Ahmed Bhat Eijaz, Sajjad Nasreena, Ahmad Tantray Javeed, Hor Yan-Yan, Rather Irfan A
Life Science Institute, Zhejiang University, Hangzhou, Zhejiang 310058, PR China.
Department of Biochemistry, University of Kashmir, Srinagar, 190006, India.
Saudi J Biol Sci. 2021 Mar;28(3):1607-1614. doi: 10.1016/j.sjbs.2020.12.049. Epub 2021 Jan 5.
The formation of inflammasome complexes contributes inactivation of inflammatory caspases caspase 1, which is generally considered essential for the innate response. Three proteins constituted this inflammasome complex, such as Nod-like receptors (NLRP or AIM2), ASC possessing caspase-recruiting domain, and caspase-1. The ASC proteins comprise two domains, the N-terminal PYD domain responsible for the interaction of various proteins, including PYD only protein 3 (POP3), and the CARD domain for association with other proteins. The PYRIN Domain-Only Protein POP3 negatively regulates responses to DNA virus infection by preventing the ALR inflammasome formation. POP3 directly interacts with ASC, therefore inhibiting ASC recruitment to AIM2-like receptors (ALRs). In the current study, we designed various constructs of the PYRIN Domain-Only Protein 3 (POP3) and ASC PYD domain to find the best-overexpressed construct for biochemical characterization as well as our complex studies. We cloned, purified, and characterized the PYD domain of pyrin only protein 3 and ASC PYD domain under physiological conditions. Our study clearly shows that the ASC PYD domain of corresponding amino acid 1-96 aa with ease self-oligomerization in physiological buffer conditions, and complex formation of POP3 PYD (1-83 aa) was inhibited by ASC PYD domain. Besides, we purified the PYD of POP3 protein in low and high salt conditions and different pH values for their biochemical characterization. Our results showed that POP3 formed a dimer under normal physiological conditions and was stable under normal buffer conditions; however, the purification in extremely low pH (pH5.0) conditions shows unstable behavior, the high salt conditions (500 mM NaCl) influence the protein aggregation. SDS PAGE arbitrated the homogeneity of the PYD domain of pyrin only protein 3 and ASC PYD domain of corresponding amino acids 1-83 and 1-96, respectively. Furthermore, our native PAGE shows the PYD domain of pyrin; only protein 3 did not form a complex with ASC PYD domain because of oligomerization mediated by the PYD domain.
炎性小体复合物的形成有助于炎症性半胱天冬酶（半胱天冬酶-1）失活，半胱天冬酶-1通常被认为是天然免疫反应所必需的。三种蛋白质构成了这种炎性小体复合物，如Nod样受体（NLRP或AIM2）、具有半胱天冬酶募集结构域的ASC和半胱天冬酶-1。ASC蛋白包含两个结构域，N端的PYD结构域负责与各种蛋白质相互作用，包括仅含PYD的蛋白3（POP3），以及用于与其他蛋白质结合的CARD结构域。仅含PYRIN结构域的蛋白POP3通过阻止ALR炎性小体形成来负向调节对DNA病毒感染的反应。POP3直接与ASC相互作用，从而抑制ASC募集到AIM2样受体（ALR）。在本研究中，我们设计了多种仅含PYRIN结构域的蛋白3（POP3）和ASC PYD结构域的构建体，以寻找用于生化特性分析以及我们的复合物研究的最佳过表达构建体。我们在生理条件下克隆、纯化并表征了仅含PYRIN的蛋白3的PYD结构域和ASC PYD结构域。我们的研究清楚地表明，相应氨基酸1 - 96位的ASC PYD结构域在生理缓冲条件下易于自我寡聚化，并且POP3 PYD（1 - 83位氨基酸）的复合物形成受到ASC PYD结构域的抑制。此外，我们在低盐和高盐条件以及不同pH值下纯化了POP3蛋白的PYD结构域以进行生化特性分析。我们的结果表明，POP3在正常生理条件下形成二聚体，并且在正常缓冲条件下稳定；然而，在极低pH（pH5.0）条件下纯化显示出不稳定行为，高盐条件（500 mM NaCl）影响蛋白质聚集。SDS - PAGE分别鉴定了仅含PYRIN的蛋白3的PYD结构域以及相应氨基酸1 - 83和1 - 96的ASC PYD结构域的均一性。此外，我们的非变性PAGE显示，由于PYD结构域介导的寡聚化，仅含PYRIN的蛋白3的PYD结构域未与ASC PYD结构域形成复合物。
