Targeting a Membrane Protease that Controls NK Cell Maturation

项目摘要/摘要 在健康和疾病中，许多重要的生物途径都是由膜内蛋白分解调节的， 在脂双层内或其附近实现蛋白质底物靶向切割的过程。四 膜内蛋白水解酶的种类已被描述，包括2位点蛋白水解酶(S2P)金属蛋白酶 家族、菱形丝氨酸蛋白酶家族、Rce1谷氨酰蛋白酶家族和天冬氨酸蛋白酶家族 这包括早老素和相关信号肽(SPP)样(SPPL)亚家族。天冬氨酸氨基 SPPL亚家族的蛋白酶，包括SPP、SPPL2a、SPPL2b、SPPL2c和SPPL3可能是最少的 了解膜内蛋白水解酶亚群。直到最近，才发现其中一些的生物学作用 蛋白水解酶出现了，相关的生物底物只有几个被鉴定出来。这些是如何 膜内蛋白水解酶对底物的识别和裂解仍然是个谜。我们发现 SPPL3在NK细胞成熟和细胞毒中的关键作用。以单元自治的方式需要SPPL3 使NK细胞从未成熟的CD27 CD11b阶段成熟到CD27 CD11b和CD27-CD22b阶段 对于正常的NK细胞对肿瘤细胞靶点的杀伤作用。只表达SPPL3基因的小鼠 在NK谱系中的D271A揭示了SPPL3的蛋白降解功能是NK细胞成熟所必需的，并且 功能。像其他SPPL亚家族的天冬氨酸膜内酶一样，人们对它是如何被激活的知之甚少。 SPPL3识别并切割其底物。所有SPPL酶都含有YD和GXGD基序，这些基序包含 催化天冬氨酸，但蛋白质中底物识别和切割所需的其他区域 还没有确定身份。此外，尽管已经确定了一些SPPL3底物，但相关的 在NK细胞成熟过程中必须被SPPL3切割的底物目前尚不清楚。为了扩展我们的基本 了解SPPL3和相关的天冬氨酸膜内酶，并促进对SPPL3的理解 由SPPL3控制的NK细胞成熟的关键检查点，我们将使用新开发的底物结合和 裂解试验确定底物选择所需的SPPL3决定因素并鉴定SPPL3底物 在NK细胞中。我们的结果将扩大对膜内天冬氨酸蛋白酶的理解，并阐明分子 控制NK细胞发育和功能的事件。

PROJECT SUMMARY/ABSTRACT A variety of important biological pathways in health and disease are regulated by intramembrane proteolysis, the process that achieves the targeted cleavage of protein substrates within or proximal to the lipid bilayer. Four classes of intramembrane proteases have been described, including the site 2 protease (S2P) metalloprotease family, the rhomboid serine protease family, the Rce1 glutamyl protease family, and the aspartyl protease family that includes presenilins and the related Signal Peptide Peptidase (SPP)-like (SPPL) subfamily. The aspartyl proteases of the SPPL subfamily, including SPP, SPPL2a, SPPL2b, SPPL2c, and SPPL3, are perhaps the least understood subset of intramembrane proteases. Only recently have the biological roles of some of these proteases emerged, and only a handful of relevant biological substrates have been identified. How these intramembrane proteases specifically recognize and cleave their substrates remains mysterious. We discovered a critical role for SPPL3 in NK cell maturation and cytotoxicity. SPPL3 is required in a cell-autonomous manner for the maturation of NK cells from the immature CD27+CD11b- stage to the CD27+CD11b+ and CD27-CD22b+ stages, and for normal NK cell cytotoxicity toward tumor cell targets. Mice engineered to express only SPPL3 D271A in the NK lineage revealed that the proteolytic function of SPPL3 is required in NK cell maturation and function. Like other aspartyl intramembrane proteases of the SPPL subfamily, very little is known about how SPPL3 recognizes and cleaves its substrates. All SPPL proteases possess YD and GXGD motifs that contain the catalytic aspartates, but other regions in the protein that are required for substrate recognition and cleavage have not been identified. Furthermore, although some SPPL3 substrates have been identified, the relevant substrate that must be cleaved by SPPL3 during NK cell maturation is currently unknown. To expand our basic understanding of SPPL3 and related aspartyl intramembrane proteases, and to advance understanding of the key checkpoint in NK cell maturation controlled by SPPL3, we will use newly developed substrate binding and cleavage assays to define SPPL3 determinants required for substrate selection and to identify SPPL3 substrates in NK cells. Our results will expand understanding of intramembrane aspartyl proteases and illuminate molecular events that control NK cell development and function.