Neurosteroid Inhibition of Pyroptotic Lysis

项目摘要 焦亡是一种细胞死亡程序，参与全球主要死亡原因的发病机制。 半胱天冬酶-1家族蛋白酶通过释放gasdermin D的成孔部分来启动焦亡， 插入质膜导致细胞溶解。热解溶解过程中释放的细胞因子导致 局部和全身病理学，但调节gasdermin D孔形成和溶解的过程并不好 明白我们最近发现神经甾体硫酸双烯醇酮作为一种新的抑制剂pyroptotic 裂解，但其作用机制尚不清楚。神经类固醇包括硫酸双烯醇酮， 亲脂性骨架，并与质膜蛋白和脂质相互作用，这表明假设， 这些分子可以破坏gasdermin D孔的形成。该提案旨在了解如何 硫酸双烯醇酮防止pyroptotic裂解，并确定是否其他类固醇共享此活动。 我们的初步数据表明，硫酸双烯醇酮在不影响细胞凋亡的情况下， 炎性体或半胱天冬酶-1的上游活化。本提案中概述的实验将测试 硫酸双烯醇酮影响焦亡期间孔形成假设和评估gasdermin D 裂解、质膜定位和寡聚化。我们还将讨论替代假设 硫酸双烯醇酮可以调节细胞体积以防止溶解或增强膜修复过程。 总之，这些实验的结果将为精确地理解细胞凋亡的机制提供信息。 作用，并揭示了一种新的策略，以破坏热解裂解。 类固醇骨架由大量分子共享，每个分子具有特定的生物活性。我们 初步数据表明，可能存在类固醇抑制热解溶解的分子决定因素， 相关类固醇显示效力降低。我们将测试一个合理选择的结构不同的库， 类固醇的能力，以防止pyroptotic溶解。这些实验将确定是否内源性 类固醇显示与潜在生理相关性一致的效力。此外，这些结果将 确定抑制热解的结构-活性关系，以促进开发有效的 特定的分子，并定义了疾病干预的新范式。

Project Summary Pyroptosis is a program of cell death involved in the pathogenesis of leading global causes of mortality. Caspase-1 family proteases initiate pyroptosis by releasing the pore-forming portion of gasdermin D, which inserts into the plasma membrane leading to cell lysis. Cellular factors released during pyroptotic lysis cause local and systemic pathology, but processes regulating gasdermin D pore formation and lysis are not well understood. We recently identified the neurosteroid pregnenolone sulfate as a novel inhibitor of pyroptotic lysis, but its mechanism of action is not yet known. Neurosteroids including pregnenolone sulfate have a lipophilic backbone and interact with plasma membrane proteins and lipids, suggesting the hypothesis that these molecules may disrupt formation of gasdermin D pores. This proposal aims to understand how pregnenolone sulfate prevents pyroptotic lysis and determine whether other steroids share this activity. Our preliminary data demonstrate that pregnenolone sulfate prevents lysis during pyroptosis without affecting upstream activation of inflammasomes or caspase-1. The experiments outlined in this proposal will test the hypothesis that pregnenolone sulfate affects pore formation during pyroptosis and assess gasdermin D cleavage, plasma membrane localization and oligomerization. We will also address the alternative hypotheses that pregnenolone sulfate may regulate cell volume to prevent lysis or potentiate membrane repair processes. Together, the results of these experiments will inform a precise molecular understanding of the mechanism of action and reveal a novel strategy to disrupt pyroptotic lysis. The steroid backbone is shared by a large number of molecules, each with specific biological activities. Our preliminary data suggest that there may be molecular determinants for steroid inhibition of pyroptotic lysis, as a related steroid demonstrates reduced potency. We will test a rationally selected library of structurally distinct steroids for the ability to prevent pyroptotic lysis. These experiments will determine whether endogenous steroids demonstrate potency consistent with potential physiologic relevance. In addition, these results will identify the structure-activity relationship for inhibition of pyroptotic lysis to facilitate development of potent and specific molecules and define a new paradigm for disease intervention.