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: 10385849
• 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/10385849
• 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; 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; base; bioinformatics pipeline; chemokine; combat; computerized tools; design; educational atmosphere; genome editing; innate immune mechanisms; insight; macrophage; 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