Deciphering the molecular mechanisms of TNT formation and function using a multi-omic approach

使用多组学方法解读 TNT 形成和功能的分子机制

基本信息

批准号：
10559527
负责人：
KARINE GOUSSET
金额：
$ 28.52万
依托单位：
CALIFORNIA STATE UNIVERSITY FRESNO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10559527
关键词：
Algorithms Alzheimer&apos s Disease American Amyloid beta-Protein Amyotrophic Lateral Sclerosis Antigens Award Binding Biological Assay Biological Markers Biological Models CD4 Positive T Lymphocytes Categories Cell Culture System Cell Line Cell membrane Cells Chemoresistance Communicable Diseases Complex Cytoplasm Detection Discrimination Disease Disparity Endocytic Vesicle Event Filopodia Flow Cytometry Fluorescence Microscopy Future Genetic Materials Genomics Goals Grant HIV-1 Health Herpesvirus 1 Human T-lymphotropic virus 1 Huntington Disease Hypoxia Immune Evasion Immunologic Surveillance In Vitro Individual Infection Influenza Injury Ischemia Light Malignant Neoplasms Malignant neoplasm of ovary Malignant neoplasm of pancreas Malignant neoplasm of urinary bladder Mass Spectrum Analysis Mediating Mediator Membrane Membrane Fusion Messenger RNA Metabolic Methods Molecular Morphology Multiple Myeloma Nanotubes Neoplasm Metastasis Neurodegenerative Disorders Neurons Organelles Parkinson Disease Pathogenicity Pathway interactions Physiological Pilot Projects Play Prions Process Proliferating Proteins Proteome Proteomics Protocols documentation Recovery Reproducibility Research Retroviridae Role Signal Pathway Signal Transduction Signaling Protein Speed Squamous cell carcinoma Stimulus Structure Subcellular structure Technology Testing Time Tissues United States National Institutes of Health Virus alpha synuclein biomarker identification cell type detection limit in vivo laser capture microdissection leukemia malignant breast neoplasm misfolded protein multiple omics neoplastic cell nervous system disorder novel novel strategies particle pathogen protein aggregation protein complex release of sequestered calcium ion into cytoplasm sample fixation tau Proteins trafficking transcriptome tumor progression

项目摘要

Our research is focused on the role of tunneling nanotubes (TNTs)—a novel mechanism of functional connectivity between cells—in the spreading of viruses, misfolded protein aggregates (leading to neurodegenerative diseases), as well as the part they may play in the proliferation and persistence of cancer. TNTs have been found in numerous cell types, allowing the transport of cytosolic and membrane-bound molecules, organelles, calcium ﬂux, and the spreading of pathogens. In vitro, these structures are very heterogeneous and numerous disparities have emerged both in their structure and functions. Similar ﬁlopodia- like structures also exist in vivo and in tissue explants. Unfortunately, little is currently known about the basic mechanism of TNT formation, their structural components, or the signaling pathways involved. Recent studies have revealed that TNTs do play an important physiological role in both health and disease. Indeed, TNTs are significant mediators of electrical, antigen, and genomic signaling, while also promoting cellular recovery after ischemic, inflamatory, and hypoxic injury. What's more, retroviruses, such as the HIV-1, HSV-1, HTLV-1, and influenza exploit these subcellular structures to facilitate infection by evading immune surveillance. Moreover, pathogenic particles and proteins, such as Aβ, prions, and HIV-1 Nef, are found to induce, and then usurp TNT-like structures to spread between cells. Spreading through TNTs is highly efficient, since it avoids diffusive transfer and evades immune detection. Finally, TNTs can mediate the direct transfer of metabolic and genetic material between tumor cells and their stroma enhancing tumor cell chemoresistance, tumor progression, and metastasis. With a previous NIH SCORE SC2 Pilot Project Award, we successfully developed a novel method to specifically isolate distinct protrusion subtypes—based on their morphology or fluorescent markers—using laser capture microdissection (LCM). Combined with a unique fixation and protein extraction protocol, we pushed the limits of microproteomics and demonstrated that proteins from LCM-isolated protrusions can successfully and reproducibly be identified by mass spectrometry using ultra-high field Orbitrap technologies. Finally, our method confirmed that different subtypes of protrusions have distinct proteomes. Therefore, our method created a unique opportunity to characterize TNTs shedding light on their role in health and disease. In this SCORE SC1 grant, we propose a three-step strategy to utilize our LCM/MS method to study TNT formation and function. This entails: 1) Expanding the TNT proteome by incorporating different cell types, induction methods, and TNT substructures using our LCM/MS method; 2) Collecting the TNT transcriptome to limit the detection bias of the individual platforms while at the same time cross-validating TNT protein/pathway identiﬁcations; and, 3) Identifying conserved TNT proteins and pathways, as well as potentially druggable proteins and biomarkers.

我们的研究重点是隧道纳米管（TNT）的作用——一种新的功能机制细胞之间的连接——在病毒传播过程中，错误折叠的蛋白质聚集体（导致神经退行性疾病），以及它们在癌症增殖和持续存在中可能发挥的作用。 TNT 已在多种细胞类型中发现，允许运输胞质和膜结合的物质分子、细胞器、钙通量和病原体的传播在体外，这些结构非常重要。它们的结构和功能都出现了异质性和巨大的差异。类似的结构也存在于体内和组织外植体中，不幸的是，目前人们对这种基本结构知之甚少。 TNT 形成的机制、其结构成分或所涉及的信号通路。最近的研究表明，TNT 确实在健康和健康方面发挥着重要的生理作用。事实上，TNT 是电、抗原和基因组信号传导的重要介质，同时也是疾病的重要介质。促进缺血、炎症和缺氧损伤后的细胞恢复此外，逆转录病毒，例如 HIV-1、HSV-1、HTLV-1 和流感利用这些亚细胞结构通过逃避来促进感染此外，Aβ、朊病毒和 HIV-1 Nef 等致病颗粒和蛋白质也受到免疫监视。发现诱导并篡夺 TNT 样结构在细胞之间传播。高效，因为它避免了扩散转移并逃避免疫检测。最后，TNT 可以介导直接作用。肿瘤细胞与其基质之间代谢和遗传物质的转移增强肿瘤细胞化疗耐药性、肿瘤进展和转移。凭借之前的 NIH SCORE SC2 试点项目奖，我们成功开发了一种新方法根据其形态或荧光标记，专门分离不同的突起亚型激光捕获显微切割 (LCM) 与独特的固定和蛋白质提取方案相结合。突破了微蛋白质组学的极限，并证明来自 LCM 分离突起的蛋白质可以使用超高场 Orbitrap 技术通过质谱法成功且可重复地识别。最后，我们的方法证实了不同亚型的突起具有不同的蛋白质组，因此，我们的方法。该方法创造了一个独特的机会来表征 TNT，揭示它们在健康和疾病中的作用。在 SCORE SC1 资助中，我们提出了一个三步策略，利用我们的 LCM/MS 方法来研究 TNT 的形成和功能需要：1) 通过整合不同的细胞类型来扩展 TNT 蛋白质组，诱导方法，以及使用我们的 LCM/MS 方法的 TNT 子结构 2) 收集 TNT 转录组；限制各个平台的检测偏差，同时交叉验证 TNT 蛋白/通路鉴定；以及，3) 鉴定保守的 TNT 蛋白和途径，以及潜在的可成药性蛋白质和生物标志物。