Biochemical mechanisms of inflammasome function

炎症小体功能的生化机制

• 批准号: RGPIN-2020-04566
• 负责人: MacDonald, Justin
• 金额: $ 2.62万
• 依托单位: University of Calgary
• 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=744270
• 关键词: Biochemical; mechanisms; inflammasome; function

The innate immune system acts as the first line of defense for animals and plants in reconciling cellular injury and infection through the early detection of impending threats, and consequential triggering of pro-inflammatory responses. The NOD-like receptors-containing pyrin (NLRP) subfamily of proteins have emerged as key sensors of intracellular danger signals. Rapidly developing biological models now indicate the various NLRPs are of paramount importance in initiating inflammatory responses to cellular injury; however, critical aspects regarding the underlying biochemistry of this protein family and their signaling complexes remain unresolved. NLRPs contain a central NACHT domain with ATP-binding and hydrolysis properties, (sometimes referred to as the nucleotide-binding oligomerization domain or NOD) that designate NLRPs as members of the larger ATPases-Associated with various cellular Activities (AAA+ ATPase) superfamily. The enzymatic properties of NACHT domains are thought to regulate NLRP oligomerization and inflammasome activation, yet much remains to be understood regarding its structure and function. NLRP3 is the most thoroughly studied and is considered to be the prototypical inflammasome-forming member, yet the basic biochemistry of NLRP3 as well as the remaining NLRP proteins (14 members in the family) remains incompletely characterized. Many assumptions regarding the enzymatic activity of NLRPs and the intrinsic regulation of inflammasome formation are based primarily upon analogy with limited corroborating evidence. Further characterization of the enzymology of inflammasome activation as well as the integration of recently revealed post-translational modifications in regulating inflammatory signaling pathways will be critical for a comprehensive understanding of this protein family. In this regard, we propose that specific biochemical properties of different NLRPs drive distinctions in catalytic activities, which in turn can impact upon oligomerization and inflammatory signaling. Thus, a definition of the enzymology of NLRP proteins and the role of ATP in driving inflammasome assembly and activation of downstream signaling pathways will be important to advance understanding of this innate immune process. It is clear that holistic studies of the entire NLRP family are necessary, and we will creatively advance understanding of the NLRP family as a whole and their propensity to assemble into functional inflammasomes. To address the knowledge deficit, systems for the effective production of all NLRP proteins will be defined, biochemical assessments of enzymatic function will be completed, novel technologies and reagents for biological interrogations will be developed, and precise structural definitions of the catalytic NACHT domain will be generated. Our technical advances in recombinant NLRP protein production support biochemical analyses of inflammasome catalytic function as well as structural biology approaches.

先天免疫系统作为动物和植物的第一道防线，通过早期检测即将发生的威胁和相应的促炎反应的触发来协调细胞损伤和感染。含有NOD样受体的pyrin（NLRP）亚家族蛋白已成为细胞内危险信号的关键传感器。快速发展的生物学模型现在表明，各种NLRP在启动细胞损伤的炎症反应中至关重要;然而，关于该蛋白家族及其信号复合物的潜在生物化学的关键方面仍然没有解决。NLRP含有具有ATP结合和水解性质的中心NACHT结构域，（有时称为核苷酸结合寡聚化结构域或NOD），其将NLRP指定为与各种细胞活性相关的较大ATP酶的成员NACHT结构域的酶性质被认为调节NLRP寡聚化和炎性小体活化，但关于其结构和功能仍有许多有待理解。NLRP 3是研究最彻底的，被认为是原型炎性小体形成成员，但NLRP 3的基本生物化学以及其余NLRP蛋白（家族中的14个成员）仍然不完全表征。关于NLRP的酶活性和炎性小体形成的内在调节的许多假设主要基于有限的确证证据的类比。进一步表征炎性小体激活的酶学以及整合最近发现的调节炎症信号传导途径的翻译后修饰对于全面了解该蛋白质家族至关重要。在这方面，我们提出不同NLRP的特定生化特性驱动催化活性的差异，这反过来又会影响寡聚化和炎症信号传导。因此，NLRP蛋白的酶学定义和ATP在驱动炎性小体组装和激活下游信号通路中的作用对于促进对这种先天免疫过程的理解将是重要的。很明显，对整个NLRP家族的整体研究是必要的，我们将创造性地推进对NLRP家族整体及其组装成功能性炎性小体的倾向的理解。为了解决知识的不足，将定义所有NLRP蛋白的有效生产系统，将完成酶功能的生化评估，将开发用于生物询问的新技术和试剂，并将生成催化NACHT结构域的精确结构定义。我们在重组NLRP蛋白生产方面的技术进步支持炎性小体催化功能的生化分析以及结构生物学方法。