Structure and mechanism of signal peptide peptidase

描述(申请人提供)：在过去的几年里，发现了一类新的蛋白酶，它可以裂解底物的跨膜结构域，并且显然包含一个膜嵌入的活性部位。这些膜内蛋白水解酶非常保守，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较远的亲缘关系，例如信号肽酶(SPP)，它在信号肽的作用下裂解残留在细胞膜上的信号肽片段。SPP对免疫监测至关重要，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。作为一种可能的天冬氨酸蛋白酶，SPP似乎与含有早老素的γ-分泌酶复合体有许多共同的生化性质。然而，与早老素不同的是，SPP显然不需要与其他蛋白质辅助因子组装，也不需要在成熟过程中将其加工成两块，成为活性蛋白酶。在这一应用中，我们建议致力于从机理和结构上理解SPP作为一种代表性的膜内蛋白酶，这是一种在生物和医学中至关重要的新兴酶类。对SPP型蛋白水解酶的详细了解将有助于我们深入了解膜内蛋白分解的机制。提出了以下具体目标：(1)SPP如何处理衬底？我们将讨论SPP是否像y-分泌酶一样在两个不同的位置切割底物，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)SPP的单体单元在二聚体中是如何排列的？利用突变和氧化交联，我们将确定SPP中哪些半胱氨酸对晚餐界面起作用，以及跨膜天冬氨酸在二聚体中是如何排列的。(3)SPP活性部位的性质是什么？我们已经发现SPP的C-末端一半单独具有催化活性；我们试图对这个催化结构域进行生化表征，并通过核磁共振对其结构进行研究。(4)极端古生菌的SPP同源基因有哪些特征？与其他全长SPP相比，嗜极端考古细菌的SPP样蛋白酶应该更稳定，更容易进行结构研究。我们将致力于古细菌SPP的表达、纯化和鉴定，为结构阐明奠定基础。摘要：信号肽(SPP)是一种蛋白质切割酶，在免疫系统中发挥重要作用，丙型肝炎病毒利用人SPP作为其感染过程的一部分。SPP还与一种复杂得多的酶有关，这种酶在阿尔茨海默病中发挥关键作用，被广泛认为是重要的潜在治疗靶点。我们的计划是更充分地了解SPP是如何工作的，并采取措施纯化SPP，以最终阐明其结构。SPP的结构将为一类不寻常的新酶的工作原理提供巨大的洞察力，这些酶对生物学和医学都是至关重要的。

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.