Regulation of Neutral Sphingomyelinase 2 by Inter-Domain Interactions

描述（由申请人提供）：中性鞘磷脂酶2 （nSMase2）是一种在细胞应激反应中产生生物活性脂质神经酰胺的酶，涉及多种重要途径和人类疾病，包括细胞凋亡、癌症、炎症反应、神经系统疾病、生长发育和骨稳态。该项目的长期目标是了解调控nSMase2活性的分子细节机制，以帮助开发新的nSMase2靶向治疗方法。虽然构成膜相关，但nSMase2显示低基底鞘磷脂酶（SMase）活性，需要阴离子磷脂（apl）激活。nSMase2的结构域结构包括两个结构域：一个将nSMase2系在膜上的疏水n端结构域和一个可溶催化c端结构域。我们现在已经确定APL结合域仅定位于n端结构域，但不了解APL结合如何激活可溶性c端结构域。初步数据表明，域间相互作用介导了这一过程。在本提案中，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。我们将使用一种改良的膜酵母双杂交（MYTH）系统来识别分子内界面上的特定残基。2)定义api在调节域间交互中的作用。我们将利用MYTH系统在缺乏主要APL（磷脂酰丝氨酸）的酵母细胞中探测缺乏APL时结构域间的相互作用。我们将确定APL结合是否促进域间相互作用，以及在APL结合时分子内界面是否发生变化。3)在分子细节上描述活化的基础。我们将确定n端肽与催化结构域的结合亲和力，并确定催化结构域单独和与n端肽结合的晶体结构。综上所述，这些结果将促进我们对nSMase2调控的理解，并促进新型nSMase2靶向治疗的开发。此外，MYTH系统的开发将为nSMase2抑制剂的高通量小分子筛选建立一个体系。这些研究与癌症和炎症直接相关。

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.