An Overview of Miami CDEIPI and a Showcase of Team Science and Cutting-Edge Research Driven by Students.

BACKGROUND: The Miami-CFAR Diversity, Equity & Inclusion Pathway Initiative (Miami CDEIPI) is designed to promote a diverse scientific workforce that reflects the communities at the highest risk of HIV in South Florida. SETTING AND METHODS: The focus of the Miami CDEIPI is to help train the next generation of Underrepresented Minorities (URM) and Black, Indigenous, People of Color (BIPOC) in HIV/AIDS-related research through a team science experience. The Miami CDEIPI objectives are to facilitate the interaction of URM/BIPOC students with the network of CFAR-affiliated investigators and to enable these students to access the cutting-edge technologies at the Miami-CFAR and the Sylvester Comprehensive Cancer Center and other resources at the University of Miami. RESULTS: Five URM/BIPOC students supported by the program in year 1 have been carrying out projects in collaboration with mentors at their parent institution and Miami-CFAR investigators. The students used the state-of-the-art laboratories and core facilities. They began their research with a proposal designed to integrate the cutting-edge technologies now available to them. Their training included participation in Miami-CFAR-sponsored activities such as seminars, an annual conference, and a national HIV workshop. Candidates in the Miami CDEIPI are in the process of developing their research proposals, integrating cutting-edge technologies into their doctoral dissertation research. Their projects are now in the completion phase. CONCLUSIONS: The Miami CDEIPI focuses its resources on one of the conspicuous gaps in the career paths of URM/BIPOC researchers-the dearth of leading URM/BIPOC scientists in the field. The Miami CDEIPI provides a professional network that supports the participation of URM/BIPOC trainees in innovative research and career skill training.

LPS modifications and AvrA activity of Salmonella enterica serovar Typhimurium are required to prevent Perforin-2 expression by infected fibroblasts and intestinal epithelial cells.

Cellular Perforin-2 (MPEG1) is a pore-forming MACPF family protein that plays a critical role in the defense against bacterial pathogens. Macrophages, neutrophils, and several other cell types that are part of the front line of innate defenses constitutively express high levels of Perforin-2; whereas, most other cell types must be induced to express Perforin-2 by interferons (α, ß and γ) and/or PAMPs such as LPS. In this study, we demonstrate that many bacterial pathogens can limit the expression of Perforin-2 in cells normally inducible for Perforin-2 expression, while ordinarily commensal or non-pathogenic bacteria triggered high levels of Perforin-2 expression in these same cell types. The mechanisms by which pathogens suppress Perforin-2 expression was explored further using Salmonella enterica serovar Typhimurium and cultured MEFs as well as intestinal epithelial cell lines. These studies identified multiple factors required to minimize the expression of Perforin-2 in cell types inducible for Perforin-2 expression. These included the PmrAB and PhoPQ two-component systems, select LPS modification enzymes and the Type III secretion effector protein AvrA.

Lymphotropic Viruses: Chronic Inflammation and Induction of Cancers.

Inflammation induced by transcription factors, including Signal Transducers and Activators of Transcription (STATs) and NF-κB, in response to microbial pathogenic infections and ligand dependent receptors stimulation are critical for controlling infections. However, uncontrolled inflammation induced by these transcription factors could lead to immune dysfunction, persistent infection, inflammatory related diseases and the development of cancers. Although the induction of innate immunity and inflammation in response to viral infection is important to control virus replication, its effects can be modulated by lymphotropic viruses including human T-cell leukemia virus type 1 (HTLV-1), Κaposi's sarcoma herpesvirus (KSHV), and Epstein Barr virus (EBV) during de novo infection as well as latent infection. These lymphotropic viruses persistently activate JAK-STAT and NF-κB pathways. Long-term STAT and NF-κB activation by these viruses leads to the induction of chronic inflammation, which can support the persistence of these viruses and promote virus-mediated cancers. Here, we review how HTLV-1, KSHV and EBV hijack the function of host cell surface molecules (CSMs), which are involved in the regulation of chronic inflammation, innate and adaptive immune responses, cell death and the restoration of tissue homeostasis. Thus, better understanding of CSMs-mediated chronic activation of STATs and NF-κB pathways in lymphotropic virus-infected cells may pave the way for therapeutic intervention in malignancies caused by lymphotropic viruses.

An Essential Role for Perforin-2 in Type I IFN Signaling.

Type I IFNs play a complex role in determining the fate of microbial pathogens and may also be deleterious to the host during bacterial and viral infections. Upon ligand binding, a receptor proximal complex consisting of IFN-α and -ß receptors 1 and 2 (IFNAR1, IFNAR2, respectively), tyrosine kinase 2 (Tyk2), Jak1, and STAT2 are assembled and promote the phosphorylation of STAT1 and STAT2. However, how the IFNARs proximal complex is assembled upon binding to IFN is poorly understood. In this study, we show that the membrane-associated pore-forming protein Perforin-2 (P2) is critical for LPS-induced endotoxic shock in wild-type mice. Type I IFN-mediated JAK-STAT signaling is severely impaired, and activation of MAPKs and PI3K signaling pathways are delayed in P2-deficient mouse bone marrow-derived macrophages, mouse embryonic fibroblasts (MEFs), and human HeLa cells upon IFN stimulation. The P2 N-glycosylated extracellular membrane attack complex/perforin domain and the P2 domain independently associate with the extracellular regions of IFNAR1 and IFNAR2, respectively, in resting MEFs. In addition, the P2 cytoplasmic tail domain mediated the constitutive interaction between STAT2 and IFNAR2 in resting MEFs, an interaction that is dependent on the association of the extracellular regions of P2 and IFNAR2. Finally, the constitutive association of P2 with both receptors and STAT2 is critical for the receptor proximal complex assembly and reciprocal transphosphorylation of Jak1 and Tyk2 as well as the phosphorylation and activation of STAT1 and STAT2 upon IFN-ß stimulation.

CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation.

Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8) is one of the oncogenic viruses responsible for human cancers, including Kaposi's sarcoma (KS), Primary Effusion Lymphoma (PEL), and the lymphoproliferative disorder multicentric Castleman's disease (MCD). Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1), is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.

TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS.

The host response to RNA virus infection consists of an intrinsic innate immune response and the induction of apoptosis as mechanisms to restrict viral replication. The mitochondrial adaptor molecule MAVS plays critical roles in coordinating both virus-induced type I interferon production and apoptosis; however, the regulation of MAVS-mediated apoptosis is poorly understood. Here, we show that the adaptor protein TAX1BP1 functions as a negative regulator of virus-induced apoptosis. TAX1BP1-deficient cells are highly sensitive to apoptosis in response to infection with the RNA viruses vesicular stomatitis virus and Sendai virus and to transfection with poly(I·C). TAX1BP1 undergoes degradation during RNA virus infection, and loss of TAX1BP1 is associated with apoptotic cell death. TAX1BP1 deficiency augments virus-induced activation of proapoptotic c-Jun N-terminal kinase (JNK) signaling. Virus infection promotes the mitochondrial localization of TAX1BP1 and concomitant interaction with the mitochondrial adaptor MAVS. TAX1BP1 recruits the E3 ligase Itch to MAVS to trigger its ubiquitination and degradation, and loss of TAX1BP1 or Itch results in increased MAVS protein expression. Together, these results indicate that TAX1BP1 functions as an adaptor molecule for Itch to target MAVS during RNA virus infection and thus restrict virus-induced apoptosis.

Elucidating dynamic protein-protein interactions and ubiquitination in NF-κB signaling pathways.

The Nuclear factor-kappaB (NF-κB) family of transcription factors plays critical roles in inflammatory responses and host defense; however, uncontrolled NF-κB activation can be deleterious by promoting autoimmune diseases and cancers. Lysine K63 (K63)-linked polyubiquitination has emerged as an important regulatory mechanism in NF-κB signaling by regulating dynamic protein-protein interactions that trigger NF-κB signaling. RIP1 and TRAF6 serve as key substrates of K63-linked polyubiquitin chains in tumor necrosis factor receptor (TNFR) and interleukin-1 receptor (IL-1R) pathways respectively as a mechanism to recruit TAK1 and IKK kinases by associated ubiquitin-binding adaptor molecules. Activation of IKKß by TAK1 induces IκBα phosphorylation, degradation, and downstream NF-κB activation. The ubiquitin-editing enzyme A20 maintains transient NF-κB activation by opposing the K63-linked polyubiquitination of RIP1 and TRAF6. A20 inducibly interacts with the adaptor molecule TAX1BP1 and the E3 ligases Itch and RNF11 to form an A20 ubiquitin-editing enzyme complex. Notably, loss-of-function somatic mutations or polymorphisms in human A20 are associated with B-cell lymphomas or a variety of autoimmune diseases as a result of dysregulated NF-κB activation. In this chapter, we summarize the protocols routinely used in our laboratories to examine ubiquitination and NF-κB signaling.

Human T-cell leukemia virus type 1 (HTLV-1) tax requires CADM1/TSLC1 for inactivation of the NF-κB inhibitor A20 and constitutive NF-κB signaling.

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.

A20-mediated negative regulation of canonical NF-κB signaling pathway.

The nuclear factor kappa B (NF-κB) plays vital role in the immune system by regulating innate and adaptive immunity, development and survival of lymphocytes, and lymphoid organogenesis. All known NF-κB activators converge on the IkappaB kinase (IKK) complex to activate the canonical and non-canonical NF-κB pathways. The IKK complex contains two catalytic subunits (IKKα and IKKß) and a regulatory subunit NEMO/IKKγ that regulates the canonical NF-κB pathway, whereas IKKα regulates the non-canonical pathway. The process of IKKα activation and its role in the regulation of canonical NF-κB activation remain elusive. The canonical pathway is rapidly activated and produces a potent inflammatory response to bacterial and viral infections as well as different types of stress; however, uncontrolled NF-κB activation can lead to autoimmune diseases and cancers. Therefore, to keep the inflammatory response in check, elaborate negative regulatory mechanisms operate to terminate NF-κB activation at multiple levels by de novo synthesis of NF-κB inhibitory proteins, and orchestration of protein ubiquitination and deubiquitination. The NF-κB target genes, IκBα and A20, play critical roles in termination of the active canonical NF-κB pathway. In this review, we discuss our recent findings describing a novel function for IKKα in nucleating the ubiquitin-editing enzyme A20 complex, a major negative regulator of canonical NF-κB signaling. Consistently with an inhibitory function of IKKα, it is targeted by the human T-cell leukemia virus 1 (HTLV-1) oncoprotein, Tax, to prevent assembly of the A20 complex to maintain persistent NF-κB activation that promotes transformation and survival of virus-transformed cells.

Regulation of NF-κB signaling by the A20 deubiquitinase.

The NF-κB transcription factor is a central mediator of inflammatory and innate immune signaling pathways. Activation of NF-κB is achieved by K63-linked polyubiquitination of key signaling molecules which recruit kinase complexes that in turn activate the IκB kinase (IKK). Ubiquitination is a highly dynamic process and is balanced by deubiquitinases that cleave polyubiquitin chains and terminate downstream signaling events. The A20 deubiquitinase is a critical negative regulator of NF-κB and inflammation, since A20-deficient mice develop uncontrolled and spontaneous multi-organ inflammation. Furthermore, specific polymorphisms in the A20 genomic locus predispose humans to autoimmune disease. Recent studies also indicate that A20 is an important tumor suppressor that is inactivated in B-cell lymphomas. Therefore, targeting A20 may form the basis of novel therapies for autoimmune disease and lymphomas.

Outfoxing FoxO transcription factors: HTLV-1 Tax oncoprotein inactivates FoxO4 via the ubiquitin-proteasome pathway.

Evaluation of: Oteiza A, Mechti N. The human T-cell leukemia virus type 1 oncoprotein tax controls forkhead box O4 activity through degradation by the proteasome. J. Virol. 85(13), 6480-6491 (2011). This study examines downstream signaling events of PI3K/AKT in the context of human T cell leukemia virus type 1 (HTLV-1) infection. The authors have demonstrated that the HTLV-1 Tax oncoprotein triggers the ubiquitination and proteasomal degradation of the FoxO4 transcription factor. Phosphorylation by AKT is requisite for Tax-induced FoxO4 degradation since mutation of the AKT phosphorylation sites abrogates FoxO4 degradation. Furthermore, Tax enhances the interaction between FoxO4 and the E3 ubiquitin ligase MDM2 which presumably leads to FoxO4 ubiquitination. Consistently, knockdown of MDM2 with a shRNA plasmid attenuates FoxO4 ubiquitination, revealing an important role for MDM2 in Tax-induced FoxO4 ubiquitination. Finally, Tax represses FoxO4 transcriptional activity in a dose-dependent manner. Taken together, the findings by Oteiza et al. suggest that Tax inactivates the tumor suppressor FoxO4 downstream of PI3K/AKT, which may play a role in HTLV-1-induced oncogenesis.

The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1.

In response to stimulation with proinflammatory cytokines, the deubiquitinase A20 inducibly interacts with the regulatory molecules TAX1BP1, Itch and RNF11 to form the A20 ubiquitin-editing complex. However, the molecular signal that coordinates the assembly of this complex has remained elusive. Here we demonstrate that TAX1BP1 was inducibly phosphorylated on Ser593 and Ser624 in response to proinflammatory stimuli. The kinase IKKα, but not IKKß, was required for phosphorylation of TAX1BP1 and directly phosphorylated TAX1BP1 in response to stimulation with tumor necrosis factor (TNF) or interleukin 1 (IL-1). TAX1BP1 phosphorylation was pivotal for cytokine-dependent interactions among TAX1BP1, A20, Itch and RNF11 and downregulation of signaling by the transcription factor NF-κB. IKKα therefore serves a key role in the negative feedback of NF-κB canonical signaling by orchestrating assembly of the A20 ubiquitin-editing complex to limit inflammatory gene activation.

Human T cell leukemia virus type 1 Tax inhibits innate antiviral signaling via NF-kappaB-dependent induction of SOCS1.

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-ß expression accompanied by reduced HTLV-1 replication and p19(Gag) levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.

Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes.

A20 negatively regulates inflammation by inhibiting the nuclear factor kappaB (NF-kappaB) transcription factor in the tumor necrosis factor-receptor (TNFR) and Toll-like receptor (TLR) pathways. A20 contains deubiquitinase and E3 ligase domains and thus has been proposed to function as a ubiquitin-editing enzyme downstream of TNFR1 by inactivating ubiquitinated RIP1. However, it remains unclear how A20 terminates NF-kappaB signaling downstream of TLRs. We have shown that A20 inhibited the E3 ligase activities of TRAF6, TRAF2, and cIAP1 by antagonizing interactions with the E2 ubiquitin conjugating enzymes Ubc13 and UbcH5c. A20, together with the regulatory molecule TAX1BP1, interacted with Ubc13 and UbcH5c and triggered their ubiquitination and proteasome-dependent degradation. These findings suggest mechanism of A20 action in the inhibition of inflammatory signaling pathways.

Role of post-translational modifications of HTLV-1 Tax in NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered, is the etiological agent of adult-T-cell leukemia/lymphoma. The HTLV-1 encoded Tax protein is a potent oncoprotein that deregulates gene expression by constitutively activating nuclear factor-κB (NF-κB). Tax activation of NF-κB is critical for the immortalization and survival of HTLV-1-infected T cells. In this review, we summarize the present knowledge on mechanisms underlying Tax-mediated NF-κB activation, with an emphasis on post-translational modifications of Tax.

The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling.

The RING domain protein RNF11 is overexpressed in breast cancers and promotes tumour growth factor-beta (TGF-beta) signalling. RNF11 has been proposed to regulate TGF-beta signalling by interacting with HECT- and SCF-type E3 ligases; however, the role of RNF11 in other signalling pathways is poorly understood. Here, we demonstrate a novel function of RNF11 as a negative regulator of NF-kappaB and jun N-terminal kinase (JNK) signalling pathways. Knockdown of RNF11 with siRNA resulted in persistent tumour necrosis factor (TNF)- and lipopolysaccharide (LPS)-mediated NF-kappaB and JNK signalling. RNF11 interacted with the NF-kappaB inhibitor A20 and its regulatory protein TAX1BP1 in a stimulus-dependent manner. RNF11 negatively regulated RIP1 and TRAF6 ubiquitination upon stimulation with TNF and LPS, respectively. Furthermore, RNF11 was required for A20 to interact with and inactivate RIP1 to inhibit TNF-mediated NF-kappaB activation. Our studies reveal that RNF11, together with TAX1BP1 and Itch, is an essential component of an A20 ubiquitin-editing protein complex that ensures transient activation of inflammatory signalling pathways.

The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin-editing enzyme A20.

The ubiquitin-editing enzyme A20 is a critical negative regulator of inflammation and cytokine-mediated activation of the transcription factor NF-kappaB; however, little is known about the mechanisms of A20-mediated inactivation of signaling intermediates such as RIP1. Here we demonstrate that the regulatory molecule TAX1BP1 recruited the E3 ligase Itch to A20 via two 'PPXY' motifs. Itch was essential for the termination of tumor necrosis factor receptor signaling by controlling A20-mediated recruitment and inactivation of RIP1. Furthermore, the Tax oncoprotein of human T cell leukemia virus type I targeted this complex for inactivation by disrupting the interaction among TAX1BP1, A20 and Itch. Thus, our studies show a previously unappreciated complexity of A20 substrate recognition and inactivation whereby TAX1BP1 and Itch function as essential subunits of an A20 ubiquitin-editing complex.

TNF pretreatment interferes with mitochondrial apoptosis in the mouse liver by A20-mediated down-regulation of Bax.

Pretreatment with low doses of the proinflammatory cytokine TNF has been shown to prevent hepatocellular apoptosis and liver damage in inflammatory as well as in ischemia/reperfusion-induced liver injury. The underlying mechanisms of protection have not been elucidated so far. In this study, these mechanisms were investigated in murine hepatocyte cultures as well as in a mouse model of TNF-dependent apoptotic liver damage (galactosamine/TNF model). Our results show that pretreatment with TNF, or application of small-interfering RNA directed against the proapoptotic Bcl2 family member Bax, interfered with the onset of mitochondrial apoptosis in vivo. Knockdown of TNF-alpha-induced-protein 3 (A20) restored mitochondrial apoptosis, Bax expression, and liver damage. The underlying mechanism of protection seems to involve a cascade of events, where TNF induces the expression of A20 in hepatocytes, A20 down-modulates Bax expression by interference with transcriptional activation, and the reduced availability of Bax interferes with the onset of mitochondrial apoptosis and the ensuing apoptotic liver damage. In conclusion, we identified Bax and A20 as key players in TNF-induced protection from apoptotic liver damage. Because treatment with TNF itself might be a risk factor for patients, we propose that overexpression of A20 might represent an alternative approach for protection from inflammation related apoptotic liver damage, as well as for TNF preconditioning during transplantation.