Targeting a Membrane Protease that Controls NK Cell Maturation

项目总结/摘要 健康和疾病中的多种重要生物学途径由膜内蛋白水解调节， 实现脂双层内或脂双层附近蛋白质底物靶向切割的过程。四 已经描述了膜内蛋白酶的种类，包括位点2蛋白酶（S2 P）金属蛋白酶 家族、菱形丝氨酸蛋白酶家族、Rce 1谷氨酰蛋白酶家族和谷氨酰蛋白酶家族 包括早老素和相关的信号肽肽酶（SPP）样（SPPL）亚家族。天冬氨酰 SPPL亚家族的蛋白酶，包括SPP、SPPL 2a、SPPL 2b、SPPL 2c和SPPL 3，可能是最少的 已知的膜内蛋白酶的子集。直到最近才发现其中一些生物学作用 蛋白酶的出现，只有少数相关的生物底物已被确定。如何将这些 膜内蛋白酶特异性地识别和切割它们的底物仍然是个谜。我们发现 SPPL 3在NK细胞成熟和细胞毒性中的关键作用。SPPL 3以小区自主的方式被要求 用于使NK细胞从未成熟的CD 27 + CD 11b-阶段成熟为CD 27 + CD 11b+和CD 27-CD 22 b + 阶段，以及针对肿瘤细胞靶点的正常NK细胞细胞毒性。仅表达SPPL 3的小鼠 NK细胞系中的D271 A揭示了SPPL 3的蛋白水解功能是NK细胞成熟所必需的， 功能像SPPL亚家族的其他胆固醇基膜内蛋白酶一样，关于如何在膜内蛋白酶中起作用知之甚少。 SPPL 3识别并切割其底物。所有SPPL蛋白酶都具有YD和GXGD基序， 除了蛋白质中底物识别和切割所需的其他区域外， 尚未被确认。此外，尽管已经鉴定了一些SPPL 3底物，但相关的SPPL 3底物的活性仍然是不稳定的。 在NK细胞成熟过程中必须被SPPL 3切割的底物目前是未知的。为了扩大我们的基础 了解SPPL 3和相关的乙酰基膜内蛋白酶，并促进对 由于SPPL 3控制NK细胞成熟的关键检查点，我们将使用新开发的底物结合， 确定底物选择所需的SPPL 3决定簇和鉴定SPPL 3底物的切割试验 在NK细胞中。我们的研究结果将扩大对膜内乙酰基蛋白酶的理解， 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.