Regulation of Neutral Sphingomyelinase 2 by Inter-Domain Interactions

说明(申请人提供)：中性鞘磷脂酶2(NSMase2)是一种在细胞应激反应中在质膜上产生生物活性脂质神经酰胺的酶，与多种重要途径和人类疾病有关，包括细胞凋亡、癌症、炎症反应、神经疾病、生长发育和骨骼稳态。该项目的长期目标是从分子上详细了解调节nSMase2活性的机制，以帮助开发新的nSMase2靶向疗法。尽管nSMase2在结构上与膜相关，但它表现出较低的基础鞘磷脂酶(SMase)活性，需要阴离子磷脂(APL)激活。NSMase2的结构域包括两个结构域：一个疏水的N-末端结构域将nSMase2连接到膜上，以及一个可溶性的催化C-末端结构域。我们现在已经确定APL结合域只定位于N-末端结构域，但不知道APL结合是如何激活可溶性C-末端结构域的。初步数据表明，域之间的相互作用调节了这一过程。在这个提案中，我们将重点研究域间相互作用在nSMase2调控中的作用，并解决以下目标：1)描述激活所需的域间接口。我们将使用一种改进的膜酵母双杂交(MYTH)系统来鉴定分子内界面的特定残基。2)明确APL在调节域间相互作用中的作用。我们将利用缺乏主要磷脂酰丝氨酸的酵母细胞中的神话系统，来探索在缺乏磷脂酰丝氨酸的情况下结构域间的相互作用。我们将确定APL结合是否促进结构域间的相互作用，以及APL结合时分子内界面是否发生变化。3)从分子细节上表征活化的基础。我们将确定N-端肽与催化域的结合亲和力，并确定单独的催化域和与N-端肽结合的晶体结构。综上所述，这些结果应该会促进我们对nSMase2调控的理解，并促进新的nSMase2靶向治疗药物的开发。此外，Myth系统的开发将建立一个高通量小分子筛选nSMase2抑制剂的系统。这些研究与癌症和炎症直接相关。 公共卫生相关性：中性鞘磷脂酶2(NSMase2)影响人体细胞的功能，与炎症和包括癌症在内的各种疾病有关。本研究旨在更详细地了解nSMase2的激活情况，为设计治疗和预防人类疾病的新药提供帮助。

DESCRIPTION (provided by applicant): Neutral sphingomyelinase 2 (nSMase2) is an enzyme that generates the bioactive lipid ceramide at the plasma membrane in response to cell stress and has been implicated in a variety of important pathways and human diseases including apoptosis, cancer, inflammatory responses, neurological disorders, growth and development, and bone homeostasis. The long-term goal of this project is to understand in molecular detail the mechanisms regulating nSMase2 activity to aid in the development of novel nSMase2 target therapeutics. Although constitutively membrane-associated, nSMase2 displays low basal sphingomyelinase (SMase) activity and requires activation by anionic phospholipids (APLs). The domain architecture of nSMase2 comprises two domains: a hydrophobic N-terminal domain that tethers nSMase2 to the membrane and a soluble catalytic C-terminal domain. We have now determined the APL binding domain to localize exclusively to the N-terminal domain but do not understand how APL binding activates the soluble C-terminal domain. Preliminary data suggests inter-domain interactions mediate this process. In this proposal we will focus our investigation on the role of inter-domain interactions in nSMase2 regulation and address the following aims: 1) To delineate the inter-domain interface required for activation. We will use a modified membrane yeast two hybrid (MYTH) system to identify specific residues at the intramolecular interface. 2) To define the role of APLs in modulating inter-domain interactions. We will utilize the MYTH system in yeast cells lacking the major APL, phosphatidylserine, to probe inter-domain interactions in the absence of APLs. We will determine if APL binding promotes inter- domain interactions and if the intramolecular interface changes upon APL binding. 3) To characterize the basis for activation in molecular detail. We will determine binding affinities of N-terminal peptides to the catalytic domain and determine crystal structures of the catalytic domain alone and bound to N-terminal peptides. Taken together, these results should advance our understanding of nSMase2 regulation and facilitate the development of novel nSMase2 targeted therapeutics. Moreover, the development of the MYTH system will establish a system for high-throughput small-molecule screening of nSMase2 inhibitors. These studies are directly relevant to cancer and inflammation. PUBLIC HEALTH RELEVANCE: The enzyme Neutral Sphingomyelinase 2 (nSMase2) affects the function of cells in the human body and is involved in inflammation and various diseases including cancer. This study aims to understand the activation of nSMase2 in great detail, to help design new drugs for the treatment and prevention of human diseases.