Structure and mechanism of signal peptide peptidase

信号肽肽酶的结构和机制

• 批准号: 7638550
• 负责人: Michael S Wolfe
• 金额: $ 33.25万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7638550
• 关键词: Active Sites; Address; Affinity; Affinity Labels; Alzheimer' s Disease; Antiviral Agents; Archaea; Aspartate; Aspartic Endopeptidases; Binding; Biochemical; Biological Assay; Biology; C-terminal; Catalytic Domain; Cells; Characteristics; Cleaved cell; Coin; Complex; Cut protein; Cysteine; Disulfides; Docking; Drug Design; Enzymatic Biochemistry; Enzymes; Escherichia coli; Family; Hepatitis C virus; Human; Immune system; Immunologic Surveillance; Infection; Label; Left; Length; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Medicine; Membrane; Mutagenesis; Mutate; Nature; Orthologous Gene; Peptide Fragments; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Play; Process; Prolactin; Property; Proteins; Proteolysis; Reagent; Recombinants; Research Personnel; Role; Serine; Serine Protease; Site; Staging; Structure; System; Testing; Transmembrane Domain; Work; analog; base; cofactor; crosslink; dimer; hepatitis C virus nucleocapsid protein; human disease; inhibitor/antagonist; insight; monomer; overexpression; presenilin; programs; protease So; research study; rhomboid; secretase; signal peptidase; signal peptide peptidase; structural biology

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.

描述（由申请人提供）：最近几年发现了一类新的蛋白酶，这些蛋白酶裂解了其底物的跨膜域，并且显然包含一个膜上的活性位点。 这些膜内蛋白酶非常保守，并在生物学和人类疾病中起许多重要作用。 该类别的一个家族与presenilins远距离相关，并用信号肽肽酶（SPP）示例，该信号肽肽酶（SPP）在信号肽酶作用后切开了残留的信号肽片段。 SPP对于免疫监测至关重要，对于乙型肝炎病毒的核心蛋白质的成熟至关重要，这表明它可能是抗病毒药物设计的值得靶标。 推定的天冬氨酸蛋白酶，SPP似乎与含有presenilin的Y-泌液酶复合物具有许多生化特性。 但是，与presenilins不同，SPP显然不需要与其他蛋白质辅助因子组装，也不需要在成熟成活性蛋白酶期间将其加工成两个部分。 在此应用中，我们建议致力于对SPP作为代表性膜内蛋白酶的机械和结构理解，这是一种新兴的生物学和医学重要性酶类别。 对SPP型蛋白酶的详细理解应提供有关膜内蛋白水解机制的深入了解。提出了以下特定目的：（1）SPP处理底物如何？我们将解决SPP是否像Y-secratase一样在两个不同的位点上裂解其底物，以及像Y-分泌酶一样的SPP是否包含一个初始的底物对接位点。 （2）如何在二聚体内排列SPP的单体单位？ 使用诱变和氧化交联，我们将确定SPP中哪种半胱氨酸有助于晚餐界面以及跨膜天冬氨酸如何在二聚体内对齐。 （3）SPP活动站点的性质是什么？ 我们发现，仅SPP的C-末端一半是催化活性的。我们试图以生化为特征，并通过NMR朝着其结构努力。 （4）来自极端嗜好古细菌的SPP直系同源物的特征是什么？与其他全长SPP相比，来自极端粒细菌的SPP样蛋白酶应比结构研究更稳定，可以更稳定。 我们将致力于对古细菌的表达，纯化和表征，为结构阐明奠定了基础。 LIE摘要：信号肽肽酶（SPP）是一种蛋白质切割酶，在免疫系统中起重要作用，丙型肝炎病毒使用人类SPP作为其感染过程的一部分。 SPP还与更复杂的酶有关，该酶在阿尔茨海默氏病中起着关键作用，并被广泛认为是治疗的重要靶标。 该计划是更充分地了解SPP的工作原理，并采取步骤净化SPP，以最终阐明其结构。 SPP的结构将为生物学和医学至关重要的不寻常的新酶的运作提供深刻的见解。