Structure and mechanism of signal peptide peptidase

描述（由申请人提供）：最近几年发现了一类新的蛋白酶，其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。 这些膜内蛋白酶是非常保守的，并在生物学和人类疾病中发挥许多重要作用。 这一类的一个家族与早老素有较远的关系，并且以信号肽肽酶（SPP）为例，其在信号肽酶作用后切割留在膜中的残余信号肽片段。 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。 SPP是一种推定的γ-淀粉酰蛋白酶，它似乎与含有早老素的γ-分泌酶复合物具有许多生物化学性质。 然而，与早老素不同，SPP显然不需要与其他蛋白质辅因子组装，也不需要在其成熟为活性蛋白酶的过程中被加工成两部分。 在这个应用程序中，我们建议工作的SPP作为一个代表性的膜内蛋白酶，在生物学和医学中至关重要的新兴酶类的机械和结构的理解。 对SPP型蛋白酶的详细了解应该为膜内蛋白水解的机制提供巨大的洞察力。提出了以下具体目标：（1）SPP如何处理衬底？我们将讨论SPP是否在两个不同的位点切割其底物，如γ-分泌酶，以及SPP是否像γ-分泌酶一样含有初始底物对接位点。(2)SPP的单体单元在二聚体中是如何排列的？ 使用诱变和氧化交联，我们将确定SPP中哪些半胱氨酸有助于晚餐界面以及跨膜的二聚体内的半胱氨酸是如何排列的。(3)SPP活动站点的性质是什么？ 我们已经发现，C-末端的一半单独的SPP是催化活性，我们试图生化表征这个催化域和工作对它的结构通过NMR。(4)极端嗜热古菌的SPP直向同源物有什么特征？极端嗜古细菌的SPP样蛋白酶应该比其他全长SPP更稳定，更适合结构研究。 我们将致力于表达，纯化和古细菌SPPs的表征，为结构阐明奠定基础。 总结：信号肽肽酶（SPP）是一种蛋白质切割酶，在免疫系统中起着重要作用，丙型肝炎病毒将人类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，并被广泛认为是治疗的重要潜在靶点。 该计划是为了更充分地了解SPP是如何工作的，并采取步骤纯化SPP，以最终阐明其结构。 SPP的结构将为了解一类对生物学和医学都至关重要的不寻常的新型酶的工作机制提供巨大的见解。

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.