Mechanism of gamma-secretase action during HPV infection

HPV感染过程中γ-分泌酶的作用机制

• 批准号: 10359822
• 负责人: Daniel C. Dimaio
• 金额: $ 60.17万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-24 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10359822
• 关键词: Address; Anogenital cancer; Binding; Biochemical; Capsid; Capsid Proteins; Cell Nucleus; Cell physiology; Cells; Cellular biology; Complex; Cytoplasm; DNA Viruses; Endocytosis; Endosomes; Event; Foundations; Golgi Apparatus; Golgi Targeting; Grant; Human; Human Papillomavirus; Human papilloma virus infection; In Vitro; Individual; Infection; Infection prevention; Integral Membrane Protein; Integration Host Factors; L2 viral capsid protein; Light; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Mass Spectrum Analysis; Mediating; Membrane; Minor; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; N-terminal; Pathway interactions; Peptide Hydrolases; Peptides; Persons; Play; Preventive vaccine; Process; Proteins; Proteomics; Publishing; Research Personnel; Role; Route; Testing; Transmembrane Domain; Vaccines; Viral; Viral Genome; Virion; Virus; base; burden of illness; combat; endosome membrane; experimental study; gamma secretase; high risk; human pathogen; improved; insight; intracellular protein transport; malignant oropharynx neoplasm; mutant; novel; novel therapeutic intervention; prevent; protein complex; receptor; reconstitution; recruit; trafficking; trans-Golgi Network; virology

Abstract This is a proposal for a new, multi-PI grant involving two senior investigators with complementary expertise to study an essential but poorly understood step in human papillomavirus (HPV) infection. HPV is responsible for approximately 5% of human cancer worldwide. During infection, HPV is internalized to the endosome from where it is targeted to the retrograde pathway for transport to the TGN en route to the nucleus for productive infection. Endosome-to-Golgi targeting represents the committed infection step and remains poorly understood. Our published and unpublished results suggest that a host factor – the transmembrane protease γ-secretase – is endowed with a novel chaperone activity that promotes insertion of an HPV capsid protein called L2 into the endosome membrane. Membrane insertion of L2 and subsequent protrusion into the cytoplasm recruits cytosolic host components that deliver HPV to the Golgi. Strikingly, we also found that HPV infection stabilizes the multi-subunit γ-secretase complex. Accordingly, the central objectives of this proposal are to elucidate, at the molecular level, how γ- secretase promotes membrane insertion of L2 and how HPV regulates the function of γ-secretase to coordinately promote HPV infection. We will identify cellular proteins that recruit γ-secretase to HPV, determine if γ-secretase-mediated capsid rearrangements allow L2 to emerge from the capsid, and attempt to reconstitute membrane insertion in vitro and determine the relevant γ-secretase subunits and activities. We will determine if γ-secretase plays a role in insertion of cell-penetrating peptides or retrograde trafficking in general. We will examine the role of the N-terminal transmembrane domain in L2 in these activities and attempt to develop viral mutants that are independent of γ-secretase. Finally, we will determine how HPV infection stabilizes the γ-secretase complex, identify other factors involved in this process, and determine if the L2 transmembrane domain is sufficient for stabilization and membrane insertion. Taken together, these experiments will shed new light on the unusual role of γ-secretase in HPV entry, improve our understanding of this crucial step in HPV infection, elucidate new aspects of general cell biology, and possibly point the way to new therapeutic approaches to prevent infection by these important human pathogens.

抽象的 这是一项新的，多PI的新赠款的建议，涉及两名高级调查员 研究人类乳头瘤病毒（HPV）感染的基本但不理解的一步的专业知识。 HPV 全球约有5％的人类癌症。在感染期间，HPV被内在化为 从靶向到逆行途径到达TGN的逆行途径的内体 生产感染的细胞核。内体到高尔基的靶向代表致命的感染 步骤，保持不当理解。我们发表的未发表的结果表明，宿主因素 - 跨膜蛋白酶γ-分泌酶 - 具有促进的新型伴侣活性 将HPV衣壳蛋白插入内体膜中。 L2的膜插入 随后向细胞质突出，募集了将HPV传递到的胞质宿主成分 高尔基。令人惊讶的是，我们还发现HPV感染稳定了多亚基γ-分泌酶复合物。 彼此之间，该提案的中心对象是在分子水平上阐明如何γ- 泌尿酶促进L2的膜插入以及HPV如何调节γ-分泌酶的功能 协同促进HPV感染。我们将确定募集γ-分泌酶至HPV的细胞蛋白， 确定γ-分泌酶介导的衣壳重排是否允许L2从衣壳中出现，并且 尝试在体外重新构建膜插入，并确定相关的γ-分泌酶亚基和 活动。我们将确定γ-分泌酶是否在插入细胞穿透肽或 一般而言，逆行贩运。我们将检查N末端跨膜结构域在L2中的作用 在这些活动中，并尝试开发独立于γ-分泌酶的病毒突变体。最后，我们 将确定HPV感染如何稳定γ-分泌酶复合物，确定涉及的其他因素 过程，并确定L2跨膜结构域是否足以稳定和膜 插入。综上所述，这些实验将为γ-分泌酶在 HPV进入，提高我们对HPV感染的关键步骤的理解，阐明 一般细胞生物学，并可能指向采用新的治疗方法的道路，以防止通过 这些重要的人类病原体。