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 - 病原体相互作用和某些病原体基因组中对去泛素化酶（DUB）的作用起着重要作用。最近将含有蛋白质的卵巢肿瘤（OTU）结构域鉴定为携带配音活性，并在整个进化过程中保守。 Crimean-Congo出血热病毒大蛋白（CCHFV-L）包含一个预测的OTU结构域，提供了一个进一步检查该复杂病毒蛋白功能的切入点。在这项研究中，将分析含有OTU结构域的CCHFV-L蛋白的亚基和可能参与蛋白酶功能的其他基序的亚基，以从不同底物中裂解泛素的潜力。从合成开放式阅读框架中用核细胞中表达的纯化蛋白质将在体外提示其蛋白酶活性。针对DUB的小型活性位点修饰符将用于识别活性位点半胱氨酸，并进一步表征真核细胞中的蛋白酶活性和特异性。活跃部位中诱捕泛素化细胞或病毒底物蛋白的非活性蛋白酶突变体将允许鉴定CCHFV-L靶蛋白，并洞悉受病毒感染损害的细胞机制。发现CCHFV-L蛋白来利用细胞功能的新机制将阐明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了一个窗口。