Structure and mechanism of signal peptide peptidase

描述（由申请人提供）：过去几年已经发现了一类新的蛋白酶，它们可以切割其底物的跨膜结构域，并且显然含有膜嵌入的活性位点。这些膜内蛋白酶非常保守，在生物学和人类疾病中发挥着许多重要作用。其中一个家族与早老素有远亲关系，信号肽肽酶（signal peptide peptidase， SPP）就是例证，它在信号肽酶作用后，将残留在膜上的信号肽片段进行切割。SPP对免疫监视至关重要，对丙型肝炎病毒核心蛋白的成熟至关重要，这表明它可能是抗病毒药物设计的一个有价值的靶点。作为一种假定的天冬氨酸蛋白酶，SPP似乎与含早老素的y分泌酶复合物具有许多生化特性。然而，与早老素不同，SPP显然不需要与其他蛋白质辅助因子组装，也不需要在其成熟为活性蛋白酶的过程中被加工成两个片段。在这个应用中，我们建议对SPP作为一种具有代表性的膜内蛋白酶的机制和结构进行理解，SPP是一种在生物学和医学中至关重要的新兴酶类。对spp型蛋白酶的详细了解将为膜内蛋白水解的机制提供巨大的见解。提出了以下具体目标：(1)SPP如何处理基板？我们将讨论SPP是否像y分泌酶一样在两个不同的位点切割底物，以及SPP是否像y分泌酶一样包含一个初始底物对接位点。(2)二聚体中SPP的单体是如何排列的？利用诱变和氧化交联，我们将确定SPP中哪些半胱氨酸参与晚餐界面，以及跨膜天冬氨酸如何在二聚体内排列。(3) SPP活性位点的性质是什么？我们发现仅SPP的C端一半具有催化活性；我们寻求生物化学表征这个催化结构域，并通过核磁共振研究它的结构。(4)嗜极古菌SPP同源物的特征是什么？来自嗜极古细菌的spp样蛋白酶应该比其他全长spp更稳定，更适合于结构研究。我们将致力于古细菌SPPs的表达、纯化和表征，为结构阐明奠定基础。摘要：信号肽肽酶（SPP）是一种在免疫系统中起重要作用的蛋白质切割酶，丙型肝炎病毒利用人SPP作为其感染过程的一部分。SPP还与一种更复杂的酶有关，这种酶在阿尔茨海默病中起着关键作用，被广泛认为是治疗的重要潜在靶点。计划是更充分地了解SPP是如何工作的，并采取步骤净化SPP，最终阐明其结构。SPP的结构将为了解一种对生物学和医学都至关重要的不寻常的新型酶的工作原理提供巨大的洞察力。

DESCRIPTION (provided by applicant): The last few years have seen the discovery of a new class of proteases that cleave the transmembrane domain of their substrates and that apparently contain a membrane-embedded active site. These intramembrane proteases are remarkably conserved and play many important roles in biology and human disease. One family of this class is distantly related to the presenilins and is exemplified by signal peptide peptidase (SPP), which cleaves remnant signal peptide fragments left in the membrane after the action of signal peptidase. SPP is critical for immune surveillance and is essential for the maturation of the core protein of the hepatitis C virus, suggesting it may be a worthy target for antiviral drug design. A putative aspartyl protease, SPP appears to share many biochemical properties with the presenilin-containing y- secretase complex. However, unlike presenilins, SPP apparently does not need to assemble with other protein cofactors nor is it processed into two pieces during its maturation into an active protease. In this application, we propose to work toward a mechanistic and structural understanding of SPP as a representative intramembrane protease, an emerging enzyme class of critical importance in biology and medicine. The detailed understanding of an SPP-type protease should provide tremendous insight into the mechanism of intramembrane proteolysis. The following specific aims are proposed: (1) How does SPP process substrates? We will address whether SPP cleave its substrates at two different sites, as does y- secretase, and whether SPP, like y-secretase contains an initial substrate docking site. (2) How are monomeric units of SPP arranged within a dimer? Using mutagenesis and oxidative crosslinking, we will determine which cysteines in SPP contribute to the dinner interface and how the transmembrane aspartates are aligned within the dimer. (3) What is the nature of the SPP active site? We have discovered that the C- terminal half of SPP alone is catalytically active; we seek to biochemically characterize this catalytic domain and work toward its structure by NMR. (4) What are the characteristics of SPP orthologs from extremophilic archaea? SPP-like proteases from extremophilic archaebacteria should be more stable and amenable to structural studies than other full-length SPPs. We will work toward expression, purification, and characterization of archaeal SPPs to set the stage for structural elucidation. LAY SUMMARY: Signal peptide peptidase (SPP) is a protein-cutting enzyme that plays an important role in the immune system, and the hepatitis C virus uses human SPP as part of its infection process. SPP is also related to a much more complicated enzyme that plays a key role in Alzheimer's disease and is widely considered an important potential target for therapy. The plan is to more fully understand how SPP works and to take steps toward purifying SPP for eventual elucidation of its structure. A structure of SPP would provide tremendous insight into the workings of an unusual new class of enzymes that are critical to both biology and medicine.