Charakterisierung der Proteaseaktivität und Spezifität der OTU-Domäne (ovarian tumour domain) des L-Proteins des Krim-Kongo-Fieber-Virus

克里米亚-刚果热病毒L蛋白OTU结构域（卵巢肿瘤结构域）的蛋白酶活性和特异性的表征

• 批准号: 59655326
• 负责人: Dr. Nicola Ternette
• 金额: --
• 依托单位: Nuffield Department of Clinical Medicine
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/59655326?language=en
• 关键词: Charakterisierung; der; Proteaseaktivit; und; Spezifit

Ubiquitin processing enzymes play an important role in host-pathogen interactions and some pathogen genomes encode for deubiquitinating enzymes (DUBs). Ovarian tumour (OTU) domain containing proteins were recently identified to harbour DUB activity and are conserved throughout evolution. Crimean-Congo hemorrhagic fever virus large protein (CCHFV-L) contains a predicted OTU domain, providing an entry point to further examine the functions of this complex viral protein. In this study, subunits of CCHFV-L protein containing the OTU domain and additional motifs possibly participating in protease function will be analysed for their potential to cleave ubiquitin from different substrates. Purified proteins expressed in prokaryotic cells from synthetic open reading frames will give first hints on their protease activity in vitro. Small active site modifiers specific for DUBs will be used to identify the active site cysteine and further characterise protease activity and specificity in eukaryotic cells. Inactive protease mutants trapping ubiquitinated cellular or viral substrate proteins within the active site will allow identification of CCHFV-L target proteins and provide insight into cellular mechanisms impaired by viral infection. The discovery of novel mechanisms of the CCHFV-L protein to exploit cellular functions will shed light on the biological role of this viral component during infection, and provide a window for potential antiviral pharmaceutical intervention.

泛素加工酶在宿主-病原体相互作用中起着重要作用，一些病原体基因组编码脱泛素化酶(DUBS)。包含卵巢肿瘤(OTU)结构域的蛋白最近被发现具有DUB活性，并且在进化过程中是保守的。克里米亚-刚果出血热病毒大蛋白(CCHFV-L)含有一个预测的OTU结构域，为进一步研究这一复杂病毒蛋白的功能提供了一个切入点。在这项研究中，CCHFV-L蛋白的亚基含有OTU结构域和其他可能参与蛋白酶功能的基序，将分析它们从不同底物中切割泛素的潜力。在原核细胞中从人工合成的开放阅读框中表达的纯化蛋白将在体外给出它们的蛋白酶活性的第一个线索。针对DUBS的小活性位点修饰物将被用于鉴定活性位点半胱氨酸，并进一步鉴定真核细胞中的蛋白酶活性和特异性。将泛素化的细胞或病毒底物蛋白捕获在活性部位内的非活性蛋白酶突变体将有助于鉴定CCHFV-L目标蛋白，并为深入了解病毒感染损伤的细胞机制提供帮助。CCHFV-L蛋白利用细胞功能的新机制的发现将揭示该病毒成分在感染过程中的生物学作用，并为潜在的抗病毒药物干预提供窗口。