Novel inhibitor of the inflammatory response induced by TNF-alpha and IL-1

[0002] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF INVENTION

[0003] The present invention relates generally to the field of NF-κB-dependent activation to regulate immune, inflammatory, and anti-apoptotic responses. In particular, the present invention describes an inhibitor of serine/threonine kinase signaling that regulates NF-κB-dependent activation, thereby regulating immune, inflammatory, and anti-apoptotic responses. The present invention describes several mutant proteins of the NF-κB-inducing kinase (NIK) that prevent activation of a downstream kinase, IκB-specific kinase (IKK). One embodiment of the present invention describes kinase-deficient mutant NIK proteins that inhibits phosphorylation and activation of IKK. Another embodiment of the present invention describes an C-terminal NIK peptide protein that effectively interacts with IKK, and blocks IKK activation by inhibiting NIK/IKK association. Yet another embodiment of the present invention describes a method for inhibiting NF-κB-dependent immune and inflammatory responses using the NIK mutant proteins.

BACKGROUND OF THE INVENTION

[0004] TNFα (tumor necrosis factor alpha) binding at the plasma membrane induces intracellular signaling events that translate into the induction of NF-κB in the nucleus. The eukaryotic NF-κB/Rel (nuclear factor-κB/Rel) family of transcription factors plays an essential role in the regulation of inflammatory, immune, and apoptotic responses (Bacuerle and Baltimore 1996; Baldwin 1996; Verma et al 1995). One of the distinguishing characteristics of the NF-κB/Rel transcription factor is its posttranslational regulation through interactions with cytoplasmic inhibitory proteins termed IκB (inhibitor-κB). NF-κB corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells, by its physical assembly with a family of cytoplasmic ankyrin-rich IκB inhibitors (Bacuerle and Baltimore 1996; Baldwin 1996; Verma et al 1995). Stimulation of cells with various pro-inflammatory cytokines, including TNFα, induces nuclear NF-κB expression. The TNFα-signaling pathway is complex and involves recruitment of at least three adapter proteins, TRADD (TNF-R1 associated death domain protein) and TRAF-2 (TNF-receptor-associated factor)-2, and the serine/threonine kinase RIP (receptor interacting protein) to the cytoplasmic tail of the type 1 TNF receptor (Hsu et al Immunity 1996; Hsu et al Cell 1996) (FIG. 1). In turn, the recruitment of these factors promotes activation of the downstream NIK (Malinin et al 1997) and IKKα and IKKβ (IκB-specific kinases) (DiDonato et al 1997; Mercurio et al 1997; Regnier et al 1997; Woronicz et al 1997; Zandi et al 1997). The activated IKKα and IKKβ directly phosphorylates the two N-terminal regulatory serines within IκBα, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome (Bacuerle and Baltimore 1996; Baldwin 1996; Verma et al 1995). Degradation of IκBα unmasks the nuclear localization signal on NF-κB, allowing the NF-κB to translocate to the nucleus where it engages cognate κB enhancer elements and activates the transcription of various κB-dependent genes involved in inflammatory, immune, and anti-apoptotic responses.

[0005] IL-1, a second proinflammatory cytokine, acts in a manner similar to TNFα. IL-1 binding to its receptor recruits the MyD88 and TRAF-6 adapter proteins and (IRAK), a serine-threonine kinase (Cao et al 1996; Muzio et al 1997). Like TRAF-2, TRAF-6 interacts with NIK. Thus, the TNFα and IL-1 signaling pathways converge at the level of NIK. The present invention provides new insights into the molecular basis for NIK regulation of NF-κB-dependent gene expression, and provides novel methods for modulating NF-κB-dependent immune, inflammatory, and anti-apoptotic responses.

SUMMARY OF THE INVENTION

[0006] The present invention provides a molecular mechanism to interfere with cytokine induction of NF-κB-dependent immune, inflammatory, and anti apoptotic responses, through interference with the assembly of NIK and the IKK proteins. A relatively small region of NIK selectively impairs the NIK-IKK interaction. The present invention provides a novel and highly specific method for modulating NF-κB-dependent immune and inflammatory responses, based on interruption of the critical protein-protein interaction of NIK and IKK. The present invention provides methods for inhibiting NF-κB-dependent gene expression, using mutant NIK proteins. One embodiment of the present invention provides kinase-deficient NIK mutant proteins that inhibit activation of IKK. Another embodiment of the invention provides N-terminus NIK mutant proteins that bind IKK, thus inhibiting NIK/IKK interaction.

BRIEF DESCRIPTION OF THE FIGURES

[0007]
FIG. 1: Schematic summary of the overlapping and unique intracellular signaling pathways including adapter molecules and specific kinases activated by the proinflammatory cytokines TNFα and IL-1.

[0008]
FIG. 2: A) Alignment of the amino acid sequences of the activation loop between subdomains VII (DFG) and VIII (M(A/S)PE) of NIK and other MAP kinases. Asterisks denote residues shown to be phosphorylated and/or implicated in the activation of these kinases. B) Biological function of wild-type NIK and kinase domain mutant forms of NIK. C) In vivo phosphorylation of wild-type NIK and the mutant forms of NIK.

[0009]
FIG. 3: A) The NIK-T559A mutant protein fails to activate IKKα- and kinase activity. B) The NIK-T559A mutant protein fails to induce κB-luciferase activity. C) The expression of NIK-T559A mutant protein dominantly interferes with TNFα-induced NF-κB activation in a dose-related manner.

[0010]
FIG. 4: A) The NIK-T559A mutant protein assembles with IKKα but fails to phosphorylate coimmunoprecipitated IKKα. B) the NIK-T559A mutant protein fails to undergo auto phosphorylation.

[0011]
FIG. 5: A) Biologically active NIK spontaneously forms homotypic oligomers in vivo. B) Myc-NIK and NIK-T7 proteins are expressed at comparable levels in transfected cultures.

[0012]
FIG. 6: A) Schematic overview of the structural organization of NIK protein. B) Myc-NIK is stably expressed in transfected 293 cells. C) Multiple domains of NIK participate in homotypic oligomerization.

[0013]
FIG. 7: A) Analysis of the functional effects of various deletion mutant forms of NIK in the presence and absence of TNFα. B) Analysis of the functional effects of various deletion mutant forms of NIK in the presence and absence of wild-type NIK protein. C) Analysis of the inhibitory effect of the C-terminus of NIK (amino acid residues 735 to 947) and shorter fragments (735-813, 814-947, 847-947) on TNFα (left graph) and NIK (right graph) induction of κB-luciferase activity.

[0014]
FIG. 8: A) All the deletion forms of Myc-NIK are comparably expressed when cotransfected with T7-IKKα. B) The C-terminus of NIK mediates heterotypic oligomerization with IKKα.

[0015]
FIG. 9: A) Expression of the C-terminal 213 amino acid segment of NIK interrupts the assembly of NIK and IKKα. B) Myc-NIK and the Myc-N1-200 and Myc-N735-947 peptides are comparably expressed in transfected cultures.

[0016]
FIG. 10: The amino acid and nucleic acid sequences of NIK.

[0017]
FIG. 11: κB-specific gene expression induce by anti-CD3-CD28 stimulation is inhibited by expression of the NIK (735-947) peptide but not by the NIK (1-220) peptide.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Terminology

[0019] The term “NIK” used herein is defined as a wild-type NF-κB inducing kinase having serine/threonine kinase activity, and having the amino acid sequence as described in FIG. 10 and SEQ ID NO.: 1.

[0020] The term “mutant NIK” used herein is defined as a NIK protein that inhibits activation of NF-κB-dependent gene expression. Two categories of mutant NIK proteins are described in the present invention and are further defined below.

[0021] The term “N-terminus deletion mutant of NIK” used herein is defined as a NIK protein lacking the N-terminus and having at least the C-terminus section that interacts with IKK protein. The N-terminus deletion mutant of NIK includes N-terminus truncation mutants of NIK, and C-terminal NIK peptides that interact with IKK protein. These mutant NIK proteins may or may not retain the catalytic kinase domain of NIK. Specific examples include, but are not limited to, the following N-terminus deletion mutants of NIK: (see FIG. 10 for amino acid sequence of NIK): N350-947 (includes amino acids His350 through Pro947), N570-947 (includes amino acids Gly570 through Pro947), N624-947 (includes amino acids Pro624 through Pro 947), N735-947 (includes amino acids Glu735 through Pro947), N735-813 (includes amino acids Glu735 through Pro813), N814-947 (includes amino acids Ser814 through Pro947), and N847-947 (includes Ala847 through Pro947).

[0022] The term “kinase-deficient mutant of NIK protein” used herein is defined as a mutant NIK protein that does not exhibit catalytic kinase activity. The terms “catalytically inactive”, “biologically inactive”, and “kinase-deficient” define a NIK protein that does not undergo auto- or trans-phosphorylation. Such phosphorylation may involve either inter- or intra-molecular phosphorylation of the activation loop of NIK which corresponds to amino acids 534-566 which controls phosphorylation activity of NIK. Additionally, the kinase-deficient mutant NIK protein does not activate IKK. The kinase-deficient phenotype of this mutant NIK protein may be due to mutations that occur within the so-called activation loop of NIK. Specific examples include, but are not limited to, the following kinase-deficient NIK mutants: a mutant NIK protein that has three amino acid substitutions Ser-549, Thr-552, and Thr-559 all replaced with alanine residues (NIKSTT549/552/559AAA); or a mutant NIK protein that has Threonine-559 substituted with alanine (NIK-T559A). A second type of kinase deficient mutant of NIK corresponds to a mutant NIK protein that has Lys-429 and Lys-430 replaced with alanine residues (NIK-KK429/430AA). This mutant is altered at the ATP binding site of the kinase and consequently cannot mediate phosphorylation.

[0023] The term “IKK” used herein is defined as the Iκ-B-specific kinases, IKKα and IKKβ.

[0024] The term “activation of an IKK protein” used herein is defined as changing an inactive IKK protein into an IKK protein that is an IκB kinase. The activated IKK phosphorylates serine 32 and serine 36 of IκB which marks this inhibitor for ubiquitination and degradation.

[0025] The term “NIK/IKK” interaction used herein is defined as NIK/IKK protein binding, a NIK/IKK protein complex and NIK/IKK protein assembly.

[0026] The term “inhibition of NIK/IKK interaction” used herein is defined as inhibiting either the formation of a NIK/IKK protein complex or disruption of a formed NIK/IKK protein complex.

[0027] The term “NF-κB” used herein is defined as a ubiquitously expressed family of eukaryotic transcription factors, comprising a homo- or hetero-dimer of DNA-binding proteins related to the proto-oncogene c-Rel, that controls the expression of many κB-dependent immmune, inflammatory, and anti-apoptotic response genes.

[0028] The term “NF-κB-dependent gene expression” used herein is defined as those immune and inflammatory genes that are under the regulatory control of the κB-enhancer. In most cells, NF-κB exists in a latent state in the cytoplasm bound to inhibitory proteins, collectively called IκB, that mask the nuclear localization signal thereby preventing nuclear translocation. The latent form of NF-κB can be induced by cytokines, such as TNFα and IL-1. Both TNFα and IL-1 signaling leads to sequential phosphorylation and activation of a series of proteins involved in a cascade pathway that requires NIK/IKK protein interaction and IKK activation, that in turn leads to phosphorylation and degradation of IκB. Degradation of the IκB inhibitor unmasks the nuclear localization signal of the NF-κB complex allowing its rapid translocation into the nucleus where it engages cognate κB-enhancer elements and activates the transcription of various NF-κB-dependent genes involved in inflammation and immune response.

[0029] Mutant NIK

[0030] The present invention provides mutant NIK proteins. In one embodiment, the NIK mutant protein is an N-terminus deletion mutant of NIK or a C-terminal NIK peptide that has at least the C-terminus section of NIK which binds to an IKK protein. The N-terminus deletion mutant NIK protein or the C-terminal NIK peptide can have some but not all of the N-terminus section. The N-terminus deletion mutant NIK protein or the C-terminal NIK peptide can also have the central sections of NIK. But the N-terminal deletion mutant NIK protein or the C-terminal NIK peptide must have at least the C-terminus section of NIK that binds to the IKK protein. In particular, one embodiment provides an N-terminus deletion mutant NIK protein or a C-terminal NIK peptide that has an amino acid sequence beginning with glutamic acid at position 735 and ending with proline at position 947 of the wild-type NIK sequence; in the present invention, this mutant is called N735-947. The present invention is not limited to only the N-terminus deletion mutant NIK protein N735-947, as the present invention also provides for other N-terminus deletion mutant proteins that create a mutant NIK protein that can interact with an IKK protein.

[0031] In another embodiment the NIK mutant protein is a kinase deficient mutant NIK protein. The kinase deficient mutant phenotype includes a NIK protein that does not undergo auto- or trans-phosphorylation. The kinase deficient phenotype also includes a NIK protein that does not phosphorylate or activate an IKK protein. Normally, a kinase-active NIK protein will phosphorylate an IKK protein which activates the IKK protein to become an IκB kinase. Therefore, an activated IKK is an IκB kinase. The present invention provides two kinase deficient mutant NIK protein sequences that differ from the wild-type NIK protein sequence. In particular, one of the kinase deficient mutant NIK proteins has the amino acid residue threonine-559 substituted with an alanine residue; in the present invention, this mutant is called NIK-T559A. A second kinase deficient NIK mutant has three amino acid residues substituted: the amino acid residues serine-549, and threonine-552, and threonine-559 are each substituted with alanine; in the present invention, this mutant is called NIK-SST549/552/559AAA. And another kinase deficient NIK mutant has two amino acid substitutions, with lysine-429 and lysine-430 substituted with an alanine residue; in the present invention, this mutant is called NIK-KK429/430AA. The present invention is not limited to only the kinase deficient NIK mutants listed above, as the present invention also provides for other amino acid substitutions that create a mutant NIK protein that has the kinase deficient phenotype.

[0032] The invention further provides nucleic acid molecules encoding the mutant NIK proteins of the present invention. In one embodiment, the nucleic acid is cDNA. Further, the invention provides a vector, which comprises the nucleic acid molecule of the present invention. Additionally, the invention provides a host vector system. This comprises the vector of the invention in a suitable host cell, e.g., a bacterial cell or eukaryotic cell.

[0033] The invention also provides methods for producing the mutant NIK proteins of the present invention. This comprises culturing the host vector system of the invention under suitable culture conditions so as to produce the mutant NIK protein in the host and recovering the mutant NIK protein so produced. The present invention also provides mutant NIK proteins produced by the method above.

[0034] The invention provides mutant NIK proteins that are labeled with a detectable marker or conjugated to a second molecule, such as a membrane permeable agent or detectable marker, and used for targeting the second molecule to NIK's target, e.g., IKK. Suitable detectable markers include, but are not limited to, a fluorescent compound, a bioluminescent compound, and chemiluminescent compound.

[0035] Methods of the Invention

[0036] Activation of NF-κB-dependent gene expression is associated with NIK/IKK interaction and activation of IKK by NIK. The invention provides methods for inhibiting activation of NF-κB-dependent gene expression, by inhibiting the NIK/IKK interaction or by inhibiting the activation of IKK by NIK, using the mutant NIK proteins of the present invention.

[0037] One embodiment of the invention provides a method for inhibiting activation of NF-κB-dependent gene expression by inhibiting NIK/IKK interaction, by contacting an IKK protein with a catalytically inactive NIK protein that continues to interact with and bind to IKK. The inhibition of the NIK/IKK interaction includes inhibiting the formation of a NIK/IKK complex, or disruption of a formed NIK/IKK complex. One particular embodiment of the present invention provides a method for inhibiting activation of NF-κB-dependent gene expression by inhibiting wild type NIK/IKK interaction, by contacting the wild type IKK/NIK protein complex with a catalytically inactive or mutant NIK protein, such that the mutant NIK competes with the wild type NIK for assembly with the IKK, thereby forming an inactive NIK/IKK protein complex. One embodiment provides using an N-terminus deletion mutant NIK protein, such as N735-947, to inhibit NIK/IKK interaction. These N-terminus mutant NIK proteins bind to IKK protein and inhibit wild type NIK/IKK interaction, thereby inhibiting activation of NF-κB-dependent gene expression. The methods of the present invention are not limited to using only the N-terminus deletion mutant NIK protein N735-947, as the present invention also provides for use of other N-terminus deletion mutant NIK proteins and other types of NIK mutant proteins that can interact with an IKK protein to inhibit activation of NF-κB-dependent gene expression.

[0038] One embodiment of the invention provides a method for inhibiting activation of NF-κB-dependent gene expression, by using a kinase-deficient NIK mutant protein. The kinase deficient phenotype includes a NIK protein that does not undergo auto- or transphosphorylation. The kinase deficient phenotype also includes a NIK protein that does not phosphorylate or activate an IKK protein. Normally, a kinase-active NIK protein will phosphorylate an IKK protein thereby activating the IKK protein to become an IκB kinase. Therefore, an activated IKK is an IκB kinase. In particular, the present invention uses NIK proteins that have the kinase deficient phenotype, such as NIK-SST549/552/559AAA or NIK-T559A to inhibit activation of NF-κB-dependent gene expression. The methods of the present invention are not limited to using only the kinase deficient NIK mutants listed above, as the present invention also provides for other mutant NIK proteins that have the kinase deficient phenotype.

[0039] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

EXAMPLE 1

[0040] Analysis of Kinase-defective Mutants of NIK

[0041] NIK protein is a member of the family of mitogen activated protein kinase kinase kinases (MAP3Ks). Three amino acid residues located within the activation loop of the NIK kinase domain are highly conserved among MAP3Ks (Alessi et al 1994; Siow et al 1997; Yan and Templeton 1994; Zheng and K. L. Guan 1994). These amino acid residues correspond to Ser-549, Thr-552 and Thr-559 in the NIK sequence (FIG. 2A). A mutant NIK, called NIK-SST549/552/559AAA, was generated in which Ser-549, Thr-552 and Thr-559 were replaced with alanine residues. A second NIK mutant, called NIK-KK429/430AA, was altered at the ATP binding site of the NIK kinase domain. These mutant NIKs and wild-type NIK were compared for their ability to stimulate κB-dependent luciferase reporter activity in transfected human 293 embryonic kidney cells (see Material and Methods section for details of transfection method).

[0042] The results demonstrated that wild-type NIK strongly stimulated luciferase activity (FIG. 2B, closed bars). Additional stimulation of these cultures with TNFα (FIG. 2B, open bars) only weakly augmented the κB-luciferase activity, suggesting that the ectopically expressed NIK was almost fully induced. In contrast, the NIK mutants NIK-SST549/552/559AAA and NIK-KK429/430AA displayed very little spontaneous functional activity (FIG. 2B, closed bars), and both mutant NIKs functioned as dominant-negative inhibitors effectively blocking NF-κB activation induced by either TNFα or wild-type NIK FIG. 2B, open bars).

[0043] To further explore the molecular basis for NIK activation, in vivo phosphorylation of wild-type and inactive mutant NIK proteins were compared by metabolic radiolabeling with 32P-labeled orthophosphoric acid. The radiolabeling procedure described in Material and methods section was used, except that after radiolabeling for 2 hours, cells were cultured in medium either in the presence or the absence of TNFα (20 ng/ml) for 15 minutes, and then NIK was immunoprecipitated with anti-Myc antibodies.

[0044] The results demonstrated significant in vivo phosphorylation of wild-type NIK but sharply diminished phosphorylation of the biologically inactive NIK-KK429/430AA and NIK-SST549/552/559AAA (FIG. 2C, top). Addition of TNFα did not enhance the level of phosphorylation of wild-type of mutant NIKs (FIG. 2C, lanes 4 through 6). The observed decrease in phosphorylation of the two mutant NIKs was not explained by marked instability of these proteins, since immunoblotting revealed only slightly lower levels of expression for each (FIG. 2C, bottom) and the half-lives of the mutant NIK proteins is similar to that of wild-type NIK.

[0045] Together, these results show that NIK induction of NF-κB correlates with its ability to undergo auto- or trans-phosphorylation in vivo. Furthermore, phosphorylation likely involves either inter- or intra-molecular phosphorylation of the activation loop of NIK. Thus, the NIK mutants NIK-KK429/430AA and NIK-SST549/552/559AAA are kinase-deficient NIK mutants.

EXAMPLE 2

[0046] Threonine 559 Plays a Critical Role in the Regulation of NIK Function

[0047] To further determine which serine or threonine residue in the activation loop of NIK (e.g. amino acids 534-566, see FIG. 2A) is critical for the regulation of its function, a set of individual mutant NIK proteins were generated. These mutant NIK proteins contained alanine substituted for either a key conserved serine or threonine residues in this loop: NIK-S549A, NIK-T552A and NIK-T559A. These mutant NIK proteins were transfected into 293 cells and evaluated for their ability to activate the downstream kinase IKKα, and to induce NF-κB-dependent transcription.

[0048] 293 cells were transfected according to the procedure described in Material and methods section below. 20 hours after transfection, in vitro kinase reactions were performed by using anti-IKKβ immunoprecipitates prepared from these cell lysates (FIG. 3A). GST-IκBα (1-62) was added as an exogenous substrate. The kinase reactions were analyzed by SDS-PAGE, followed by transfer to a nitrocellulose membrane and autoradiography. The phosphorylated GST-IκBα substrate is indicated on the right of FIG. 3A. The lower panels show the amounts of immunoprecipitated IKKα and expressed NIK present in each of the cell lysates.

[0049] For the functional assays, 293 cells were co-transfected (see Material and methods section below) with 200 ng of κB-luciferase and 100 ng of β-galactosidase reporter plasmids (6RZ), together with 1 μg of expression vector (pRK6) containing either wild-type NIK or mutant NIK-SST549/552/559/AAA, NIK-S549A, NIK-T552A or NIK-T59A. All transfections included the 6RZ plasmid to normalize for differences in gene transfer efficiency by assay of β-galactosidase activity. Twenty hours after transfection, the cultures were stimulated with or without TNFα (20 ng/ml) for 6 hours. Cell lysates were prepared from the cultures, and the luciferase activity present in these lysates was determined as described in Material and methods section below.

[0050] The results show that mutants NIK-S549A and NIK-T552A functioned similarly to wild-type NIK. However, the mutant NIK-T559A failed to induce IKKα activity (FIG. 3A) or κB-luciferase activity (FIG. 3B).

[0051] To determine whether the inability of the NIK-T559A mutant protein to activate IKKα and NF-κB-dependent transcription was due to its failure to phosphorylate IKKα, vectors encoding kinase-deficient IKKα (K44M) were co-transfected with vectors encoding wild type NIK or the inactive T559A mutant of NIK (FIG. 4A). Then, IKKα was immunoprecipitated and subjected to an immunocomplex kinase reaction to test the ability of co-immunoprecipitated NIK to phosphorylate IKKα.

[0052] The results demonstrated that wild-type NIK and mutant NIK-S549A and NIK-T552A effectively phosphorylated the coimmunoprecipitated IKKα (K44M) protein in vitro (FIG. 4A). In contrast, the mutant NIK-T559A assembled normally with IKKα (FIG. 3A, bottom) but failed to phosphorylate the coprecipitating IKKα (FIG. 4A, lane 5). In addition, NIK-T559A also lacks the ability for auto-phosphorylation compared to the other NIK activation loop mutant proteins (FIG. 4B, lane 5).

[0053] Together, these results show that Thr-559 corresponds to a key regulatory residue within the activation loop of NIK that is essential for NIK function. Thus, NIK-T559A is a kinase-deficient NIK mutant protein.

EXAMPLE 3

[0054] Ectopically Expressed NIK Spontaneously Forms Homotypic Oligomers in vivo

[0055] To determine if expressed NIK exists as monomeric or assembled homotypic oligomers in vivo, two different epitope-tagged versions of NIK, Myc-NIK and NIK-T7, were cotransfected into HeLa cells. Lysates from these transfected cells were then sequentially immunoprecipitated with anti-T7 antibodies and immunoblotted with anti-Myc antibodies.

[0056] HeLa cells were transiently transfected with plasmids encoding T7- and Myc-epitope-tagged versions of NIK, as described later in Material and Methods. After 24 hours of culture, a portion of the cells were treated with TNFα for 5 or 10 minutes, as indicated in FIG. 5A. Cell lysates were prepared from the cultures and subjected to immunoprecipitation (IP) with anti-T7 antibody conjugated to agarose beads. The immunoprecipitates were then analyzed by immunoblotting with anti-Myc antibodies. Aliquots of the whole-cell lysates (10 μl) were subjected to SDS-PAGE and immunoblotted with anti-T7 or anti-Myc antibodies to determine the levels of Myc-NIK and NIK-T7 expression. In addition, the biological activity of the added TNFα was confirmed by induced degradation of endogenous IκBα detected by immunoblotting with antibodies specific for the C-terminus of IκBα.

[0057] The results demonstrated that, when NIK-T7 and Myc-NIK were coexpressed, the two proteins were effectively coimmunoprecipitated (FIG. 5A, lane 2). In addition, stimulation of these cultures for 5 or 10 minutes with TNFα did not further enhance NIK oligomerization (FIG. 5A, lanes 3 and 4). However, the added TNFα was biologically active, since it induce the partial degradation of endogenous IκBα observed at 10 minutes (FIG. 5B). The immunoblotting results demonstrated that the Myc-NIK and NIK-T7 protein were expressed at comparable levels in the transfected cultures (FIG. 5B, lane 2).

[0058] Collectively, these results show that biologically active NIK forms dimers or higher order oligomers in vivo.

EXAMPLE 4

[0059] Mapping the NIK Domains Involved in Homotypic Oligomerization

[0060] To determine which subregions of NIK mediate the homotypic interaction, a series of nested deletion mutant forms of NIK protein were prepared and tested. Each of the deletion mutant NIKs contained an N-terminal Myc epitope tag, and each was stably expressed in 293 cells cotransfected with NIK-T7 (FIG. 6B, lanes 3-11). Cell lysates were immunoblotted with anti-Myc and anti-T7 to verify protein expression levels.

[0061] 24 hours after transfection, cell lysates were prepared, subjected to SDS-PAGE, and blotted with anti-Myc or anti-T7 antibodies to determine protein expression levels (FIG. 6B). To detect the presence of oligomers formed between mutant NIK proteins and NIK-T7, aliquots of cell lysates were subjected to immunoprecipitation (IP) with anti-T7 antibody conjugated to agarose beads. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies (FIG. 6C).

[0062] The results demonstrated that, with the exception of Myc-N1-200 and Myc-N570-947, the deletion mutant forms of NIK protein formed oligomers with NIK-T7, as indicated by their coimmunoprecipitation with anti-T7 antibodies (FIG. 6C, lanes 2-11). The results also demonstrated that NIK amino acid residues 570 to 974 failed to oligomerize, although a shorter NIK fragment containing amino acid residues 735 to 947 did form oligomers. It is possible that the 570-947 NIK fragment has an altered protein conformation due to the presence of multiple proline residues between 672 and 759.

EXAMPLE 5

[0063] Functional Effects of the C-terminus of NIK on TNFα and Wild-type NIK-induced NF-κB Activation

[0064] To determine the intrinsic biological function of deletion mutant forms of NIK proteins and their effects on TNF-α and NIK activation of NF-κB, 293 cells were cotransfected with a NIK deletion mutant and the κB luciferase reporter plasmid, using the method described in the Material and Methods section below. Briefly, 3 μg of expression vectors encoding a NIK deletion mutant was cotransfected into 293 cells, together with κB-luciferase and β-galactosidase reporter plasmids. 20 hours after transfection, the cultures were stimulated with or without TNFα (20 ng/ml) (FIG. 7A) or 0.3 μg of a wild-type NIK expression vector (FIG. 7B) for 6 hours (TNFα induction) or 24 hours (wild-type NIK induction). Cell lysates were prepared and the luciferase activities of these lysates were determined.

[0065] The results demonstrated that only the N200-947 mutant form of NIK retained significant, albeit weak, stimulatory activity, in the absence of induction from TNFα or wild-type NIK (FIGS. 7A and B, closed bars). When tested in the presence of TNF-α or wild type NIK as an inducer, the C-terminal deletion mutant forms of NIK (N1-366, N1-578 and N1-624) produced only modest and various degrees of inhibition (FIGS. 7A and B, open bars). In contrast, each of the N-terminus deletion mutant forms of NIK (N350-947, N578-947, N624-947 and N735-947) markedly inhibited both TNF-α and wild-type NIK induction of κB-luciferase activity.

[0066] These results confirm and extend previous studies (Malinin et al 1997) that showed residues 624 to 947 of NIK exert inhibitory effects on TNF-α signaling. Malinin previously showed that TRAF-2 interaction with NIK involves C-terminal sequences located between amino acids 624 and 947. The results presented here show that NIK interaction with IKKα similarly involves the C-terminus of NIK, specifically, an overlapping subregion encompassing residues 735 to 947.

[0067] To determine which sequences of the 213-amino acid C-terminal region of NIK are required for inhibition of κB-activity, additional N-terminus deletion mutant forms of NIK (N735-813, N814-947 and N847-947) were prepared and analyzed for their ability to induce κB-luciferase expression (FIG. 7C). The co-transfection and induction methods used for this experiment are described for FIGS. 7A and B above.

[0068] The results demonstrated that none of the smaller C-terminal fragments of NIK induced an increase in κB-luciferase activity when added alone (FIG. 7C). Furthermore, none of the smaller C-terminal fragments exerted potent inhibitory effects on TNF-α-induced (FIG. 7C, left graph) or on wild type NIK-induced NF-κB-driven luciferase activity (FIG. 7C, right graph).

[0069] Thus a 213 amino acid fragment of NIK, containing the carboxyl terminus of NIK including amino acids 735 to 947, represents the smallest fragment thus far identified which potently inhibits TNF-α mediated activation on NF-κB.

EXAMPLE 6

[0070] The C-terminus of NIK Mediates Binding to IKKα, the IκB-specific Kinase

[0071] To further explore the biological basis for the significant inhibitory effects of the C-terminal region of NIK (N735-947) on TNF-α signaling, the potential role of this region in NIK binding to IKKα was examined.

[0072] IKKα-T7 was coexpressed with the various Myc-NIK deletion mutants followed by coimmunoprecipitation with anti-T7 antibodies and immunoblotting with anti-Myc antibodies (FIG. 8). In this experiment, the kinase-deficient mutant, NIK-KK429/430AA (see Example 1, FIG. 2B), was used in place of the wild-type NIK to avoid potential phosphorylation of IKKα which might weaken the NIK-IKKα interaction. 293 cells were cotransfected with IKKα-T7 and Myc-NIK or a deletion form of NIK (Myc-NIK), using the method described in Material and Methods section below. Briefly, the immunoblotting experiment was performed as follows. 24 hours after transfection, cell lysates were prepared, subjected to SDS-PAGE, and immunoblotted with anti-Myc antibodies or anti-T7 antibodies to determine the overall level of expression of the individual proteins (FIG. 8A). Aliquots of the cell lysates were subjected to immunoprecipitation (IP) with anti-T7 antibodies conjugated to agarose beads. The immunoprecipitates were then subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies (FIG. 8B).

[0073] The results demonstrated that all of the deletion mutant forms of NIK and T7-IKKα were comparably expressed in these transfection experiments (FIG. 8A). The results also demonstrated that the kinase-deficient NIK mutant (Myc-NIK-KK429/430A) interacts with IKKα, as shown by the coimmunoprecipitation results (FIG. 8B, lane 2). Furthermore, the results demonstrated that none of the C-terminus deletion mutant forms of NIK assembled with IKKα (FIG. 8B, lanes 3-7), while all of the N-terminus deletion mutant proteins, including N735-947, effectively associated with IKKα (FIG. 8B, lanes 8-11).

EXAMPLE 7

[0074] Expression of the C-terminal 213-amino-acid Segment of NIK Interrupts the Assembly of NIK and IKKα.

[0075] To determine if expression of the 213-amino-acid C-terminal fragment of NIK (N735-947) is sufficient to disrupt the interaction of NIK and IKKα, cells were cotransfected with Myc-NIK and IKKα-T7, and with either Myc-N735-947 or Myc-N1-220. The Myc-N1-220 construct expresses a comparably-sized peptide containing the N-terminus of NIK (residues 1 to 220), and was used as a control in this experiment. 293 cells were cotransfected using the method described in the Material and Methods section below. Briefly, the immunoblotting experiment was performed as follows. 24 hours after transfection, cell lysates were prepared from the transfected cultures. Aliquots of the cell lysates were subjected to immunoprecipitation (IP) with anti-T7 antibodies conjugated to agarose beads. The immunoprecipitates were then subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies (FIG. 9A). Aliquots of the cell lysates were subjected to SDS-PAGE and immunoblotted with anti-Myc antibodies to assess the expression of the individual proteins (FIG. 9B).

[0076] The results demonstrated that the N-terminus deletion NIK mutant, containing NIK residues 735 through 947 (Myc-N735-947) effectively blocked the physical association of IKKα (IKKα-T7) with NIK (Myc-NIK) (FIG. 9A, lane 3), since a NIK/IKKα complex did not immunoprecipitate when the N-terminal fragment of NIK was coexpressed. The results also demonstrated that full-length NIK (Myc-NIK) and the two NIK fragments (Myc-N1-220 and Myc-N735-947) were comparably expressed in these transfected cultures (FIG. 9B). Furthermore, the results demonstrated that the N-terminal fragment of NIK (Myc-N1-200) did not block NIK/IKKα association.

[0077] Collectively, the results presented in FIGS. 8 and 9 show that the C-terminus of NIK, containing amino acids 735 through 945, corresponds to a binding domain for IKKα that is both necessary and sufficient for NIK binding to IKKα. Additionally, in TNFα signaling assays, the C-terminal fragment of NIK prevents effective assembly of NIK and IKKα, resulting in inhibition of NF-κB-dependent activation.

EXAMPLE 8

[0078] Expression of the C-terminal 213 Amino Acid Segment of NIK Interrupts CD3-CD2 Costimulatory Signaling

[0079] The activation of T cells requires two signals involving binding of antigen to the T cell receptor and the binding of B7 ligands to CD28 receptors on T cells. Binding of these receptors promotes the activation of L-2 gene expression which is dependent on NF-κB/c-Rel induction. Binding of these receptors includes binding of the receptor to its cognate ligand, or ligation of the receptor with antibody. Our recent studies now demonstrate that the NIK 735-947 effectively inhibits CD3-CD28 mediated costimulation of a composite enhancer element present in the IL-2 gene (FIG. 11). In contrast, a similarly sized N-terminal peptide, NIK 1-200, was without effect. These findings indicate that the C-terminal domain of NIK forms not only an effective inhibitor of TNFα stimulation, but also of immune activation mediated through the T cell receptor CD3 complex and the CD28 co-receptor.

[0080] Material and Methods

[0081] Expression Vector, Biological Reagents, and Cell Lines

[0082] Plasmid κB-TATA-luciferase has been previously described (S. -C. Sun et al 1996). The LacZ reporter construct containing the Rous sarcoma virus long terminal repeat (6RZ) was obtained from D. Pearce (University of California, San Fransisco).

[0083] The full-length NIK cDNA was generated in three fragments by reverse transcription-PCR using Jurkat E6-1 mRNA and three pairs of primers (see Lin et al 1998 for sequence of primers) based on the sequence published by Malinin et al (Malinin et al 1997). The PCR fragments were subcloned into pBluescript, and the full-length NIK cDNA was reconstituted by sequentially subcloning the fragments into the pRK vector (provided by Allan Hall, University College London) in frame with an N-terminal Myc epitope tag. The resulting full-length NIK cDNA was designated pRK-Myc-NIK. The full-length NIK cDNA was further subcloned into pEV3S in frame with a C-terminal T7 epitope tag and was designated pEV-NIK-T7. The two kinase domain NIK mutants, NIK-KK429/430AA and NIK-S549A/T552A/T559A, were generated by overlapping PCR. Murine IKKα/CHUK has been described previously (Connelly and Marcu 1995) and was subcloned into pEV3S in frame with a C-terminal T7 epitope to generate pEV-IKKα-T7. Recombinant human TNFα was purchased from Endogen (Cambridge, Mass.) HeLa and 293 cells were maintained in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin.

[0084] Transfections and Reporter Gene Assays

[0085] 293 cells (5×105 cells/well) were seeded into six-well (35 mm diameter) plates and transfected the following day with 4 μg of DNA by the calcium phosphate precipitation method (Sambrook, Fritz and Maniatis 1989). Cells were co-transfected with plasmid κB-TATA-luciferase (S. -C. Sun et al 1996), the LacZ reporter construct containing the Rous sarcoma virus long terminal repeat (6RZ) (obtained from D. Pearce University of California, San Francisco), and an expression vector (pEV3S) containing wild-type or mutant NIK subcloned in-frame with a C-terminal T7 epitope tag. All transfections included the 6RZ plasmid to normalize for differences in gene transfer efficiency by assay of β-galactosidase activity. The mutant NIKs were generated by overlapping PCR. After 15 to 20 hours, selected cultures were stimulated with TNFα (20 ng/ml) for 6 hours. Luciferase activity was typically measured 20 to 25 hours after transfection using the enhanced luciferase assay kit and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, Mich.). Data were presented as fold induction of luciferase activity ± the standard deviations derived from independent triplicate transfections.

[0086] Immunoprecipitation

[0087] Immunoprecipitation was performed by lysing the transfected cells 24 hours later in ELB buffer containing 1.5% Nonidet P-40, 250 mM NaCl, 50 mM HEPES (pH=7.4), 1 mM EDTA, and the following protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml antipain, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 0.5 μg/ml pepstatin, 7.5 μg/ml bestatin, 4 μg/ml phosphoroamidon, and 5 μg/ml trypsin inhibitor. Lysates were immunoprecipitated with anti-T7-Tag antibody linked to agarose beads (Novagen, Madison, Wis.). Immunoprecipitates were washed three times in lysis buffer and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose membranes and immunoblotting with anti-Myc-Tag antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.).

[0088] Immune Complex Kinase Assays

[0089] For the immune complex kinase assays, transfected cells were lysed 12 to 18 hours posttransfection, and immunoprecipitated with antibodies and protein A-agarose beads, as described above. Immunoprecipitated beads were further washed with kinase buffer containing 10 mM HEPES (pH=7.4), 1 mM MnCl2, 5 mM MgCl2, 12.5 mM β-glycero-2-phosphate, 50 μM Na3VO4, 2 mM NaF, and 50 μM dithiothreitol. After suspension in 20 μl of kinase buffer, the immunoprecipitates were incubated with 5 μCi of [γ-32P]ATP (6000 Ci/mmol) with or without 1 μg of recombinant glutathione S-transferase (GST)-IκBα (1-62) as an exogenous substrate for 30 minutes at 30° C. The reaction was terminated by the addition of SDS sample buffer. The samples were analyzed by SDS-PAGE, followed by transfer to nitrocellulose membranes and exposure to Hyperfilm MP (Amersham Life Sciences). The membranes were subsequently probed with antibodies to determine the amount of immunoprecipitated quinces.

[0090] In vivo Radiolabeling with 32P-labeled Orthophosphoric Acid

[0091] 24 hours after transfection, the cells were washed once in phosphate-free Dulbecco modified Eagle medium (Life Technologies) supplemented with 10% dialyzed, heat-inactivated fetal bovine serum and starved for 1 hour in the same medium. 32P-labeled orthophosphoric acid (0.5 mCi) was then added to the cells. After incubation for 2 hours, the cells were lysed in ELB buffer and immunoprecipitated with anti-Myc-Tag antibody and protein A-agarose as described above. Immunoprecipitates were analyzed by SDS-PAGE, followed by transfer to nitrocellulose membranes and exposure to Hyperfilm MP. Immunoblotting was performed with anti-Myc-Tag antibodies to assess the levels of immunoprecipitated NIK proteins.

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	Number	Date	Country
Parent	09257703	Feb 1999	US
Child	09871889	Jun 2001	US

Novel inhibitor of the inflammatory response induced by TNF-alpha and IL-1

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