Oct. 7, 1997 at the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209, and given accession
number 209340. The deposited clone is inserted in the pBluescript
SK(-) plasmid (Stratagene, LaJolla, Calif.) using the EcoRI and
XhoI restriction endonuclease cleavage sites.
[0030] Nucleic Acid Molecules
[0031] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer, and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0032] Using the information provided herein, such as the
nucleotide sequence in SEQ ID NO: 1 or SEQ ID NO:3, a nucleic acid
molecule of the present invention encoding an IRAK-2 polypeptide
may be obtained using standard cloning and screening procedures,
such as those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule described
in SEQ ID NO:1 was discovered in a cDNA library derived from HUVEC
cells. The determined nucleotide sequence of the IRAK-2 cDNA of SEQ
ID NO:1 contains an open reading frame encoding a protein of about
590 amino acid residues and a deduced molecular weight of about 65
kDa. The nucleic acid molecule described in SEQ ID NO:3 was
discovered in cDNA libraries derived from HUVEC cells and activated
neutrophils. The determined nucleotide sequence of the IRAK-2 cDNA
of SEQ ID NO:3 contains an open reading frame encoding a protein of
about 625 amino acids. The IRAK-2 proteins shown in SEQ ID NO:2 and
SEQ ID NO:4 are about 35-40% identical and about 50-60% similar to
IRAK (SEQ ID NO:5).
[0033] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors, the predicted IRAK-2
polypeptide encoded by the deposited cDNA comprises about 625 amino
acids, but may be anywhere in the range of 600-650 amino acids.
[0034] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0035] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0036] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) shown
in SEQ ID NO:1 or SEQ ID NO:3; and DNA molecules which comprise a
sequence substantially different from those described above but
which, due to the degeneracy of the genetic code, still encode an
IRAK-2 protein. Of course, the genetic code is well known in the
art. Thus, it would be routine for one skilled in the art to
generate such degenerate variants.
[0037] In addition, the present inventors have identified the
following cDNA clones related to extensive portions of SEQ ID NO:1
and SEQ ID NO:3: HPMCW18R (SEQ ID NO:7), HTADQ88R (SEQ ID NO:8),
HNFEL57R (SEQ ID NO:9), HAPCM54R (SEQ ID NO:10), HNFFX36R (SEQ ID
NO:11), HNFHL91R (SEQ ID NO:12), and HCE5L53R (SEQ ID NO:13).
[0038] The following public EST, which relates to portions of SEQ
ID NO: 1 and SEQ ID NO:3, has also been identified: Genbank
Accession No. N52479, (SEQ ID NO: 14).
[0039] In another aspect, the invention provides isolated nucleic
acid molecules encoding the IRAK-2 polypeptide having an amino acid
sequence as encoded by the cDNA clone contained in the plasmid
deposited as ATCC Deposit No. 209340 on Oct. 7, 1997. In a further
embodiment, nucleic acid molecules are provided encoding the
full-length IRAK-2.alpha. or IRAK-2.beta. polypeptide lacking the
N-terminal methionine. The invention also provides an isolated
nucleic acid molecule having the nucleotide sequence shown in SEQ
ID NO:1 or SEQ ID NO:3 or the nucleotide sequence of the IRAK-2
cDNA contained in the above-described deposited clone, or a nucleic
acid molecule having a sequence complementary to one of the above
sequences. Such isolated molecules, particularly DNA molecules, are
useful as probes for gene mapping, by in situ hybridization with
chromosomes, and for detecting expression of the IRAK-2 gene in
human tissue, for instance, by Northern blot analysis.
[0040] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA or the nucleotide sequence shown in SEQ ID
NO:1 or SEQ ID NO:3 is intended fragments at least about 15 nt, and
more preferably at least about 20 nt, still more preferably at
least about 30 nt, and even more preferably, at least about 40 nt
in length which are useful as diagnostic probes and primers as
discussed herein. Of course, larger fragments 50, 100, 150, 200,
250, 300, 350,400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450,
1500, 1550, 1600, 1650, or 1700 nt in length are also useful
according to the present invention as are fragments corresponding
to most, if not all, of the nucleotide sequence of the deposited
cDNA or as shown in SEQ ID NO:1 or SEQ ID NO:3. By a fragment at
least 20 nt in length, for example, is intended fragments which
include 20 or more contiguous bases from the nucleotide sequence of
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO:1 or SEQ ID NO:3.
[0041] Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding epitope-bearing portions of
the IRAK-2 protein. In particular, such nucleic acid fragments of
the present invention include nucleic acid molecules encoding: a
polypeptide comprising amino acid residues from about 96 to about
193 in SEQ ID NO:2 or SEQ ID NO:4; a polypeptide comprising amino
acid residues from about 207 to about 254 in SEQ ID NO:2 or SEQ ID
NO:4; a polypeptide comprising amino acid residues from about 293
to about 316 in SEQ ID NO:2 or SEQ ID NO: 4; a polypeptide
comprising amino acid residues from about 416 to about 472 in SEQ
ID NO:2 or SEQ ID NO:4; a polypeptide comprising amino acid
residues from about 487 to about 541 in SEQ ID NO:2 or SEQ ID NO:4;
and a polypeptide comprising amino acid residues from about 559 to
about 619 in SEQ ID NO:4. The inventors have determined that the
above polypeptide fragments are antigenic regions of the IRAK-2
polypeptides. Methods for determining other such epitope-bearing
portions of the IRAK-2 protein are described in detail below.
[0042] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in ATCC
Deposit 209340. By "stringent hybridization conditions" is intended
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0043] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0044] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in SEQ ID
NO:1 or SEQ ID NO:3). Of course, a polynucleotide which hybridizes
only to a poly A sequence (such as the 3' terminal poly(A) tract of
the IRAK-2 cDNA shown in SEQ ID NO:1 or SEQ ID NO:3), or to a
complementary stretch of T (or U) resides, would not be included in
a polynucleotide of the invention used to hybridize to a portion of
a nucleic acid of the invention, since such a polynucleotide would
hybridize to any nucleic acid molecule containing a poly (A)
stretch or the complement thereof (e.g., practically any
double-stranded cDNA clone).
[0045] As indicated, nucleic acid molecules of the present
invention which encode an IRAK-2 polypeptide may include, but are
not limited to those encoding the amino acid sequence of the
full-length polypeptide, by itself; the coding sequence for the
full-length polypeptide and additional sequences, such as those
encoding a leader or secretory sequence, such as a pre-, or pro- or
prepro-protein sequence; the coding sequence of the full-length
polypeptide, with or without the aforementioned additional coding
sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities. Thus, the sequence encoding the polypeptide may be
fused to a marker sequence, such as a sequence encoding a peptide
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (Qiagen, Inc.), among others, many of
which are commercially available. As described in Gentz et al.,
Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin protein, which has been described by Wilson et al.,
Cell 37:767-778 (1984). As discussed below, other such fusion
proteins include the IRAK-2 fused to Fc at the N- or
C-terminus.
[0046] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the IRAK-2 protein. Variants
may occur naturally, such as a natural allelic variant. By an
"allelic variant" is intended one of several alternate forms of a
gene occupying a given locus on a chromosome of an organism. Genes
II, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0047] Such variants include those produced by nucleotide
substitutions, deletions or additions, which may involve one or
more nucleotides. The variants may be altered in coding regions,
non-coding regions, or both. Alterations in the coding regions may
produce conservative or non-conservative amino acid substitutions,
deletions or additions. Especially preferred among these are silent
substitutions, additions and deletions, which do not alter the
properties and activities of the IRAK-2 protein or portions
thereof. Also especially preferred in this regard are conservative
substitutions.
[0048] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:2; (b) a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:2, but lacking the N-terminal
methionine; (c) a nucleotide sequence encoding the polypeptide
having the amino acid sequence in SEQ ID NO:4; (d) a nucleotide
sequence encoding the polypeptide having the amino acid sequence in
SEQ ID NO:4, but lacking the N-terminal methionine; (e) a
nucleotide sequence encoding the polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
209340; (f) a nucleotide sequence encoding the IRAK-2 polypeptide
having the amino acid sequence encoded by the cDNA clone contained
in ATCC Deposit No. 209340, but lacking the N-terminal methionine;
or (g) a nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e), or (f).
[0049] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a IRAK-2 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the IRAK-2 polypeptide. In other
words, to obtain a polynucleotide having a nucleotide sequence at
least 95% identical to a reference nucleotide sequence, up to 5% of
the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up
to 5% of the total nucleotides in the reference sequence may be
inserted into the reference sequence. These mutations of the
reference sequence may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0050] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID
NO:3 or to the nucleotides sequence of the deposited cDNA clone can
be determined conventionally using known computer programs such as
the Bestfit program (Wisconsin Sequence Analysis Package, Version 8
for Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0051] The present application is directed to nucleic acid
molecules at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or to the
nucleic acid sequence of the deposited cDNA, irrespective of
whether they encode a polypeptide having IRAK-2 activity. This is
because even where a particular nucleic acid molecule does not
encode a polypeptide having IRAK-2 activity, one of skill in the
art would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having IRAK-2 activity
include, inter alia, (1) isolating the IRAK-2 gene or allelic
variants thereof in a cDNA library; (2) in situ hybridization
(e.g., "FISH") to metaphase chromosomal spreads to provide precise
chromosomal location of the IRAK-2 gene, as described in Verma et
al., Human Chromosomes: A Manual of Basic Techniques, Pergamon
Press, New York (1988); and (3) Northern Blot analysis for
detecting IRAK-2 mRNA expression in specific tissues.
[0052] Preferred, however, are nucleic acid molecules having
sequences at least 95%, 96%, 97%, 98% or 99% identical to a nucleic
acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or to a nucleic
acid sequence of the deposited cDNA which do, in fact, encode a
polypeptide having IRAK-2 protein activity. By "a polypeptide
having IRAK-2 activity" is intended polypeptides exhibiting IRAK-2
activity in a particular biological assay. For example, IRAK-2
protein activity can be measured using the luciferase assay
described in Cao et al., Nature 383: 443-446 (1996) and below in
Example 1.
[0053] Briefly, cells which have been transfected with a nucleic
acid encoding for a candidate polypeptide, such as human 293 cells,
are transfected with an ELAM-1-luciferase reporter plasmid.
Luciferase activity is measured in these cells and compared to
cells which have been transfected with the luciferase construct,
but not with the candidate polypeptide. A higher level of
luciferase activity in cells with the candidate polypeptide is
indicative of IRAK-2 activity.
[0054] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of
the deposited cDNA or a nucleic acid sequence shown in SEQ ID NO:1
or SEQ ID NO:3 will encode a polypeptide "having IRAK-2 protein
activity." In fact, since degenerate variants of these nucleotide
sequences all encode the same polypeptide, this will be clear to
the skilled artisan even without performing the above described
comparison assay. It will be further recognized in the art that,
for such nucleic acid molecules that are not degenerate variants, a
reasonable number will also encode a polypeptide having IRAK-2
protein activity. This is because the skilled artisan is fully
aware of amino acid substitutions that are either less likely or
not likely to significantly effect protein function (e.g.,
replacing one aliphatic amino acid with a second aliphatic amino
acid).
[0055] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that proteins are surprisingly tolerant of amino
acid substitutions.
[0056] Vectors and Host Cells
[0057] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of IRAK-2 polypeptides or fragments thereof by
recombinant techniques.
[0058] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0059] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be
translated.
[0060] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0061] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 available from Pharmacia. Among preferred eukaryotic vectors
are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0062] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0063] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as, hIL5-receptor has been fused with Fc portions for the purpose
of high-throughput screening assays to identify antagonists of
hIL-5. See, D. Bennett et al., Journal of Molecular Recognition,
Vol. 8:52-58 (1995) and K. Johanson et al., The Journal of
Biological Chemistry, Vol. 270, No. 16:9459-9471 (1995).
[0064] The IRAK-2 protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the
host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may
also include an initial modified methionine residue, in some cases
as a result of host-mediated processes.
[0065] IRAK-2 Polypeptides and Fragments
[0066] The invention further provides an isolated IRAK-2
polypeptide having the amino acid sequence encoded by the deposited
cDNA, or the amino acid sequence in SEQ ID NO:2, or the amino acid
sequence in SEQ ID NO:4, or a peptide or polypeptide comprising a
portion of the above polypeptides.
[0067] It will be recognized in the art that some amino acid
sequences of the IRAK-2.alpha. or IRAK-2.beta. polypeptides can be
varied without significant effect of the structure or function of
the protein. If such differences in sequence are contemplated, it
should be remembered that there will be critical areas on the
protein which determine activity.
[0068] Thus, the invention further includes variations of the
IRAK-2.alpha. or IRAK-2.beta. polypeptide which show substantial
IRAK-2 polypeptide activity or which include regions of IRAK-2
protein such as the protein portions discussed below. Such mutants
include deletions, insertions, inversions, repeats, and type
substitutions. As indicated above, guidance concerning which amino
acid changes are likely to be phenotypically silent can be found in
Bowie, J. U., et al., "Deciphering the Message in Protein
Sequences: Tolerance to Amino Acid Substitutions," Science
247:1306-1310 (1990).
[0069] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2 or SEQ ID NO:4, or that encoded by the deposited
cDNA, may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid
residues includes a substituent group, or (iii) one in which the
mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as an IgG Fc fusion
region peptide or leader or secretory sequence or a sequence which
is employed for purification of the mature polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0070] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the
IRAK-2 protein. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but
can also be problematic when preparing pharmaceutical formulations,
because they can be immunogenic. (Pinckard et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845
(1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993)).
[0071] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
1TABLE 1 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0072] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of amino acid
substitutions for any given IRAK-2 polypeptide will not be more
than 50, 40, 30, 20, 10, 5 or 3.
[0073] Amino acids in the IRAK-2 proteins of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as in vitro proliferative
activity.
[0074] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell or a native source. For example, a recombinantly produced
version of the IRAK-2 polypeptide can be substantially purified by
the one-step method described in Smith and Johnson, Gene 67:31-40
(1988).
[0075] The polypeptides of the present invention include the a
polypeptide comprising the polypeptide encoded by the deposited
cDNA; a polypeptide comprising the polypeptide encoded by the
deposited cDNA, but minus the N-terminal methionine; a polypeptide
comprising amino acids about 1 to about 590 in SEQ ID NO:2; a
polypeptide comprising amino acids about 2 to about 590 in SEQ ID
NO:2; a polypeptide comprising amino acids about 1 to about 625 in
SEQ ID NO:4; a polypeptide comprising amino acids about 2 to about
625 in SEQ ID NO:4; as well as polypeptides which are at least 95%
identical, and more preferably at least 96%, 97%, 98% or 99%
identical to those described above and also include portions of
such polypeptides with at least 30 amino acids and more preferably
at least 50 amino acids.
[0076] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
IRAK-2 polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the IRAK-2
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0077] As a practical matter, whether any particular polypeptide is
at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or to the
amino acid sequence encoded by deposited cDNA clone can be
determined conventionally using known computer programs such the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0078] The polypeptide of the present invention are useful as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0079] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide described herein.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0080] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983) Antibodies that react with predetermined sites on proteins.
Science 219:660-666. Peptides capable of eliciting protein-reactive
sera are frequently represented in the primary sequence of a
protein, can be characterized by a set of simple chemical rules,
and are confined neither to immunodominant regions of intact
proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl
terminals.
[0081] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. See, for instance, Wilson et al., Cell 37:767-778
(1984) at 777.
[0082] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about at least
about 15 to about 30 amino acids contained within the amino acid
sequence of a polypeptide of the invention. Non-limiting examples
of antigenic polypeptides or peptides that can be used to generate
IRAK-2-specific antibodies include: a polypeptide comprising amino
acid residues from about 96 to about 193 in SEQ ID NO:2 or SEQ ID
NO:4; a polypeptide comprising amino acid residues from about 207
to about 254 in SEQ ID NO:2 OR SEQ ID NO:4; a polypeptide
comprising amino acid residues from about 293 to about 316 in SEQ
ID NO:2 or SEQ ID NO: 4; a polypeptide comprising amino acid
residues from about 416 to about 472 in SEQ ID NO:2 or SEQ ID NO:4;
a polypeptide comprising amino acid residues from about 487 to
about 541 in SEQ ID NO:2 or SEQ ID NO:4; and a polypeptide
comprising amino acid residues from about 559 to about 619 in SEQ
ID NO:4. As indicated above, the inventors have determined that the
above polypeptide fragments are antigenic regions of the IRAK-2
protein.
[0083] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. Houghten, R.
A. (1985) General method for the rapid solid-phase synthesis of
large numbers of peptides: specificity of antigen-antibody
interaction at the level of individual amino acids. Proc. Natl.
Acad. Sci. USA 82:5131-5135. This "Simultaneous Multiple Peptide
Synthesis (SMPS)" process is further described in U.S. Pat. No.
4,631,211 to Houghten et al. (1986).
[0084] As one of skill in the art will appreciate, IRAK-2
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. This has been shown, e.g., for
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EPA 394,827;
Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG part can
also be more efficient in binding and neutralizing other molecules
than the monomeric IRAK-2 protein or protein fragment alone
(Fountoulakis et al., J. Biochem 270:3958-3964 (1995)).
[0085] Screening Assays
[0086] The present inventors have shown that IRAK-2 mediates NF-kB
activation induced by IL-1R stimulation. NF-kB is an ubiquitous
transcription factor which has been shown to activate transcription
of enzymes, such as cyclooxygenase-2 (Newton et al., Biochem.
Biophys. Res. Commun. 237(1):28-32 (1997)); cytokines, such as
RANTES (Moriuchi et al., J. Immunol. 158(7):3483-3491 (1997));
adhesion molecules, such as E-selectin (ELAM-1) (Read et al., J.
Biol. Chem. 272(5):2753-2761 (1997)); and other molecules. The
normal functions of NF-kB include communication between cells,
embryonal development, the response to stress, inflammation and
viral infection, and the maintenance of cell type specific
expression of genes (for review, see Wulczyn et al., J. Mol. Med.
74(12):749-769 (1996)). Upregulation of NF-kB could be used to
treat viral infections, such as HIV ((Moriuchi et al., J. Immunol.
158(7):3483-3491 (1997)), and damage caused by oxidative stress
(Renard et al., Biochem. Pharmacol. 53:149-160 (1997)).
Disregulation of NF-kB activation has been linked to adult
respiratory distress syndrome, sepsis syndrome, asthma, rheumatoid
arthritis, inflammatory bowel disease, malignant transformation and
hyperplasia (Blackwell et al., Am. J. Respir. Cell. Mol. Biol.
17(1):3-9 (1997); Barnes, Int. J. Biochem. Cell. Biol.
29(6):867-870 (1997); and Gilmore et al., Oncogene 9:2391-2398
(1996)). Accordingly, inhibitors of NF-kB could be used to treat
these disorders. Several inhibitors of NF-kB have been identified,
including antioxidants such as alpha-tocopherol (Erl et al., Am. J.
Physiol. 273:H634-H640 (1997)), and glucocorticoids, such as
dexamethasone (Wang et al., J. Immunol. 159:534-537 (1997))).
[0087] Thus, the present invention also provides a screening method
for determining whether a compound of interest is an agonist or
antagonist of the IRAK-2 pathway. This method involves contacting
cells which express IRAK-2, either exogenously or endogenously,
with a compound of interest, assaying NF-kB mediated transcription,
and comparing the NF-kB mediated transcription to a standard
response. The standard response is the level of NF-kB mediated
transcription in cells expressing IRAK-2 that have not been
contacted with the compound of interest, whereby an increase in
NF-kB mediated transcription over the standard indicates that the
compound of interest is an agonist of the IRAK-2 pathway and a
decrease in NF-kB mediated transcription under the standard
indicates that the compound of interest is an antagonist of the
IRAK-2 pathway.
[0088] By "assaying NF-kB mediated transcription" is intended
qualitatively or quantitatively measuring NF-kB mediated
transcription. By the invention, the compound of interest is an
agonist of the IRAK-2 pathway if NF-kB mediated transcription is
enhanced over that observed due to IRAK-2 in the absence of the
compound of interest and the compound of interest is an antagonist
of the IRAK-2 pathway if NF-kB mediated transcription is diminished
compared to that observed due to IRAK-2 in the absence of the
compound of interest. Since IRAK-2 activates NF-kB transcription,
any in vitro or in vivo assay which measures NF-kB activity can be
used in this method.
[0089] For example, a construct encoding for IRAK-2 is transfected
into a cell, along with a construct containing a reporter gene
which is under the control of a promoter which is activated in the
presence of NF-kB. Any reporter gene which is known in the art can
be used in this assay. Examples of reporter genes useful in this
assay include, but are not limited to, luciferase,
.beta.-galactosidase, and chloramphenicol acetyltransferase.
NF-kB-responsive promoters can include one or more binding sites
for NF-kB. Examples of promoters which are sensitive to NF-kB
include, but are not limited to, the promoter for ELAM-1 and the
promoter for RANTES. After transfection of the constructs, the cell
is contacted with a compound of interest, and the reporter gene
expression is measured and compared to the reporter gene expression
seen in cells which have not been contacted with the compound of
interest. An increase in reporter gene expression in cells which
have been contacted with the compound of interest indicates that
the compound is an agonist of the IRAK-2 pathway. A decrease in
reporter gene expression in cells which have been contacted with
the compound of interest indicates that the compound is an
antagonist of the IRAK-2 pathway.
[0090] IRAK-2 Related Disorder Diagnosis
[0091] For IRAK-2 related disorders, it is believed that
substantially altered (increased or decreased) levels of IRAK-2
gene expression can be detected in tissues taken from a mammal
having such a disorder, relative to a "standard" mammal, i.e., a
mammal of the same species not having the disorder. Thus, the
invention provides a diagnostic method useful during diagnosis of
an IRAK-2 related disorder, which involves assaying the expression
level of the gene encoding the IRAK-2 protein in mammalian cells or
body fluid and comparing the gene expression level with a standard
IRAK-2 gene expression level, whereby an increase in the gene
expression level over the standard is indicative of certain
disorders.
[0092] IRAK-2 related disorders are believe to include, but are not
limited to, leukemia, lymphoma, rheumatoid arthritis, sarcoidosis,
tuberculosis, onchocerciasis, allergies, various bacterial
infections, arteriosclerosis, autoimmune diseases, and inflammatory
diseases.
[0093] Where a diagnosis has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting enhanced IRAK-2
gene expression will experience a worse clinical outcome relative
to patients expressing the gene at a lower level.
[0094] By "assaying the expression level of the gene encoding the
IRAK-2 protein" is intended qualitatively or quantitatively
measuring or estimating the level of the IRAK-2 protein or the
level of the mRNA encoding the IRAK-2 protein in a first biological
sample either directly (e.g., by determining or estimating absolute
protein level or mRNA level) or relatively (e.g., by comparing to
the IRAK-2 protein level or mRNA level in a second biological
sample).
[0095] Preferably, the IRAK-2 protein level or mRNA level in the
first biological sample is measured or estimated and compared to a
standard IRAK-2 protein level or mRNA level, the standard being
taken from a second biological sample obtained from an individual
not having the disorder. As will be appreciated in the art, once a
standard IRAK-2 protein level or mRNA level is known, it can be
used repeatedly as a standard for comparison.
[0096] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source which contains IRAK-2 protein or mRNA. Biological samples
include mammalian body fluids (such as sera, plasma, urine,
synovial fluid and spinal fluid) which contain IRAK-2 protein, and
ovarian, prostate, heart, placenta, pancreas liver, spleen, lung,
breast and umbilical tissue.
[0097] Preferred mammals include monkeys, apes, cats, dogs, cows,
pigs, horses, rabbits and humans. Particularly preferred are
humans.
[0098] Total cellular RNA can be isolated from a biological sample
using the single-step guanidinium-thiocyanate-phenol-chloroform
method described in Chomczynski and Sacchi, Anal. Biochem.
162:156-159 (1987). Levels of mRNA encoding the IRAK-2 protein are
then assayed using any appropriate method. These include Northern
blot analysis (Harada et al., Cell 63:303-312 (1990)), S1 nuclease
mapping (Fujita et al., Cell 49:357-367 (1987)), the polymerase
chain reaction (PCR), reverse transcription in combination with the
polymerase chain reaction (RT-PCR) (Makino et al., Technique
2:295-301 (1990)), and reverse transcription in combination with
the ligase chain reaction (RT-LCR).
[0099] Assaying IRAK-2 protein levels in a biological sample can
occur using antibody-based techniques. For example, IRAK-2 protein
expression in tissues can be studied with classical
immunohistological methods (Jalkanen, M., et al., J. Cell. Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.
105:3087-3096 (1987)).
[0100] Other antibody-based methods useful for detecting IRAK-2
protein gene expression include immunoassays, such as the enzyme
linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA).
[0101] Suitable labels are known in the art and include enzyme
labels, such as, Glucose oxidase, and radioisotopes, such as iodine
(.sup.125I, .sup.121I), carbon (.sup.14C), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.112In), and technetium
(.sup.99mTc), and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0102] Modes of Administration
[0103] It will be appreciated that conditions caused by a decrease
in the standard or normal level of IRAK-2 activity in an individual
can be treated by administration of IRAK-2 protein. Thus, the
invention further provides a method of treating an individual in
need of an increased level of IRAK-2 activity comprising
administering to such an individual a pharmaceutical composition
comprising an effective amount of an isolated IRAK-2 polypeptide of
the invention effective to increase the IRAK-2 activity level in
such an individual.
[0104] As a general proposition, the total pharmaceutically
effective amount of IRAK-2 polypeptide administered parenterally
per dose will be in the range of about 1 .mu.g/kg/day to 10
mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. More preferably, this
dose is at least 0.01 mg/kg/day, and most preferably for humans
between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, the IRAK-2 polypeptide is typically administered at a
dose rate of about 1 .mu.g/kg/hour to about 50 .mu.g/kg/hour,
either by 1-4 injections per day or by continuous subcutaneous
infusions, for example, using a mini-pump. An intravenous bag
solution may also be employed.
[0105] Pharmaceutical compositions containing the IRAK-2 of the
invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. By "pharmaceutically acceptable carrier" is
meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
[0106] Chromosome Assays
[0107] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0108] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a IRAK-2 protein
gene. This can be accomplished using a variety of well known
techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome
mapping using well known techniques for this purpose.
[0109] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes.
[0110] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
probes from the cDNA as short as 50 or 60 bp. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual Of Basic
Techniques, Pergamon Press, New York (1988).
[0111] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0112] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0113] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1:
Characterization of IRAK-2.alpha.
[0114] A novel partial human cDNA was identified that showed
significant homology to both IRAK and Pelle. Screening of a human
HUVEC cDNA library resulted in the isolation of a full length cDNA
clone; analysis of the nucleotide sequence revealed an open reading
frame encoding a 590 amino acids (aa) protein with a calculated MW
of 65 kDa (FIG. 1). Clustall alignment analysis showed significant
homology to both IRAK and Pelle (FIG. 3). Given its sequence and
functional similarity to IRAK the molecule was designated IRAK-2.
Northern blot analysis revealed a single IRAK-2 transcript
expressed in a variety of tissues whose size (about 4 Kbp) was
consistent with that of the cDNA.
[0115] Ectopic expression of IRAK-2 in human 293 cells induced
NF-kB activation as determined by relative luciferase activity of a
NF-kB responsive construct. Truncated versions of IRAK-2 encoding
amino acid residues 1 to 96 of SEQ ID NO:2 [IRAK-2 (1-96)] or amino
acid residues 97 to 590 of SEQ ID NO:2 [IRAK-2 (97-590)] failed to
induce any luciferase activity suggesting that integrity of the
molecule was essential for its function (FIG. 4A). Deletional
analysis has previously shown that a mutant version of Pelle
analogous to IRAK-2 (97-590) is also inactive leading to the
suggestion that Pelle's recruitment to the plasma membrane through
its N-terminal domain is necessary for its subsequent function
(Galindo, R. L., et al., Development 121:2209-2218 (1995)). Given
this, it was tested whether IRAK-2 (1-96) or IRAK-2 (97-590) could
act as dominant negative inhibitors of IL-1R-induced NF-kB
activity. Coexpression of IL-1RI and IL-1RAcP (L-1Rs for clarity)
strongly induced NF-kB activity. Surprisingly, both IRAK-2 (1-96)
and IRAK-2 (97-590) inhibited IL-1Rs-induced NF-kB activity. A
dominant negative mutant version of the downstream kinase NIK that
is implicated in IL-1R-induced NF-kB activation was used as a
positive control; the unrelated adapter molecule TRAF2 (298-522)
was used as a negative control (FIG. 4B).
[0116] Given the sequence similarity shared by IRAK and IRAK-2, and
the functional involvement of IRAK-2 in IL-1Rs-induced NF-kB
activity, it was analyzed whether IRAK-2 was recruited to the IL-1R
signaling complex. Interestingly, while IRAK preferentially
coprecipitated with IL-1RAcP, IRAK-2 preferentially bound to the
IL-1RI. In contrast, a mutant version of IRAK-2 lacking the first
96 amino acid residues [IRAK-2 (97-590)] failed to associate with
IL-1RI suggesting that its N-terminal domain docks with the
cytoplasmic domain of IL-1RI. Confirming this was the finding that
a truncated form of IRAK-2 coding for the first 96 amino acid
residues [IRAK-2 (1-96)] specifically coprecipitated with
IL-1RI.
[0117] Certain members of the TRAF adapter family mediate NF-kB
activation induced by a number of cytokine receptors. TRAF2, for
example plays a critical role in TNFR1 and -2 mediated NF-kB
activation. TRAF6 has recently been implicated in the IL-1
signaling pathway and shown to complex with IRAK (Cao, Z., et al.,
Nature 383:443-6 (1996)). It was therefore determined if IRAK-2
interacted with TRAF6 when coexpressed in 293T cells. Both IRAK and
IRAK-2 coprecipitated with TRAF6 but not with the related TRAF2. A
dominant negative version of TRAF6 [TRAF6 (298-522)] which inhibits
IL-1-induced NF-kB activity, also bound both IRAK and IRAK-2.
Further, IRAK-2-induced NF-kB activity was specifically inhibited
by dominant negative TRAF6 (298-522) but not by a dominant negative
version of TRAF2 [TRAF2 (87-501)] (FIG. 5). These data are in
keeping with TRAF6 acting downstream of IRAK-2, in the IL-1
mediated NF-kB signaling pathway.
[0118] Additional putative proximally participating
adapters/regulators were sought by systematically looking for
proteins showing homology to either Tube or IL-1RAcP. BLAST
searches of the public data base revealed the cytoplasmic domain of
the IL-1RAcP to possess significant homology to MyD88 (Lord, K., et
al., Oncogene 5:1095 (1990)). Sequence similarity between MyD88,
IL-1RI and Toll has previously been reported, but the functional
significance of this homology has been obscure. Interestingly, the
MyD88 polypeptide has a modular structure composed of two fused
module types: a N-terminal "interaction domain" (or DD for Death
Domain that was initially defined in proteins involved in
programmed cell death), (Feinstein, E., et al., Trends Biochem.
Sci. 20:342-4 (1995); and Hofmann, K. & Tschopp, J., et al.,
FEBS Letters 371:321 (1995)) and a C-terminal domain related to the
cytoplasmic region of IL-1RAcP, IL-1RI, Toll and the recently
identified human Toll homologue (Hardiman, G., et al., Oncogene
13:2467-75 (1996); Hultmark, D., Biochem. Biophys. Res. Commun.
199:144 (1994); Bonnert, T., et al., FEBS lett. 402:81-84(1997);
and Medzhitov, R., et al., Nature 388:394 (1997)). Given the
presence of these two distinct domains it was hypothesized that
MyD88 might simultaneously connect a transmembrane receptor
belonging to the IL-1R family with a downstream signaling mediator.
To test this, the role of human MyD88 was functionally
characterized.
[0119] Ectopic expression of MyD88 in 293 cells strongly induced
NF-kB activity in a dose dependent manner. Similarly, a truncated
version of MyD88 encoding the N-terminal domain (DD), MyD88
(1-151), activated NF-kB albeit to a lesser extent. In contrast,
the C--terminal region, MyD88 (152-296) did not induce any
luciferase activity (FIG. 6A). Significantly, MyD88-induced NF-kB
activity was specifically inhibited by TRAF6 but not TRAF2 dominant
negative expression constructs suggesting that TRAF6 and MyD88
likely participate in the same signaling pathway and that TRAF6
functions downstream of MyD88 (FIG. 6B). It was next tested whether
MyD88 (152-296) could act as a dominant negative inhibitor of
IL-1Rs-induced NF-kB activity; MyD88 (152-296) specifically
inhibited IL-1Rs-induced but not TNFR2-induced NF-kB activation. A
dominant negative version of TRAF6 [TRAF6 (289-522)] similarly
inhibited IL-1Rs-induced but not TNFR2-induced NF-kB activation; in
contrast, a dominant negative version of TRAF2 [TRAF2 (87-501)]
abrogated TNFR2-induced, but not IL-1Rs-induced, NF-kB activity
confirming the specificity of effects observed with MyD88
(152-296).
[0120] Given the significant sequence homology existing between
MyD88 and the IL-1RAcP, it was investigated whether the two could
interact. Upon coexpression in 293T cells, MyD88 and IL-1RAcP
formed an immunoprecipitable complex. IL-1RI, which shows weaker
sequence similarity to MyD88, did not associate with MyD88 under
these experimental conditions. Domain mapping studies revealed that
the sequence homologous C-terminal region of MyD88 was sufficient
for binding to the IL-1RAcP cytoplasmic domain (FIGS. 7A-7B)
consistent with a hemophilic interaction.
[0121] In an effort to molecularly order the proximal components of
the IL-1R signaling complex identified herein, it was tested
whether the dominant negative mutant versions of MyD88 and IRAK-2
could inhibit the active forms of the others. A dominant negative
version of MyD88 completely abrogated IL-1Rs-induced NF-kB
activation but failed to inhibit IRAK-2-induced NF-kB activation
(FIGS. 8A-8C). On the other hand, dominant negative versions of
IRAK-2, significantly inhibited both IL-1Rs- and MyD88-induced
NF-kB activity. These results are consistent with MyD88 acting
upstream of IRAK-2 in the IL-1R signaling pathway.
[0122] Given the presence of a N-terminal "interaction domain" (DD)
in both MyD88 and IRAK-2 (Feinstein, E., et al., supra; and
Hofmann, K. & Tschopp, J., supra)) it was tested whether these
two proteins could interact. It was found that MyD88 specifically
coprecipitated with IRAK-2. Significantly a truncated version of
IRAK-2 lacking the N-terminal domain (DD) [IRAK-2 (97-590)], that
failed to induce NF-kB activation, also failed to associate with
MyD88; similarly, the version of MyD88 (152-296) that was unable to
induce NF-kB activity, was also impaired in its ability to bind
IRAK-2 lending functional credence to this interaction.
[0123] Taken together these results support a model wherein MyD88
acts as an adapter/regulator in the IL-1R signaling complex by
independently interacting with IL-1RAcP and IRAK-2. However, we
were unable, under these experimental conditions, to assemble a
multimolecular complex between MyD88, IRAK-2 and the IL-1Rs. This
is consistent with the possibility that MyD88 is only transiently
recruited to the IL-1R signaling complex where it subsequently
regulates IRAK-2's activity.
[0124] Methods
[0125] cDNA Cloning and Analysis.
[0126] A partial cDNA clone was used to screen a human HUVEC cDNA
library. Hybridizing clones were characterized by automated DNA
sequencing. Alternatively the sequence corresponding to aa, 391 to
570 of IL-1RAcP was used to search the NCBI Gene Bank nr database.
Human and murine MyD88 cDNAs were identified as having
statistically significant homology to IL-1RAcP. Sequence assembly,
comparison and alignment were performed using DNASTAR software.
[0127] Expression Vectors.
[0128] Mammalian expression vectors encoding Flag-TRAF6, Flag-TRAF6
(289-522), Flag-TRAF2, Flag-TRAF2 (87-501), NIK (KK429-430AA),
ELAM-Luciferase reporter plasmid, Flag-IL-1RAcP and IRAK have been
previously described ((Cao, Z., et al., Nature 383:443-6 (1996);
Chinnaiyan, A., et al., Science 274:990-92 (1996); Malinin, N. L.,
et al., Nature 385:5:540-4 (1997); and Rothe, M., et al., Science
269:1424-7 (1995)). AU1-IRAK-2 (1-96), AU1-MyDS88, AU-1-MyD88
(152-296) and HA-MyD88 (1-151) were PCR amplified from a HUVEC cDNA
library using custom-made oligonucleotide primers encoding the AU1
or HA epitope tag. Amplified fragments were cloned into the
mammalian expression vector pCDNA3 (Invitrogen). IRAK-2-MyC and
IRAK-2 (97-590)-MyC were obtained hy PCR amplification and cloned
in frame into pCDNA3-MyC-His vector (Invitrogen). Flag-IL-1RI and
Flag-.DELTA.IL-1RI were similarly obtained by PCR amplification
from the HUVEC cDNA library and sub cloned in frame into
pCMV-1-Flag expression vector.
[0129] Transfection and Coimmunoprecipitation.
[0130] Human embryonic 293 or 293T cells were transiently
transfected by calcium phosphate method with the indicated
plasmids. The total amount of DNA was kept constant. 24-36 hours
after transfection, cells were lysed in 0.5 ml buffer (1% NP40, 150
mM NaCl, 50 mM Tris, 1 mM EDTA and protease inhibitors cocktail).
Cell lysates were adjusted to 0.7 M NaCl and the indicated
antibodies were added for 1 to 4 hours. Immune complexes were
precipitated by the addition of protein-G-Sepharose (Sigma). After
extensive washing, the Sepharose heads were boiled in sample buffer
and the eluted proteins fractionated by SDS-PAGE. Subsequent
protein immunoblotting was performed as described (Chinnaiyan, A.,
et al., Cell 81:505-12 (1995)).
[0131] NF-kB Luciferase Assay.
[0132] Cells were transfected with 0.1 .mu.g ELAM-Luciferase
reporter plasmid, 0.2 .mu.g pCMV-.beta.Gal and the indicated
expression vectors; total amount of transfected DNA was kept
constant by supplementation with empty vector. Relative NF-kB
activity was calculated by normalizing relative luciferase activity
with .beta.Gal activity as previously described (Cao, Z. et al.,
Nature 383:443446 (1996).
Example 2
Tissue Distribution of IRAK-2 mRNA Expression
[0133] Northern blot analysis is carried out to examine IRAK-2 gene
expression in human tissues, using methods described by, among
others, Sambrook et al., cited above. A cDNA probe containing the
nucleotide sequence corresponding to the open reading frame of the
IRAK-2.alpha. protein (SEQ ID NO: 1) is labeled with .sup.32P using
the rediprime.TM. DNA labeling system (Amersham Life Science),
according to manufacturer's instructions. After labeling, the probe
is purified using a CHROMA SPIN-100.TM. column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number
PT1200-1. The purified labeled probe is then used to examine
various human tissues for IRAK-2 mRNA.
[0134] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) are obtained from Clontech and examined with the
labeled probe using ExpressHyb.TM. hybridization solution
(Clontech) according to manufacturer's protocol number PT1190-1.
Following hybridization and washing, the blots are mounted and
exposed to film at -70.degree. C. overnight, and films developed
according to standard procedures.
[0135] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0136] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0137] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
Sequence CWU 1
1
* * * * *