Investigating the fundamental mechanisms of immune cell exocytosis

研究免疫细胞胞吐作用的基本机制

• 批准号: RGPIN-2020-07139
• 负责人: Sugita, Shuzo
• 金额: $ 3.64万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=763305
• 关键词: Investigating; fundamental; mechanisms; immune; cell

Vesicular exocytosis is a fundamental cellular process that plays an imperative role in both brain and immune system function. In the brain, synaptic vesicle exocytosis is the basis for interneuronal communication, enabling a diversity of functional and cognitive processes. In the immune system, specialized immune cells such as mast cells, natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) undergo secretory granule exocytosis, which facilitates the eradication of infected and malignant cells. Neurons and immune cells use paralogous sets of proteins to control Ca2+-dependent exocytosis. However, the functional machinery involved in immune cell exocytosis is much less characterized and understudied compared to the intensively studied neuronal exocytosis. My lab has developed extensive knowledge and expertise in studying both neuronal and immune cell exocytosis, which has led to a synergy in understanding common and distinct aspects between the two processes. For example, although SNARE proteins play essential roles in both types of exocytosis, each type appears to utilize different isoforms of SNARE proteins. Furthermore, neuronal and immune cell exocytosis also employ distinct proteins for inducing exocytosis, although common C2 domains seem to be used for the Ca2+ sensing action that is required. The long-term objective of the present research program is to elucidate the mechanisms of immune cell exocytosis and ultimately, to modify and enhance immune cell exocytosis to generate a more robust immune system. The short-term objective is to determine the key SNARE proteins and their regulators (or priming proteins) that mediate immune cell exocytosis and to investigate the underlying biochemical mechanisms of immune cell exocytotic regulation. Based on our recent studies, we propose the following hypotheses: 1) Syntaxin-3 and SNAP-23 are the key t-SNARE proteins involved in immune cell exocytosis. 2) Munc13-4 plays a dual role in secretory granule exocytosis by acting as a Ca2+ sensor for exocytosis and facilitating exocytotic priming along with Munc18-2. 3) The Ca2+ sensing function of Munc13-4 is mediated via its C2 domains, while the priming function of Munc13-4 and Munc18-2 is facilitated through interactions with the SNARE complexes containing syntaxin-3 and SNAP-23. We will explore these hypotheses by focusing on the following Specific Aims: Aim 1. What are the critical t-SNARE proteins for immune cell exocytosis? Aim 2. Can potential gain-of-function Munc13-4 and Munc18-2 mutants enhance exocytosis from immune cells as well as increase the cytotoxicity of NK cells? Aim 3. What is the biochemical basis for the regulatory functions of Munc13-4? Our research will provide fundamental insight into the mechanisms of granule exocytosis in immune cells. Importantly, the knowledge that would be obtained from this study has the potential to reveal novel strategies for improving the effectiveness of the immune system.

囊泡胞吐作用是一个基本的细胞过程，在大脑和免疫系统功能中起着至关重要的作用。在大脑中，突触囊泡胞吐作用是神经元间通讯的基础，使功能和认知过程的多样性成为可能。在免疫系统中，特化免疫细胞如肥大细胞、自然杀伤（NK）细胞和细胞毒性T淋巴细胞（CTL）经历分泌颗粒胞吐作用，这有助于根除感染的和恶性的细胞。神经元和免疫细胞使用旁系同源蛋白质组来控制Ca 2+依赖性胞吐作用。然而，与深入研究的神经元胞吐作用相比，免疫细胞胞吐作用所涉及的功能机制的特征要少得多，而且研究不足。 我的实验室在研究神经元和免疫细胞胞吐方面积累了丰富的知识和专业知识，这导致了对这两个过程之间共同和不同方面的理解。例如，尽管SNARE蛋白在两种类型的胞吐中发挥重要作用，但每种类型似乎利用不同的SNARE蛋白亚型。此外，神经元和免疫细胞的胞吐作用也采用不同的蛋白质诱导胞吐作用，虽然共同的C2域似乎是用于所需的Ca 2+传感行动。 本研究计划的长期目标是阐明免疫细胞胞吐作用的机制，并最终修改和增强免疫细胞胞吐作用，以产生更强大的免疫系统。短期目标是确定介导免疫细胞胞吐的关键SNARE蛋白及其调节剂（或引发蛋白），并研究免疫细胞胞吐调节的潜在生化机制。基于我们最近的研究，我们提出以下假设：1）Syntaxin-3和SNAP-23是参与免疫细胞胞吐的关键t-SNARE蛋白。 2)Munc 13 -4在分泌颗粒胞吐中起双重作用，作为胞吐的Ca 2+传感器，并与Munc 18 -2一起沿着促进胞吐引发。3)Munc 13 -4的Ca 2+感应功能是通过其C2结构域介导的，而Munc 13 -4和Munc 18 -2的引发功能是通过与含有syntaxin-3和SNAP-23的SNARE复合物的相互作用来促进的。 我们将通过关注以下具体目标来探索这些假设：目标1。免疫细胞胞吐作用的关键t-SNARE蛋白是什么？目标二。潜在的功能获得性Munc 13 -4和Munc 18 -2突变体能否增强免疫细胞的胞吐作用并增加NK细胞的细胞毒性？ 目标3. Munc 13 -4调控功能的生物化学基础是什么？我们的研究将为免疫细胞中颗粒胞吐作用的机制提供基本的见解。重要的是，从这项研究中获得的知识有可能揭示改善免疫系统有效性的新策略。