Hepatitis B virus (HBV) causes an estimated 600,000 deaths annually, largely due to hepatocellular carcinoma (HCC). HBx, a promiscuous transactivator, is a viral oncoprotein, but its exact functions are poorly understood. Many studies have suggested that NF-κB signaling mediates HBx functions, but the underlying molecular mechanisms remain yet to be elucidated. Here, we provide evidence that HBx-mediated NF-κB activation depends on the physical interaction between HBx and a transcription factor, p65. In the cytoplasm, HBx-p65 interaction may promote IκBα phosphorylation and subsequent p65 nuclear localization. A cytokine assay using qPCR and RT-PCR indicates that HBx is associated with a unique profile of cytokine mRNA expression. As shown by chromatin immunoprecipitation (ChIP), HBx in the nuclues can be recruited to the gene promoter by p65. These findings support the importance of HBx-p65 interaction and suggest that it is potentially a promising target of novel therapeutics for HBV-associated liver diseases, including HCC.

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

Type 1 Diabetes (T1D) is an autoimmune disease affecting around 0.1-0.8% of the population worldwide and is characterized by a progressive destruction of insulin-producing beta cells. Pro-inflammatory cytokines released by immune cells around the islets contribute for the “first wave” of beta cell apoptosis. Cytokine-mediated activation of the transcription factor nuclear factor kappa (NF-κB) contributes to beta cell demise in T1D. This is unusual, since NF-κB has anti-apoptotic effects in other cells. NF-κB is activated in most cells via the canonical pathway, while its activation via the non-canonical NF-κB pathway is restricted to few cell types, such as maturing/differentiating immune cell and osteoclasts. We have now observed that IL-1β+IFN-γ induces an atypical activation of the non-canonical NF-κB pathway in beta cells. This activation depends on different crosstalk mechanisms between the canonical and non-canonical NF-κB pathways, including the down-regulation of the E3 ligase Fbw7, which targets the p100 for proteasomal degradation, and up-regulation of another E3 ligase, βTrCP, which in turn induces cleavage of p100 to p52, a hallmark step in the non-canonical NF-κB activation. Importantly, cytokine-mediated activation of the non-canonical pathway regulates the expression of late NF-κB dependent genes, such as Ccl5, Ccl19, Ccl12, Fas that regulate both pro-inflammatory and pro-apoptotic responses and are implicated in beta cell loss in T1D. This atypical activation of the non-canonical NF-κB pathway by pro-inflammatory cytokines in beta cells constitutes a novel “feed-forward” mechanism that may explain the particular pro-apoptotic effect of this transcription factor in beta cells. Besides regulation of pro-death responses, NF-κB activation in beta cells triggers the expression of the ubiquitin-editing protein A20, encoded by TNFAIP3. A20 restricts NF-κB signalling and possess anti-apoptotic activities in beta cells. Importantly, genome-wide association studies have identified TNFAIP3 as a candidate gene for T1D. We presently demonstrated that A20 effects in beta cells are not restricted to inhibition of NF-κB. Thus, A20 also suppresses the pro-apoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK), and activates the survival signaling mediated via the v-akt murine thymoma viral oncogene homolog (Akt), thus inhibiting the intrinsic pathway of apoptosis. Finally, a cohort study of T1D children indicated that the risk allele of the rs2327832 single nucleotide polymorphism of TNFAIP3 predict lower C-peptide and higher hemoglobin A1c (HbA1c) levels 12 months after disease onset, indicating that this candidate gene contributes for reduced residual beta-cell function and impaired glycemic control in early T1D. In conclusion, our results indicate a critical role for A20 in the regulation of beta cell survival and unveil novel mechanisms by which A20 controls beta-cell fate. Moreover, we identified the single nucleotide polymorphism rs2327832 of TNFAIP3 as a prognostic marker for diabetes outcome in children with T1D.We have also observed that A20 protects beta cells against the pro-apoptotic effects of cytokines by preventing the degradation of the anti-apoptotic protein Mcl-1. Mcl-1 belongs to the Bcl-2 family of proteins that regulate the intrinsic apoptotic pathway. It was previously shown that Mcl-1 depletion contributes to apoptosis in rat beta cells and that its expression is downregulated in islets from T1D individuals infected by enteroviruses. We have now confirmed in human beta cells that decreased Mcl-1 expression contributes to cytokine-mediated beta cell death. We generated a beta cell specific Mcl-1 knockout mouse line (βMcl-1 KO) and observed that islets derived from these mice were more susceptible to pro-apoptotic stimuli exposure ex vivo. Of note, βMcl-1 KO mice were more vulnerable to multiple low dose streptozotocin-induced diabetes than their wild type littermates. One of the mechanisms by which cytokines mediate Mcl-1 degradation is via its phosphorylation by GSK3β. Overexpression of A20 increased AKT phosphorylation, providing a negative feedback on GSK3β activity and preventing Mcl-1 degradation. Cytokines also increase Mcl-1 ubiquitination, a process regulated by the E3 ligases Mule and βTrCP and the deubiquitinase USP9X. The present findings indicate that pro-inflammatory cytokines trigger post-translational modifications of Mcl-1 leading to its degradation. This contributes to exacerbation of pro-death responses and beta cell demise in T1D, but it can be prevented, at least in part, by A20. As a whole, our data unveil novel post-translational mechanisms and different ubiquitin editing proteins that regulate beta cell fate in T1D and are modulated by pro-inflammatory cytokines. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Please read the abstract in the section of 00front of this document / Thesis (PhD (Medical Plant Science))--University of Pretoria, 2008. / Plant Science / unrestricted

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23440号 / 生博第461号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 朝長 啓造, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Corals are critical to marine biodiversity and human welfare. Coral reefs cover <1% of the seafloor but support ~1/3 of all marine species. Approximately 1.5 billion people live within 100 km of coral reefs, relying upon them for food, income from tourism, and protection from storms. Their economic value has been estimated at $375 billion annually. The foundation of coral reefs is the intracellular symbiosis between corals and photosynthetic dinoflagellates of the family Symbiodiniaceae. Tropical corals satisfy up to 95% of their nutritional requirements through photosynthesis, and their ability to construct reefs is biochemically coupled to photosynthesis. While permitting corals to thrive, photosymbiosis also increases their exposure to environmental stressors and vulnerability to climate change. Reliance on photosynthesis restricts reef-building corals to shallow, clear, tropical waters, where they experience higher temperatures and UV exposure. The generation of reactive oxygen species by the symbiont also exposes corals to greater oxidative stress. The symbiosis is particularly sensitive to climate change: all of the mass coral bleaching events have occurred since 1982, driven by elevated ocean temperatures. Molecular cross-talk between host and symbiont impacts resilience of the coral holobiont and resistance to bleaching. Unfortunately, we know little about how photosymbiosis impacts expression or activity of coral genes. Tropical corals engage in an obligate symbiosis with Symbiodiniaceae, so we cannot study their gene expression in a stable aposymbiotic state. However, the northern star coral, Astrangia poculata, engages in a facultative symbiosis with Symbiodiniaceae. I used RNA sequencing to investigate how symbiosis impacts gene expression in A. poculata, focusing on genes implicated in photosymbiosis: antioxidant enzymes (specifically superoxide dismutases) and the NF-κB signaling pathway. From an improved transcriptome assembly, I recovered core elements of a primitively simple NF-κB signaling pathway and a rich complement of SOD proteins. 273 coral transcripts—many associated with protein metabolism and vesicle-mediated transport— were differentially expressed in symbiotic versus aposymbiotic corals. Unlike in the facultatively symbiotic sea anemone Exaiptasia, symbiosis was not associated with depressed NF-κB transcript levels. IKKε, a potential positive regulator of NF-κB activity, was strongly up-regulated, as was one particular superoxide dismutase.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are among the most commonly diagnosed forms of cancer in the United States. Due to their widespread prevalence and high mortality rate, it is vital to develop effective therapeutic drugs to combat these deadly diseases. In both PDAC and CRC, the multifunctional factor nuclear factor kappa B (NF-kB), a central coordinator of cellular immune responses, is activated abnormally, leading to tumorigenesis and cancer progression. Therefore, controlling NF-kB activity is critical in the treatment of these cancers. In a previous study, we identified a new mechanism by which NF-kB activity is regulated by an epigenetic enzyme known as protein arginine methyltransferase 5 (PRMT5). We showed that overexpression of PRMT5 not only activated NF-kB, but also significantly promoted several characteristics associated with cancer, including increased cell proliferation, migration, and anchorage-independent growth in both PDAC and CRC cells. Moreover, in order to examine the therapeutic potential of PRMT5 in these cancers, we adapted the state-of-the-art AlphaLISA technique into a high throughput screen (HTS) platform to screen for PRMT5 inhibitors. As a result, we successfully identified the small molecule PR5-LL-CM01 as our lead hit. Further validation experiments confirmed that PR5-LL-CM01 is a potent and specific PRMT5 inhibitor that exhibits significant anti-tumor efficacy in both in vitro and in vivo models of PDAC and CRC. Additionally, in a second screen, we discovered two natural compounds, P1608K04 and P1618J22, that can also function as the PRMT5 inhibitors. These findings further highlight the robustness of the PRMT5- specific AlphaLISA HTS technique. To conclude, we describe here for the first time a novel role of PRMT5 as a tumor-promoting factor in PDAC and CRC through NF-kB activation. By successfully developing and applying an innovative AlphaLISA HTS technique, we discovered PR5-LL-CM01, P1608K04, and P1618J22 as novel PRMT5 inhibitors, with PR5-LL-CM01 showing the strongest potency in both PDAC and CRC models. Therefore, we demonstrated that PRMT5 is a promising therapeutic target in PDAC and CRC, and the novel PRMT5 inhibitor PR5-LL-CM01 could serve as a promising basis for new drug development in PDAC and CRC.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer predisposition in which loss of heterozygosity at the NF2 gene locus leads to the development of tumors of neural crest derived origin, most commonly bilateral vestibular schwannomas. There are currently no FDA approved chemotherapeutic agents for treatment in patients with NF2. Development of therapeutic agents has been hampered by our incomplete knowledge of how Merlin, the protein product of the NF2 gene, functions as a tumor suppressor. In order develop a deeper understanding for how loss of Merlin leads to oncogenic transformation in Schwann cells we have developed a genetically engineered mouse model (GEMM) of Neurofibromatosis Type 2 in which functional expression of Merlin is lost in Schwann cell precursors. In parallel studies utilizing these mice, we have sought to understand the pathophysiology driving tumor formation in Merlin deficient Schwann cells. In Chapter 1, we explore the role of Merlin as a negative regulator of the Group A p21 activated kinases, PAK1 and PAK2. We demonstrate that PAK1, a previously well established oncogene in solid tumors and Merlin binding partner, is hyperactivated in Merlin deficient schwannomas. Through therapeutic interventions and genetic manipulations we demonstrate that inhibition of PAK1 was capable of reducing tumor formation and alleviating sensorineural hearing loss in our NF2 GEMM. In Chapter 2, we investigate the role of NF-kB inducing kinase (NIK) and NF-kB signaling in the formation and growth of Merlin deficient Schwann cell tumors. Prior work in our lab as well as by others demonstrated elevated NF-kB signaling in Merlin deficient Schwann cell tumors. We observed accumulation of a catalytically active fragment of NF-kB inducing kinase and present data that accumulation of a 55Kd constitutively active fragment of NIK is sufficient trigger wild type Schwann cells to form tumors. In vivo however, Schwann cell intrinsic expression of NIK is not required for tumor formation or growth. / 2 years (2021-05-24)

Indiana University-Purdue University Indianapolis (IUPUI) / The overexpression of the multi-domain enzyme fatty acid synthase (FASN) has long been associated with poor clinical prognosis and treatment outcome in various cancers. Previous research in the Zhang lab has determined a role for FASN in mediating increases in non-homologous end-joining (NHEJ) DNA double-strand break repair activity allowing for increased cancer cell survival, and this mechanism was found to involve inhibition of NF-kB/p65. The mechanism responsible for the regulation of NF-kB/p65 by FASN in cancer cells, however, remains unknown. To this end, I was able to determine that FASN negatively regulates both the expression and activity of NF-kB/p65 in breast cancer cells, and that this effect was likely mediated by the 16-carbon saturated fatty acid palmitate, the end product of FASN catalytic activity. Specifically, FASN was found to negatively regulate p65 expression by disrupting its protein stability as a result of an increase in poly-ubiquitination of p65 protein and subsequent proteasomal degradation. Further, I found that the phosphorylation site Thr254 of p65 is involved in the regulation of p65 protein stability by FASN, in that mutation of this residue resulted in a disruption in p65 stability. Finally, I was able to determine that FASN likely inhibits the ability of the peptidyl-prolyl cis/trans isomerase Pin1 to assist in maintaining p65 stability, in that both siRNA knockdown and pharmacological inhibition of Pin1 resulted in a reduction of p65 expression in FASN shRNA knockdown cells. The determination of this signaling mechanism serves to expand our understanding of the role of FASN in breast cancer cells and has the potential to assist in uncovering more effective ways to target the oncogenic FASN pathway to kill breast tumor cells and to overcome resistance to drug treatment.

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