Shao-Cong Sun, Ph.D. - US grants

Adult T-cell leukemia (ATL) is a very aggressive and often fatal T-cell malignancy that is caused by infection of the type I human T-cell leukemia virus (HTLV-I). The HTLV-I encoded Tax protein appears to play a central role in the initiation of this virally induced T-cell malignancy. Tax acts by activating cellular transcription factors, including members of the NF- 1kappaBalpha/Rel family, which in turn induces various cellular genes involved in lymphocyte activation and growth. In resting T cells, NF- kappaB/Rel factors are sequestered m the cytoplasm by various inhibitory proteins including I-kappaB-alpha, a 37 kD protein containing multiple ankyrin-like repeats. HTLV-I Tax expression results in the constitutive nuclear expression of these kappaB-enhancer binding proteins, leading to the deregulated expression the various cellular growth-related genes, which have been proposed to induce the polyclonal T-cell proliferation, a prelude to the establishment of ATL. Our recent studies suggest that Tax activation of NF-kappaB is associated with the phosphorylation and degradation of I-kappaB-alpha and the activated nuclear NF-kappaB seems, together with other yet to be identified transcriptional enhancers or silencers, to mediate the Tax-induced transcriptional induction of the c- rel proto-oncogene. We have further observed that these Tax-induced cellular events can be blocked by various inhibitors of cellular signal transduction. Together, these results raise the possibility that Tax may induce a specific cellular signal transduction pathway leading to a cascade of both cytoplasmic and nuclear reactions with phosphorylation of IkBa serving as a molecular trigger. This specific action of Tax results in the nuclear expression of both NF-kB and c-Rel. Based on these results, the overall objective of this grant proposal is a fundamental understanding of the Tax-mediated NF-kappaB/Rel induction signaling pathway. To approach this overall objective, a set of biochemical studies will be undertaken to precisely map the sites of Tax-induced phosphorylation within I-kappaB-alpha. In turn, site-directed mutations will be sequentially introduced into these sites to examine the functional significance of these phosphorylation events on Tax-mediated I-kappaB- alpha degradation. In conjunction with these site-directed mutagenesis, progressive deletional analyses will also be performed to completely define the sequences within I-kappaB-alpha that are required for protease attack. To explore the Tax-mediated signaling pathway, different strategies will be used to identify and characterize the cellular molecular components, including I-kappaB-alpha-specific kinases and the more proximal signaling molecules, involved in the Tax-mediated activation of NF-kappaB/Rel. Finally, the precise mechanism of Tax-induced transcriptional induction of c-rel gene will be explored by isolation and functional analyses of the human c-rel gene promoter.

Adult T-cell leukemia (ATL) is a very aggressive and often fatal T-cell malignancy that is caused by infection of the type I human T-cell leukemia virus (HTLV-I). The HTLV-I encoded Tax protein appears to play a central role in the initiation of this virally induced T-cell malignancy. Tax acts by activating cellular transcription factors, including members of the NF- 1kappaBalpha/Rel family, which in turn induces various cellular genes involved in lymphocyte activation and growth. In resting T cells, NF- kappaB/Rel factors are sequestered m the cytoplasm by various inhibitory proteins including I-kappaB-alpha, a 37 kD protein containing multiple ankyrin-like repeats. HTLV-I Tax expression results in the constitutive nuclear expression of these kappaB-enhancer binding proteins, leading to the deregulated expression the various cellular growth-related genes, which have been proposed to induce the polyclonal T-cell proliferation, a prelude to the establishment of ATL. Our recent studies suggest that Tax activation of NF-kappaB is associated with the phosphorylation and degradation of I-kappaB-alpha and the activated nuclear NF-kappaB seems, together with other yet to be identified transcriptional enhancers or silencers, to mediate the Tax-induced transcriptional induction of the c- rel proto-oncogene. We have further observed that these Tax-induced cellular events can be blocked by various inhibitors of cellular signal transduction. Together, these results raise the possibility that Tax may induce a specific cellular signal transduction pathway leading to a cascade of both cytoplasmic and nuclear reactions with phosphorylation of IkBa serving as a molecular trigger. This specific action of Tax results in the nuclear expression of both NF-kB and c-Rel. Based on these results, the overall objective of this grant proposal is a fundamental understanding of the Tax-mediated NF-kappaB/Rel induction signaling pathway. To approach this overall objective, a set of biochemical studies will be undertaken to precisely map the sites of Tax-induced phosphorylation within I-kappaB-alpha. In turn, site-directed mutations will be sequentially introduced into these sites to examine the functional significance of these phosphorylation events on Tax-mediated I-kappaB- alpha degradation. In conjunction with these site-directed mutagenesis, progressive deletional analyses will also be performed to completely define the sequences within I-kappaB-alpha that are required for protease attack. To explore the Tax-mediated signaling pathway, different strategies will be used to identify and characterize the cellular molecular components, including I-kappaB-alpha-specific kinases and the more proximal signaling molecules, involved in the Tax-mediated activation of NF-kappaB/Rel. Finally, the precise mechanism of Tax-induced transcriptional induction of c-rel gene will be explored by isolation and functional analyses of the human c-rel gene promoter.

DESCRIPTION (Adapted from Investigator's Abstract): The long-range goal of this laboratory is to understand how signals initiated from the TCR and CD28 are integrated and transduced to downstream transcription factors. This knowledge is important for rational development of strategies and therapies to modulate immune responses in the treatment of cancer and various immunological disorders such as autoimmune diseases. The objective of this application is to elucidate the signaling function of a recently cloned MAP3K, NIK, in transducing the T cell co-stimulatory signals. Preliminary studies demonstrate, by both genetic and biochemical approaches, that NIK plays a critical role in T cell co-stimulation. It was shown that NIK is essential for TCR/CD28- mediated activation of different transcription factors, including NFkB and AP-1, both serving as key regulators of a CD28-responsive IL-2 gene enhancer, CD28RE/AP-1. This finding indicates that NIK may serve as a key signaling component transducing the TCR/CD28 signals to different downstream signaling pathways. As seen with many signaling molecules, NIK contains a number of structural motifs known to mediate protein- protein interactions. The central hypothesis to be tested is that NIK interacts with multiple signaling molecules in the T cell co-stimulatory pathway, mediating activation of different downstream kinases and transcription factors. There are three specific aims: (1) Investigate the signaling mechanisms through which NIK regulates downstream transcription factors; (2) Define the structural basis for the signaling function of NIK; and (3) Clone and characterize novel NIK-interacting proteins. At the completion of the project, it is expected that it will be known how NIK functions in signal transduction pathways connecting the TCR/CD28 signals to activation of downstream CD28RE/AP-1 regulatory factors. It is also expected that novel signaling molecules will have been isolated which function in the T cell co-stimulatory pathway.

The long-range goal of this research project is to understand how HTLV-I Tax protein deregulates the cellular NF-kappaB transcription factor pathway. This knowledge is critical for understanding how HTLV-I induces human T-cell transformation and, thus, is important for rational development of anti-HTLV therapies. Our studies performed during the current granting period demonstrate that Tax activates NF-kappaB by inducing phosphorylation and subsequent degradation of the NF-kappaB inhibitor IkappaB. Tax triggers IkappaB phosphorylation by stimulating the activity of a cellular IkappaB kinase (IKK), which is assembled in a large (greater than 700kD), multicomponent complex named IKK signalsome. The overall objective of the studies proposed in this continuation application is to elucidate the mechanisms by which Tax persistently activates the IKK signaling machinery. As will be presented under Progress Report, we have recently demonstrated that Tax physically interacts with the IKK complex via direct binding to the IKK regulatory subunit, IKKgamma. The IKKgamma- directed Tax/IKK association occurs in both transfected and HTLV- I infected T cells and is correlated with persistent activation of IKK. Additional studies demonstrate that Tax activation of IKK also requires upstream kinases. Interestingly, one of the putative IKK upstream kinases, Cot, physically interacts with Tax. These findings indicate that Tax may stimulate the chronic activity of IKK by forming stable complexes with both IKK and its upstream activators. We have also found that Tax-stimulated IKK activation is associated with phosphorylation of the IKK subunits, a modification that often triggers the catalytic activity of protein kinases. Thus, the central hypothesis to be tested is that Tax stimulates persistent activity of IKK via a mechanism involving its physical interaction with IKK and upstream activators and phosphorylation-dependent IKK activation. To accomplish the objective of this application, we will pursue four specific aims: (1) determination of the biochemical mechanism and functional significance of Tax/IKKgamma physical interaction in Tax activation of IKK; (2) investigation of the role of Cot and other upstream kinases in Tax-mediated IKK activation; (3) dissecting the molecular components of the IKK signalsomes in normal versus HTLV1-infected T cells; and (4) determination of the functional significance of IKK subunit phosphorylations in Tax-stimulated IKK activation.

DESCRIPTION (provided by applicant): Signal-induced processing of the nfkb2 gene product, pl00, is a critical mechanism of NF-kB regulation. The full-length pl00 functions as a potent inhibitor of NF-kB, sequestering various NF-kB members in the cytoplasm. Upon processing, the C-terminal half of pl00 is degraded by the proteasome, leading to generation of an active NF-kB component, p52, which is required for peripheral B cell growth and function and the formation of germinal centers in lymphoid organs. Emerging evidence suggests that defect in p52 generation causes deficiencies in humeral immune responses, while overproduction of p52 or the loss of intact pl00 is associated with abnormal lymphocyte growth and development of lymphoid malignancies. Since the processing of pl00 serves to both generate p52 and interrupt the inhibitory function of pl00, deregulation of this proteolytic event may have profound effect on lymphocyte growth and function. The overall objective of this application is to understand the molecular mechanism regulating pl00 processing. This knowledge is important for rational development of strategies and therapies to modulate immune responses and treat lymphoid malignancies. We have recently shown that the processing of pl00 is tightly suppressed by its C-terminal sequences and that the active pl00 processing can be induced through its phosphorylation and ubiquitination. Based on these findings, we hypothesize that the signal for constitutive pl00 processing is normally masked by its negative-regulatory sequences and that the inducible processing of pl00 is triggered by its site-specific phosphorylation and ubiquitination. To accomplish the objective of this application, we will pursue four specific aims: (i) define the negative- and positive-regulatory sequences of pl00 processing; (ii) identify and characterize cellular factors regulating the processing of pl00; (iii) investigate how the GRR regulates pl00 processing; (iv) investigate pl00 processing in vivo using transgenic mice.

DESCRIPTION (provided by applicant): Our long-range goal is to dissect the signaling pathways mediating microbial induction of inflammatory mediators in macrophages. This knowledge is important for rational design of strategies to modulate immune responses against infections and to treat inflammation and septic shock. The overall objective of this application is to elucidate the molecular mechanisms regulating the signaling function of a protein kinase, Tpl2. Recent genetic studies have revealed a central role for Tpl2 in mediating macrophage activation by lipopolysaccharide (LPS), a potent inflammatory elicitor from Gram-negative bacteria. However, how the function of Tpl2 is regulated remains unknown. The studies proposed in this grant application are based on a large body of preliminary data generated by the applicants. We have demonstrated that in macrophages, Tpl2 forms a latent complex with the nfkbl gene product p105. Genetic and biochemical evidence suggests that both the stability and kinase function of Tpl2 are tightly controlled by its partner p105. Interestingly, LPS-stimulated Tpl2 activation is correlated with its phosphorylation and release from p105. We have further observed that LPS also stimulates the degradation of Tpl2, which may serve as a negative feedback mechanism to prevent uncontrolled activation of this key inflammatory mediator. Based on these findings, the central hypothesis to be tested is that the signaling function of Tpl2 is regulated by both positive and negative mechanisms, which involve its modification by upstream signals and dynamic interaction with p105. To accomplish the objective of this application, we will pursue four specific aims: (1) define the biochemical mechanisms mediating Tpl2 activation; (2) investigate how Tpl2 is targeted for degradation; (3) determine the mechanisms by which p105 regulates the stability and function of Tpl2; and (4) identify upstream signaling molecules involved in Tpl2 activation. At the completion of this research, we expect to have determined how the signaling function of Tpl2 is positively and negatively regulated in the LPS signaling cascade. We also expect to have isolated novel signaling molecules participating in this important innate immune response.

[unreadable] DESCRIPTION (provided by applicant): CYLD is a recently identified deubiquitinating enzyme (DUB) that negatively regulates the signaling events mediated by immune receptors, such as TNF receptors (TNFRs). To understand the physiological function of CYLD, we have generated CYLD knockout mice and undertaken extensive characterization analyses. These genetic studies have revealed a critical role for CYLD in regulating T-cell development and activation. CYLD-deficient mice have a severe defect in generating CD4 and CD8 mature thymocytes, resulting in more than 50% reduction in peripheral T-cell numbers. Interestingly, despite their lower numbers, the CYLD-/- peripheral T cells are hyper-responsive to TCR stimulation. These findings suggest that CYLD plays critical but distinct roles in regulating thymocyte development and peripheral T-cell activation. The overall objective of this revised application is to understand the molecular mechanism by which CYLD regulates T-cell development and activation. As a critical step towards achieving this objective, we have identified the protein tyrosine kinase LCK as a specific target of CYLD. CYLD physically interacts with LCK in thymocytes in response to TCR stimulation and inhibits the ubiquitination of LCK. CYLD regulates the inducible binding of LCK to its target ZAP-70, thereby participating in TCR-proximal signaling events. These findings provide an important insight into the mechanism by which CYLD positively regulates thymocyte development. Since CYLD negatively regulates peripheral T-cell activation, these findings also raise a number of intriguing questions. Does CYLD possess opposing functions in regulating TCR signaling of developing and peripheral T cells? Does CYLD negatively regulate T-cell costimulatory receptors, especially TNFR family members? Does CYLD serve as an intrinsic negative regulator of peripheral T cells or act indirectly through regulating thymocyte development? Another important question is how the signaling function of CYLD is regulated. In this regard, we have shown that CYLD is phosphorylated along with the activation of both thymocytes and peripheral T cells, thus suggesting the intriguing possibility that the signaling function of CYLD is subject to regulation by its phosphorylation. We will perform three specific aims to address these questions and to achieve our overall objective. (1) Characterize the molecular mechanism by which CYLD regulates thymocyte development and TCR signaling. (2) Examine how CYLD negatively regulates peripheral T-cell activation and whether the CYLD deficiency causes autoimmunity. (3) Investigate the biochemical mechanism and functional significance of CYLD phosphorylation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The noncanonical NF-?B signaling pathway mediates activation of specific NF-?B members, p52 and RelB, which regulate important biological functions such as osteoclastogenesis, lymphoid organogenesis, lymphocyte development and activation, and generation of immunological tolerance. This novel NF-?B pathway relies on processing of the p52 precursor protein, p100. Since p100 functions as a cytoplasmic inhibitor of RelB, the p100 processing not only generates p52 but also causes nuclear translocation of active p52/RelB NF-?B complex. Over the past few years, we have made seminal pioneer findings demonstrating that the processing of p100 is tightly regulated through its site-specific phosphorylation by a novel kinase complex composed of NIK and IKK1. Our work also reveals the aberrant p100 processing in leukemia T cells transformed by the human T-cell leukemia virus (HTLV). The overall objective of this continuation application is to elucidate the molecular mechanisms mediating normal and deregulated noncanonical NF-?B signaling. The proposed project is based on strong preliminary data and published work from our laboratory. In particular, our recent work suggests a novel mechanism of NIK regulation, which appears to involve its dynamic interaction with a negative regulator, TRAF3, and modulation of its expression level. Intriguingly, induction of noncanonical NF-?B signaling is associated with TRAF3 degradation, although how this intermediate signaling step is regulated remains unclear. Our preliminary studies also reveal the involvement of novel regulators of noncanonical NF-?B signaling. Moreover, we have made significant progress towards understanding the pathological activation of noncanonical NF-?Bs by the leukemia virus HTLV. Based on these findings, we hypothesize that noncanonical NF-?B signaling is tightly controlled by negative and positive regulators, the deregulation of which may contribute to both immunological disorders and HTLV-induced T-cell malignancies. We will perform three specific aims to examine our hypotheses. (1) Elucidate the biochemical mechanisms that regulate the signaling function of NIK. (2) Characterize the intermediate signaling steps and molecular components of the noncanonical NF-?B pathway. (3) Investigate the role of noncanonical NF-?B pathway in normal and pathological T-cell activation. PUBLIC HEALTH RELEVANCE: The NF-?B family of transcription factors regulates diverse biological processes, most notably immune and inflammatory responses. The focus of this research project is to understand a noncanonical (or atypical) signaling pathway of NF-?B activation. This pathway leads to activation of a sub-group of NF-?B members and is required for specific adaptive immune functions, including lymphoid organ formation, lymphocyte development and activation. Uncontrolled noncanonical NF-?B signaling is linked to chronic inflammation and autoimmunity, whereas defect in this pathway causes immune deficiencies. The studies proposed in this application address the molecular mechanisms of noncanonical NF-?B signaling and will be important for the development of new and effective immune therapies.

DESCRIPTION (provided by applicant): T cells effectively respond to foreign antigens but are tolerant to self-tissues and normal enteric flora. Inappropriate activation or differentiation of T cells can lead to severe immunological disorders, including autoimmunity and inflammation. Thus, a better understanding of the molecular mechanisms regulating T-cell activation and tolerance is important for rational design of therapies for immunological diseases. The overall objective of this application is to understand the molecular and cellular mechanisms by which a newly identified deubiquitinase (DUB), CYLD, regulates T-cell function and autoimmune inflammation. During the current funding period, we have made seminal findings that establish CYLD as a pivotal regulator of T-cell activation and autoimmune inflammation. CYLD-deficient T cells are hyper-responsive to TCR stimulation, and the CYLD knockout (CYLD-/-) mice spontaneously develop intestinal inflammation with major features of human inflammatory bowel disease (IBD). The CYLD-/- mice also experience severe bone loss, which is known as a major extra-intestinal complication of IBD and animal model of colitis. Moreover, our preliminary studies reveal that loss of CYLD renders mice hypersensitive to the induction of experimental autoimmune encephalomyelitis (EAE). Thus, CYLD is a master regulator of T-cell function and autoimmune inflammatory diseases. The studies proposed in this continuation application are based on strong preliminary and published data from our laboratory. In particular, we have shown that loss of CYLD in T cells causes constitutive activation of NF-kB, a transcription factor mediating T-cell activation and survival and being involved in many inflammatory disorders. The CYLD-/- T cells also display marked attenuation of the costimulatory molecule ICOS, which is crucial for the induction of T-cell tolerance and regulation of T-cell differentiation. Consistent with these molecular studies, we have found that the CYLD-/- T cells appear to be defective in tolerance to enteric microbes, since their adoptive transfer into lymphocyte-deficient Rag1-/- mice induces severe colitis. Furthermore, the CYLD-/- mice produce aberrantly high levels of inflammatory Th17 and Th1 cells, coupled with heightened EAE response. We have obtained preliminary evidence that CYLD regulates macrophage response to TLR-stimulated expression of a specific subset of cytokines known to regulate Th1 and Th17 differentiation. Based on these findings, we hypothesize that CYLD regulates key signaling events involved in T-cell activation and differentiation as well as tolerance induction. We will perform three specific aims to accomplish our overall objective. (1) Examine the molecular mechanism and functional significance of CYLD-mediated NF-kB regulation in T cells. (2) Characterize the molecular and cellular mechanisms by which CYLD regulate T-cell tolerance and inflammatory T-cell differentiation. (3) Examine the immunological and osteoclast-intrinsic mechanisms by which CYLD regulates bone erosion.

DESCRIPTION (provided by applicant): Recognition of microbes by Toll-like receptors (TLRs) triggers the induction of proinflammatory cytokines and other immune mediators, which serves as an important innate immune mechanism against infections. However, uncontrolled secretion of inflammatory mediators causes chronic inflammatory diseases or even a devastating acute illness, septic shock. The long-range goal of this project is to dissect the signaling pathways involved in TLR-mediated induction of inflammatory mediators. This knowledge is important for rational design of more effective anti-inflammatory therapies. Over the past few years, we have made seminal findings demonstrating a pivotal TLR signaling axis: the IKK/Tpl2 axis. IKK is known as a kinase that activates the transcription factor NF-(B by mediating phosphorylation and degradation of the NF-(B inhibitor I(B, whereas Tpl2 is a MAP3K that activates the MAP kinase ERK through phosphorylating the ERK kinase, MEK1. Interestingly, Tpl2 is sequestered by an I(B-like molecule, p105, and the activation of Tpl2 requires IKK-mediated p105 phopshorylation and degradation. Thus, the IKK/Tpl2 signaling axis controls two major TLR pathways, the NF-(B and ERK pathways. The overall objective of this continuation application is to understand how IKK regulates Tpl2 and how the IKK/Tpl2 signaling axis is activated by upstream TLR signals. The proposed project is based on strong preliminary data from our laboratory. In particular, our studies have revealed the involvement of IKK/Tpl2 functional interplay and novel signaling factors in TLR-stimulated p105 degradation and Tpl2 activation. We have further shown that in addition to mediating Tpl2 activation, IKK controls the fate of activated Tpl2, a function that may prevent prolonged Tpl2 activation. Additionally, we made significant progress toward understanding the mechanism that connects the IKK/Tpl2 axis to upstream TLR signals. Our genetic evidence suggest that a recently identified E3 ubiquitin ligase, Pellino1, mediates IKK/Tpl2 activation by specific TLRs. Together, these innovative preliminary results form a solid foundation for the studies proposed in this application. We will perform three specific aims to accomplish our overall objective. (1) Examine the molecular mechanism of IKK- dependent Tpl2 activation. (2) Examine the role of IKK in regulating the fate of Tpl2. (3) Examine how the IKK/Tpl2 signaling axis is regulated by upstream TLR signals

DESCRIPTION (provided by applicant): Ubiquitination has emerged as a pivotal mechanism that regulates signal transduction in the immune system. Deregulated ubiquitination events are associated with severe immunological disorders, such as autoimmunity and chronic inflammation. A critical component of the ubiquitination system is E3 ubiquitin ligase, a superfamily (more than 600 members) of enzymes that confer specificity of ubiquitination by recognizing substrates. Since each E3 targets a small number of proteins for ubiquitination, characterization of the physiological targets of specific E3s represents a challenging and highly significant task. This information is critical for rational design of therapeutic approaches. The long-range goal of this project is to understand the immunoregulatory functions of a newly identified family of E3 ubiquitin ligases, Peli (also called Pellino). Peli proteins conjugate both lysine (K) 63- and K48-linked polyubiquitin chains, although their in vivo biological functions remains poorly understood. By gene targeting, our preliminary studies and recently published work revealed a critical, and seemingly complex, role for Peli1 in the regulation of immune receptor signaling and autoimmunity. Peli1 negatively regulates T-cell activation and maintains T-cell tolerance, and Peli1 deficiency causes systemic autoimmune symptoms. Paradoxically, the Peli1 knockout (KO) mice are refractory to the induction of experimental autoimmune encephalomyelitis (EAE), an organ-specific autoimmune disease of the central nervous system (CNS). Interestingly, although the Peli1 KO mice have hyper-production of inflammatory T cells in the peripheral lymphoid organs, these immune cells failed to migrate to the CNS. We have obtained genetic evidence that Peli1 is required for innate immune receptor signaling and induction of proinflammatory cytokines and chemokines in the CNS-resident microglial cells. These innovative findings demonstrate a pivotal and paradoxical role for Peli1 in the regulation of T-cell activation and CNS innate immune receptor signaling. Elucidation of the underlying mechanism is highly important for therapeutic approaches. Thus, the overall objective of this grant application is to understand how Peli1 exerts its immunoregulatory functions. Our hypothesis is that Peli1 targets different signaling factors for ubiquitination, thereby regulating both innate immune cell activation and T-cell tolerance. To achieve our overall objective, we will (1) examine how Peli1 regulates T-cell activation and tolerance; (2) examine how Peli1 regulates innate immune receptor signaling and CNS inflammation; and (3) elucidate the biochemical mechanisms regulating the activation and function of Peli1.

? DESCRIPTION (provided by applicant): Signal transduction from toll-like receptors (TLRs) mediates innate immune cell activation in host defense against infections; however, deregulated TLR signaling also contributes to the development of inflammatory diseases. The long-range goal of this research project is to understand the molecular mechanisms regulating TLR signaling in macrophages, a major type of innate immune cells involved in the regulation of immune responses, inflammation, as well as tumor microenvironment. During the previous funding cycles, the PI's laboratory has made seminal discoveries in this area. Moreover, we have generated a large body of innovative preliminary data that form a solid foundation for this continuation application. In particular, our preliminary studies identified a novel TLR signaling mediator, Zranb1 (also called Trabid). Zranb1 is a deubiquitinase with poorly defined physiological functions due to the lack of an animal model. Using newly generated Zranb1 conditional KO mice, we found that Zranb1 deficiency in macrophages specifically attenuates TLR-stimulated expression of a subset of proinflammatory cytokines, including IL-12 and IL-23. We have obtained preliminary evidence that Zranb1 regulates the fate of c-Rel, a transcription factor involved in IL-12/IL-23 gene induction. Along the same line, our preliminary studies led to the unexpected observation that a TRAF family member, TRAF2, serves as a pivotal negative regulator of the proinflammatory axis of TLR signaling. TRAF2 aberrant expression is associated with human inflammatory bowel disease (IBD), but it has been unclear whether TRAF2 positively or negatively regulates colon inflammation. By generating myeloid cell-conditional Traf2 KO (Traf2-MKO) mice, we demonstrated that TRAF2 ablation greatly promotes TLR-stimulated proinflammatory cytokine expression in macrophages and sensitizes mice for colitis induction in an animal model of IBD. We further showed that TRAF2 deficiency in macrophages leads to marked accumulation of c-Rel and IRF5, major transcription factors mediating induction of proinflammatory cytokines. Interestingly, our data suggest the regulation of c-Rel and IRF5 by a novel ubiquitin/proteasome-dependent mechanism relying on both TRAF2 and a related TRAF member, TRAF3. Collectively, these findings establish TRAF2 and Zranb1 as pivotal regulators of TLR signaling and highlight a novel signaling mechanism. Our hypothesis is that TRAF2 and Zranb1 regulate the proinfammatory axis of TLR signaling through controlling the fate of important signaling molecules, including c-Rel and IRF5. To test this hypothesis, we will (1) elucidate the mechanism by which TRAF2 regulates TLR signaling in macrophages; (2) define the molecular mechanism by which Zranb1 mediates TLR signaling; and (3) investigate the in vivo pathophysiological functions of TRAF2 and Zranb1 in myeloid cells. We believe that the proposed project addresses a unique and novel aspect of TLR signaling and will yield innovative results that substantially advance the field.

? DESCRIPTION (provided by applicant): Ubiquitination is a crucial mechanism that regulates T-cell activation and T cell-mediated immune responses, and deregulated ubiquitination events are linked to human immunological disorders including autoimmunity and inflammation. A well-known function of ubiquitination is to control T cell receptor (TCR)-proximal signaling events by mediating ubiquitin-dependent degradation of CD3¿ and key signaling factors. Ubiquitination also regulates the fate or activation of downstream signaling molecules in the TCR pathway, in a positive or negative manner. Like phosphorylation, ubiquitination is a reversible process, in which ubiquitin chains are conjugated and deconjugated by ubiquitinating enzymes (E1, E2, E3) and deubiquitinases (DUBs), respectively. Despite the extensive studies of E3 ubiquitin ligases, the DUBs that regulate TCR signaling and T-cell functions are still poorly defined. The PI's laboratory has been in a leading position in this research area. During the previous funding cycles, we have made seminal findings demonstrating a crucial role for the DUB CYLD in regulating T-cell activation and autoimmune inflammation. Moreover, our recently published work and preliminary studies have led to the identification of several novel T cell-regulatory DUBs, which forms a solid foundation for this renewal application. Using biochemical and mouse genetic approaches, we have identified Otud7b as a DUB that positively regulates TCR-proximal signaling. Otud7b deficiency attenuates the activation of the TCR-proximal kinase Zap70 as well as multiple downstream signaling factors. Interestingly, in response to TCR/CD28 stimulation, Otud7b is rapidly recruited to CD3¿ and appears to regulate the stability of this TCR signaling chain. Based on these findings, we hypothesize that Otud7b may facilitate TCR signaling by regulating the ubiquitination of CD3¿ and possibly key TCR-proximal signaling factors. Our studies also led to the identification of two homologous DUBs of the USP family, Usp4 and Usp15, as negative regulators of T-cell activation. Interestingly, despite the strong homology of Usp4 and Usp15, genetic ablation of either of them causes hyper-activation of T cells. This finding raises the question of how these two highly homologous DUBs exert non-redundant signaling functions and whether they also have redundancies in some signaling functions. The overall objective of this continuation application is to understand the molecular mechanisms by which Otud7b and Usp4/Usp15 regulate TCR signaling and T cell-mediated immunity and autoimmune inflammation. To accomplish our overall objective, we will (1) define the molecular mechanism by which Otud7b mediates TCR signaling, (2) examine how Usp4 and USP15 negatively regulate TCR signaling, and (3) investigate in vivo functions of Otud7b and Usp4 in regulating T-cell function and autoimmune inflammation. We believe that these proposed studies are highly innovative and will yield high-impact findings that lead to a major advancement of the field. With the establishment of newly generated mouse models and our extensive experience, we are in a unique position to carry out the proposed project.

Project Summary/Abstract The NF-?B family of transcription factors regulates diverse biological processes, including immune and inflammatory responses. NF-?B activation involves two major pathways: the canonical and noncanonical pathways. A central step in the noncanonical pathway is ubiquitin-dependent processing of the NF-?B2 precursor protein p100, which generates mature NF-?B2 p52 and cause nuclear translocation of the p100- sequestered noncanonical NF-?B members, p52 and RelB. During the previous funding cycles, the PI's laboratory has made pivotal contributions to the advancement of this relatively new field, including discovery of NIK as a central signaling component and identification of TRAF3 as a primary negative regulator that controls the fate of NIK. It is now known that TRAF3 functions as the substrate binding subunit of a NIK-specific E3 ubiquitin ligase complex, TRAF3-TRAF2-cIAP. Despite these progresses, our knowledge on noncanonical NF- ?B signaling is still quite limited. In particular, only few regulators of this pathway have been characterized, and the function of noncanonical NF-?B in different types of immune cells is poorly defined. The overall objective of this continuation application is to characterize novel regulators and functions of the noncanonical NF-?B pathway. The proposed studies are based on innovative preliminary data from the PI's laboratory. Using newly generated NIK-conditional KO mice, we demonstrated a dendritic cell (DC)- specific function of noncanonical NF-?B in regulating mucosal immunity and obtained important clues to the underlying mechanism. To identify novel regulators of the noncanonical NF-?B pathway, we employed a recently developed approach, BioID, to screen for proteins interacting with major components of this pathway. After extensive characterizations, we have identified a TRAF3-binding protein, TFG, and a p100-binding protein, Otub1, which are critically involved in noncanonical NF-?B regulation. TFG is crucial for preventing abnormal NIK accumulation, whereas Otub1 is a deubiquitinase (DUB) that inhibits p100 ubiquitination and processing. Our gene knockdown and KO mouse studies suggest important functions of TFG and Otub1 in immune regulation. These innovative findings form a solid foundation for this continuation application. To accomplish our overall objective, we will (1) elucidate the mechanism by which noncanonical NF-?B functions in DCs to regulate mucosal immunity; (2) characterize novel factors that regulate noncanonical NF-?B signaling; and (3) investigate the immunoregulatory functions of novel noncanonical NF-?B regulators. The PI's laboratory pioneered the discovery of noncanonical NF-?B pathway and has been in a leading position in this area. During the previous funding cycles, we have made seminal discoveries that have been instrumental for the advancement of the filed. We believe that the proposed studies will once again lead to high-impact findings that substantially advance the field. With our extensive experience and the innovative preliminary data, we are in a unique position to carry out the proposed studies.

PROJECT SUMMARY/ABSTRACT Toll-like receptor (TLR) signaling plays a crucial role in mediating innate immunity and, when deregulated, also contributes to the pathogenesis of inflammatory diseases. Better understanding of the molecular mechanisms regulating TLR signaling and inflammatory responses is highly significant for improving the therapeutic approaches in the treatment of inflammatory diseases. During the previous funding cycles, the PI?s laboratory has made seminal discoveries in this area. Moreover, we have generated a large body of innovative preliminary data that form a solid foundation for this continuation application. In particular, our preliminary studies demonstrated a crucial role for the protein kinase, TBK1, in controlling TLR signaling and preventing inflammatory disorders. Although TBK1 is known as a kinase that mediates type I interferon (IFN) induction and antiviral innate immunity, its in vivo functions have been poorly studied due to the lack of a viable mouse model. Using newly generated TBK1 conditional knockout (cKO) mice, we have discovered novel functions of TBK1 in the regulation of immune and inflammatory responses. Our preliminary studies have demonstrated a crucial role for TBK1 in controlling inflammatory responses by functioning in both innate immune cells and intestinal epithelial cells (IECs). Myeloid cell-conditional TBK1 KO (Tbk1-MKO) mice are hypersensitive to colitis induction and spontaneously develop aberrant adipose tissue expansion and inflammation. TBK1 negatively regulates TLR signaling and TLR-stimulated expression of proinflammatory cytokines in macrophages. We have further demonstrated that conditional deletion of TBK1 in IECs increases proinflammatory cytokine production and Th17 cell generation in the intestine, sensitizing mice for intestinal tumorigenesis. Based on these innovative findings, we hypothesize that TBK1 functions in both innate immune cells and IECs to regulate proinflammatory TLR signaling and inflammatory disorders. The overall objective of this continuation application is to elucidate the mechanism underlying the anti-inflammatory functions of TBK1. To accomplish this overall objective, we will perform two specific aims. In Aim 1, we will examine how myeloid cell TBK1 regulates TLR signaling and inflammation. In Aim 2, we will elucidate the mechanism by which TBK1 functions in IECs to regulate intestinal immune homeostasis and tumorigenesis. We believe that these proposed studies address novel mechanisms that regulate TLR signaling and inflammatory responses and will lead to high-impact results that substantially advance the field.

PROJECT SUMMARY/ABSTRACT Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) causes coronavirus disease 2019 (COVID-19), which is characterized by acute inflammation in the lung and other organs, such as the heart and intestine. It is increasingly clear that the pathogenesis of SARS-CoV2 involves suppression of antiviral innate immunity and induction of inflammatory responses. SARS-CoV2 suppresses induction of the antiviral type I interferons (IFNs) and, thereby, escapes from destruction by the early phase antiviral immunity. Subsequent induction of inflammatory responses drives the development of COVID-19. Understanding how SARS-CoV2 suppresses type I IFN expression and induces inflammatory responses, is crucial for designing therapeutic approaches. Based on the studies of SARS-CoV, the close homolog of SARS-CoV2, several viral proteins have been implicated in the interplay with host immune system, contributing to the suppression of type I IFN responses and induction of proinflammatory cytokines. The major goal of this supplementary application is to understand the mechanisms by which SARS-CoV2 proteins suppress antiviral innate immunity and stimulate exacerbated inflammatory responses. We will perform two specific aims. In Specific Aim 1, we will examine how SARS-CoV2 proteins suppress TBK1 signaling and antiviral innate immunity. As described in the parent grant, TBK1 is a kinase that responds to signals from the toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) during viral infections and mediates induction type I IFNs. At the same time, TBK1 negatively regulates proinflammatory cytokine induction to prevent exacerbated inflammation. Our parental grant focuses on the elucidation of how TBK1 regulates TLR signaling and intestinal inflammation caused by gut microbes. In this supplementary application, we will specifically address how SARS-CoV2 proteins modulate TBK1 signaling in the suppression of antiviral immunity and stimulation of inflammation. We will examine our hypothesis that suppression of TBK1 signaling by SARS- CoV2 proteins not only inhibits type I IFN production but also promotes inflammatory responses. In Specific Aim 2, we will systematically define the mechanisms by which SARS-COV2 proteins induce inflammatory responses using both cell culture and mouse models. We will examine the signaling pathways involved in SARS-CoV2-induced expression of proinflammatory cytokines in macrophages and epithelial cells. We will also examine how the Spike protein of SARS-CoV2 downregulates its cellular receptor, angiotensin- converting enzyme 2 (ACE2). Since ACE2 is a pivotal anti-inflammatory factor, we hypothesize that Spike protein-induced ACE2 downregulation critically contributes to the induction of lung and intestinal inflammation. We believe that these proposed studies address novel mechanisms that mediate the pathogenesis of COVID19 and will have important implications for COVID19 therapies.