Membrane adaptor proteins are characterized by the lack of enzymatic activity and the presence of various interaction sites for other proteins and cellular membranes. They typically function as scaffolds connecting receptors or other adaptors with proximal signaling molecules at cellular membranes. Their overall effects on signaling can be activating or inhibiting depending on the nature of the effector molecules they recruit. SCIMP is one of the membrane adaptors discussed in this thesis. It is expressed in antigen- presenting cells and it has been previously shown to enhance MHCII signaling in B cells. This thesis covers the analysis of SCIMP functions beyond B cells and describes the first analysis of SCIMP deficient mice. Although the results of this analysis did not show any alterations in immune cell populations, the novel function of SCIMP in dendritic cell signaling downstream of DECTIN- 1 was uncovered. DECTIN-1 is a pattern recognition receptor involved in antifungal immunity. The data presented in this thesis describe the role of SCIMP in sustaining DECTIN-1 signaling over relatively long periods of time and the contribution of SCIMP signaling to maintaining prolonged production of pro-inflammatory cytokines. PSTPIP2 is another interesting adaptor discussed in this thesis. It is...

14-3-3 proteins are among a family of phospho-binding proteins that are known to regulate many essential cellular mechanisms. By binding to sites of phosphorylation, 14-3-3s are integrated into multiple signaling pathways that govern critical processes, such as apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. These processes are crucial for tumorigenesis and 14-3-3 proteins are known to play a central role in facilitating cancer progression. In this study, my colleagues and I discover two novel 14-3-3 interacting proteins, ATG9A and PTOV1, that are both vital to essential cellular functions and describe various mechanisms that these two proteins regulate. ATG9A is a multi-pass transmembrane lipid scramblase that is found primarily as a homotrimer in the ER or small ATG9A vesicles. It is essential in the cellular recycling process called autophagy and is believed to act at the earliest stages of autophagy by providing the seed for the growth of the double membrane vesicle called an autophagosome. Previous work in our lab demonstrated that upon hypoxic stress, AMPK, the master nutrient-sensing kinase, phosphorylates S761 on the C-terminus of ATG9A. This triggers the binding of 14-3-3ζ to contribute to ATG9A function in hypoxia induced autophagy. Despite this revelation, the exact function of ATG9A is still poorly understood, especially in unstimulated conditions where autophagy functions at a basal level and AMPK is inactive. In this study, we sought to understand ATG9A function more broadly by identifying novel interactors of ATG9A and the role ATG9A plays in basal autophagy. To do this, we employed BioID mass spectrometry and various biochemical approaches to identify LRBA as a bona fide ATG9A interactor and autophagy regulator. Furthermore, using deuterium labeling and quantitative whole proteome mass spectrometry, and various other biochemical techniques, we show that ATG9A regulates the basal degradation of p62 and is recruited to sites of basal autophagy by active poly-ubiquitination to initiate basal autophagy. PTOV1 is an oncogenic protein that is poorly understood. Our current understanding of PTOV1 is limited to a few studies, which demonstrate that PTOV1 is highly expressed in primary prostate tumor samples and is correlated with metastasis, drug resistance, and poor clinical outcomes. In this study, we identify a mechanism by which SGK2, a poorly understood kinase, phosphorylates PTOV1 at S36 to trigger 14-3-3 binding at that site to increase PTOV1 stability in the cytosol and increase c-Jun expression. Upon SGK2 inhibition, 14-3-3 releases PTOV1 and PTOV1 is shuttled into the nucleus where HUWE1, an E3 ubiquitin ligase, ubiquitinates PTOV1 and initiates PTOV1 degradation by the proteasome. This is the first detailed mechanism of regulation identified for the poorly understood oncogene, PTOV1, and sheds light on potential therapeutic targets for cancer treatments.

<p>  </p> <p>Ubiquitination is a post-translational modification that activates a variety of signaling pathways. The process of tagging ubiquitin (Ub) onto a substrate protein requires three proteins. First, the E1-activating protein primes Ub for attachment to the E2-conjugating enzymes. The E2-conjugating enzyme then brings Ub to E3 ligases, which also recruit the substrate proteins. The final step of this cascade is the transfer of Ub onto the substrate protein. More commonly, ubiquitinated proteins are then degraded via the proteasome. This cascade to downregulate proteins is employed as a cellular adaptation mechanism in response to various threats, including bacterial and viral pathogens. Although the Ub system exists exclusively in eukaryotes, in recent years many bacterial effector proteins and viral factors have been shown to hijack the system through highly regulated mechanisms. In my Ph.D. work, I characterized the hijacking mechanism of a protein produced by human papillomavirus (HPV) that causes downregulation of p53. Downregulation of p53 leads to the oncogenic effects of HPV infection. A strain of oncogenic HPV, HPV-16, produces the E6 protein, which forms a complex with the human ubiquitin E3 ligase, E6AP. This allows E6AP to recognize p53 for ubiquitination. Furthermore, the ability of E6 to act as an adaptor protein to target unnatural substrate proteins has been employed by medicinal chemists as the basis of <u>pro</u>teolysis <u>ta</u>rgeting <u>c</u>himeras (PROTACs). To this extent, my thesis covers three broad ideas that will add to our understanding of <strong>1) Cellular adaptor protein regulation, 2) viral adaptor protein hijacking, and 3) PROTAC ligand development.</strong></p>

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25399号 / エネ博第478号 / 新制||エネ||89(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 片平 正人, 教授 佐川 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25400号 / エネ博第479号 / 新制||エネ||89(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 片平 正人, 教授 佐川 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Alternativ splicing av pre-mRNA ger upphov till proteindiversitet. Histonmodifieringar kopplas till den alternativa splicingens reglering genom adaptorsystem som overfor den epigenetiska informationen direkt till splicingfaktorerna. De cis- agerande RNA- elementen pa exoner och introner med tillhorande trans- reglerande splicingfaktorer paverkas darfor direkt av specifika histonmodifieringar. En sammankopplande integrerad modell over en rad DNA- baserade processer foreslas. Denna komplexa modell ger en bild av interaktioner och paverkan mellan dessa delar. Kromatin remodellering kravs for bildandet av eukromatin. Nukleosomers placering vid exonrika regioner med specifika modifieringsmonster pekar ut exonerna samt mojliggor inbindning av RNA polymeras II som med sin CTD doman rekryterar bade splicing- och modifieringsfaktorer. Transkriptionshastigheten paverkas av nukleosomplaceringen vilket i sin tur paverkar rekrytering av spliceosomens komponenter, andra trans- agerande regulatorer och aven pre-mRNA sekvensens sekundarstruktur. Kromatin- adaptorkomplex laser av specifika histonmodifieringar och overfor informationen till splicingapparaten. Detta skapar mojlighet till den viktiga cell- och vavnadsspecifika alternativa splicingens reglering. I den integrerade modellen blir komplexiteten tydligare dar alla dessa processer interagerar med varandra och de cis- regulatoriska sekvenserna pa premRNA transkriptet. / Alternative splicing of pre-mRNA generates protein diversity. Histone modifications are connected to the regulation of alternative splicing through adaptor systems that transfers the epigenetic information directly to the splicing factors. The cis- acting RNA elements on the exons and introns together with the trans- regulating splicing factors are therefore directly affected of specific histone modifications. An integrated model over several DNA process mechanisms is suggested. This complex model explains the interactions of the different parts and how they affect each other. Chromatin remodelers are required to obtain euchromatin. Nucleosome positioning at exon rich regions with a specific modification pattern point out where the exons are, and this enable the RNA polymerase II to find and bind to the DNA. It’s CTD domain recruits both splicing- and modifications factors. The transcription rate is also affected of the nucleosome positioning and that in turn affects the recruitment of the components of the spliceosomen, other trans- acting regulators and even the formation of the secondary structure of the pre-mRNA transcript. Chromatin- adaptor complex reads specific histone modifications and transfers this information to the splicing apparatus. All this creates the possibility to regulate important cell- and tissue specific alternative splicing patterns. The integrated model makes the complex processes more clearer when all these integrates with each other and the cis- acting regulating elements on the pre-mRNA transcript.

No description available.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

The process of HIV-1 particle production is a multi-step process directed by the viral structural protein Gag. As Gag is the only viral protein required to form virus-like particles, it presents a viable target for anti-viral therapeutics of which there are currently none. Although the functions of Gag during the particle assembly process have been well characterized, one of the least known parts of the assembly process is how Gag is targeted to the site of virus assembly. Two main virus assembly sites have been identified in cells that support HIV-1 replication: the plasma membrane or multivesicular bodies (MVBs). However the mechanism by which Gag is targeted to either of these sites remains unknown. The δ subunit of Adaptor Protein Complex 3 has previously been identified as a cellular co-factor for HIV-1 Gag and was reported to mediate Gag trafficking to MVBs, providing a mechanism for Gag targeting to this assembly site. Additionally, AP-3δ was reported to be required for HIV-1 production, suggesting that Gag to MVB targeting is also required for HIV-1 production. The work presented in this thesis further investigates the role of AP-3δ in Gag trafficking to MVBs and its role in HIV-1 production in previously unexplored host environments. Through the use of RNA interference-mediated depletion of AP-3δ, we determined that AP-3δ is dispensible for virus replication in infected HeLa cells, chronically infected HeLa-LAV cells and infected primary human monocyte-derived macrophages. We concomitantly disrupted AP-3 function by disrupting its association with membranes and observed no effect on virus production. Collectively, these results demonstrate that AP-3δ is not required for HIV-1 replication. However, AP-3δ was demonstrated to be required for Gag targeting to MVBs thus presenting a new model for the function of AP-3δ in the context of HIV-1 replication.

Beclin 1 is a haplo-insufficient tumor suppressor that is decreased in many human tumors. The function of Beclin 1 in cancer has been attributed primarily to its role in the degradative process of autophagy. However, the role of autophagy itself in tumorigenesis is context-dependent and can be both preventive and promoting. Due to its dual function in cancer a better understanding of this process is necessary to develop potential novel cancer therapies. To gain insight into the role of autophagy in breast carcinoma, I analyzed the autophagydependency of different subtypes of breast cancer. My results implicate that triple-negative breast carcinoma cells are more dependent on autophagy than luminal breast carcinoma cells. Chemical inhibition of autophagy decreased the tumorigenicity of triple-negative breast carcinoma cells with regard to proliferation and anchorage-independent growth. However, RNAi-mediated suppression of two autophagy genes, ATG5 and Beclin 1, revealed different outcomes. While suppression of ATG5 decreased glycolysis, Beclin 1 depletion did not affect the glycolytic rates. These results suggest autophagy-independent pro-tumorigenic effects of loss of Beclin 1 in cancer. Beclin 1 is a core component of the Vps34/Class III PI3K (PI3KC3) and Vps15/p150 complex that regulates multiple membrane trafficking events. I describe a novel mechanism of action for Beclin 1 in breast cancer involving its control of growth factor receptor signaling. I identify a specific stage of early endosome maturation that is regulated by Beclin 1, the transition of APPL1- containing phosphatidyIinositol 3-phosphate-negative (PI3P-) endosomes to PI3P+ endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P-/APPL+ signaling competent compartment. As a result, suppression of BECN1 sustains growth factor stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, Beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Taken together my data identify a novel role for Beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of Beclin 1 expression would enhance breast cancer progression independent of its impact on autophagy.