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.

摘要 结核病是感染性疾病死亡的主要原因， 结核分枝杆菌感染的治疗具有挑战性。为了开发替代疗法，本建议 旨在寻找新的免疫途径，负责控制结核病在巨噬细胞中的存活。一个天生的 可以抗菌免疫反应是自噬。在选择性自噬中， 病原体进入细胞质并触发激酶的激活，激酶使自噬磷酸化 适配器，从而在溶酶体中靶向微生物进行破坏。TANK结合激酶1（TBK 1）和 PTEN诱导的推定激酶1（PINK 1）是两种涉及自噬的巨噬细胞激酶， 细菌，但其磷酸化底物的身份和PINK 1在TB自噬中的潜在作用是 不清楚为了确定激酶底物，我们将完成我们的分析， 肺结核感染时激酶缺陷小鼠骨髓源性巨噬细胞磷酸化蛋白质组的研究（目的 1）。磷酸化蛋白质组学和免疫荧光显微镜显示四种自噬受体共定位 其中三个是磷酸化的为了确定这些以前未被承认的 自噬衔接子在自噬体靶向结核病中的应用，我们将获得靶向敲除小鼠或使用 CRISPR/Cas9创建具有自噬适配器突变的巨噬细胞并量化自噬靶向， 炎症反应和衔接子缺陷细胞中的细菌生长（Aim 2）。通过了解 自噬靶向结核病的机制，我们可能能够设计针对结核病的宿主导向疗法。 在正式课程和导师的指导下，Budzik博士的目标是了解激酶的机制， 结核病的自噬依赖靶向，并发展技能和填补知识空白，以成为一个 独立科学家Budzik博士的多元化指导团队包括初级导师Jeffery考克斯博士，加州大学 伯克利分校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，UCSF医学教授，内科科学家， 具有结核病临床试验和诊断相关转化研究的专业知识，委员会成员Dr. Nevan Krogan，UCSF细胞和分子药理学教授，该提案的合作者， 在应用高通量网络生物学对微生物发病机理的机理见解的专业知识，博士。 Jayanta Debnath，UCSF教授兼病理学主席，其研究包括 自噬靶向，大卫尔勒博士，加州大学旧金山分校医学教授，真核基因组编辑专家， 丹尼尔波特诺伊博士，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， 李斯特菌先天免疫国际专家。Budzik博士的研究计划侧重于激酶介导的 将结核病定位于自噬，再加上他有组织的训练计划，将使他能够获得数据， R 01申请的先决条件技能，并作为独立调查员发展独特的科学利基。