Deciphering the role of autophagy receptor phosphorylation and host kinases in the targeting of M. tuberculosis to autophagy

解读自噬受体磷酸化和宿主激酶在结核分枝杆菌靶向自噬中的作用

• 批准号: 10617222
• 负责人: Jonathan M Budzik
• 金额: $ 19.91万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10617222
• 关键词: Adaptor Signaling Protein; Alternative Therapies; Anti-Bacterial Agents; Anti-Inflammatory Agents; Antibiotic Therapy; Autophagocytosis; Autophagosome; Bacteria; Binding; Biochemistry; Biological; Biology; Biophysics; Bone Marrow; CRISPR/Cas technology; Cause of Death; Cells; Clinical Trials; Committee Members; Communicable Diseases; Cytosol; DNA receptor; Data; Diagnostic; Environment; Equilibrium; Event; Genetic; Goals; Growth; Host Defense Mechanism; Immune; Immunofluorescence Microscopy; Immunology; Infection; Inflammation; Inflammatory; Inflammatory Response; Innate Immune Response; Interferons; International; Knockout Mice; Knowledge; Link; Listeria; Lysosomes; MAPK8 gene; Macrophage; Mediating; Medicine; Mentors; Mentorship; Microbe; Mitochondria; Molecular; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Multiple drug resistant Mycobacteria Tuberculosis; Mus; Mutation; Mycobacterium tuberculosis; NF-kappa B; Natural Immunity; PINK1 gene; Pathogenesis; Pathology; Pathway interactions; Patients; Pharmacology; Phosphorylation; Phosphotransferases; Physicians; Play; Process; Proteomics; Research; Research Personnel; Research Proposals; Resistance; Role; Scientist; Signal Transduction; Sterilization; TANK-binding kinase 1; Techniques; Testing; Time; Training; Translating; Translational Research; Tuberculosis; Ubiquitin; Viral; bioinformatics pipeline; chemokine; clinical translation; combat; computerized tools; design; educational atmosphere; genome editing; innate immune mechanisms; insight; microbial; mortality; novel; novel therapeutics; pathogen; phosphoproteomics; professor; receptor; recruit; response; skills; structural biology; tool; tuberculosis treatment; ubiquitin ligase

ABSTRACT Tuberculosis is a major cause of mortality from infectious disease, and multi-drug resistant (MDR) Mycobacterium tuberculosis infections are challenging to treat. To develop alternative therapies, this proposal seeks to find new immune pathways responsible for controlling TB survival in macrophages. One innate immune response that can be anti-bacterial is autophagy. In selective autophagy, macrophages sense pathogens that access the cytosol and trigger the activation of kinases, which phosphorylate autophagy adaptors and thereby target the microbe for destruction in the lysosome. TANK-binding kinase 1 (TBK1) and PTEN-induced putative kinase 1 (PINK1) are two macrophage kinases implicated in autophagy targeting of bacteria, but the identity of their phosphorylated substrates and potential role of PINK1 in autophagy of TB are unclear. To determine the kinase substrates, we will complete our analysis of changes in the phosphoproteome during TB infection in bone marrow-derived macrophages from kinase-deficient mice (Aim 1). Phosphoproteomics and immunofluorescence microscopy revealed four autophagy receptors colocalize with TB and three of them are phosphorylated. To determine the role of these formerly unrecognized autophagy adaptors in autophagosomal targeting of TB, we will obtain targeted knockout mice or use CRISPR/Cas9 to create macrophages with autophagy adaptor mutations and quantify autophagy targeting, inflammatory responses, and bacterial growth in the adaptor deficient cells (Aim 2). By understanding the mechanism of autophagy targeting of TB, we may be able to design host-directed therapies for TB. Guided by formal coursework and mentorship, Dr. Budzik’s goals are to understand the mechanism of kinase- dependent targeting of TB to autophagy, and develop skills and fill knowledge gaps in order to become an independent scientist. Dr. Budzik’s diverse mentoring team includes primary mentor Dr. Jeffery Cox, UC Berkeley Professor of Immunology and Pathogenesis, an internationally recognized expert on TB host- pathogen interactions, co-mentor Dr. Payam Nahid, UCSF Professor of Medicine, a physician scientist with expertise on TB clinical trials and translational research relating to diagnostics, and committee members Dr. Nevan Krogan, UCSF Professor of Cellular and Molecular Pharmacology, a collaborator on this proposal with expertise in applying high throughput network biology to mechanistic insights on microbial pathogenesis, Dr. Jayanta Debnath, UCSF Professor and Chair of Pathology, whose research encompasses mechanisms of autophagy targeting, Dr. David Erle, UCSF Professor of Medicine, an expert on eukaryotic genome editing, and Dr. Daniel Portnoy, UC Berkeley Professor of Biochemistry, Biophysics, and Structural Biology, an international expert on innate immunity to Listeria. Dr. Budzik’s research proposal focusing on kinase-mediated targeting of TB to autophagy together with his organized training plan will allow him to obtain data and prerequisite skills for a R01 application and develop a unique scientific niche as an independent investigator.

抽象的 结核病是传染病和多重耐药性 (MDR) 死亡的主要原因 结核分枝杆菌感染的治疗具有挑战性，该提案提出了开发替代疗法的建议。 寻求找到负责控制巨噬细胞中结核病生存的新免疫途径。 可以抗菌的免疫反应是自噬，在选择性自噬中，巨噬细胞有意义。 病原体进入细胞质并触发激酶激活，从而磷酸化自噬 接头，从而靶向微生物并在溶酶体中进行破坏。 PTEN 诱导的推定激酶 1 (PINK1) 是两种与自噬靶向相关的巨噬细胞激酶 细菌，但其磷酸化底物的身份以及 PINK1 在 TB 自噬中的潜在作用尚不清楚 为了确定激酶底物，我们将完成对变化的分析。 激酶缺陷型小鼠骨髓源性巨噬细胞结核感染期间的磷酸化蛋白质组（目的 1) 磷酸蛋白质组学和免疫荧光显微镜显示四种自噬受体共定位。 与结核病和其中三个被磷酸化确定这些以前未被认识到的作用。 自噬接头在自噬体靶向结核病中，我们将获得靶向敲除小鼠或使用 CRISPR/Cas9 创建具有自噬接头突变的巨噬细胞并量化自噬靶向， 通过了解适配器缺陷细胞中的炎症反应和细菌生长（目标 2）。 通过自噬靶向结核病的机制，我们也许能够设计针对结核病的宿主导向疗法。 在正式课程和指导的指导下，Budzik 博士的目标是了解激酶- 结核病对自噬的依赖性靶向，并发展技能并填补知识空白，以便成为 Budzik 博士的多元化指导团队包括主要导师 Jeffery Cox 博士（加州大学）。 伯克利免疫学和发病机制教授，国际公认的结核病宿主专家 病原体相互作用，共同导师 Payam Nahid 博士，加州大学旧金山分校医学教授，医学科学家 结核病临床试验和诊断相关转化研究方面的专业知识，委员会成员 Dr. Nevan Krogan，加州大学旧金山分校细胞和分子药理学教授，该提案的合作者 博士拥有将高通量网络生物学应用于微生物发病机制机制见解的专业知识。 Jayanta Debnath，加州大学旧金山分校教授兼病理学系主任，他的研究包括 自噬靶向，David Erle 博士，UCSF 医学教授，真核基因组编辑专家， Daniel Portnoy 博士，加州大学伯克利分校生物化学、生物物理学和结构生物学教授， 对李斯特菌先天免疫的国际专家 Budzik 博士的研究提案侧重于激酶介导。 将结核病靶向自噬以及他组织的培训计划将使他能够获得数据和 R01 申请的必备技能，并作为独立研究者开发独特的科学领域。