Expression, purification, structural and biochemical characterization of the hemagglutinin-cleaving proprotein convertase PC5

血凝素裂解前蛋白转化酶 PC5 的表达、纯化、结构和生化表征

• 批准号: 309089047
• 负责人: Dr. Kornelia Hardes
• 金额: --
• 依托单位: Leibniz-Institut für Alternsforschung - Fritz-Lipmann-Institut e.V. (FLI)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/309089047?language=en
• 关键词: Expression; purification; structural; biochemical; characterization

The family of proprotein convertases (PCs) comprises nine members, whose catalytic domains are highly conserved. PCs are involved in the processing of several precursor proteins of hormones or growth factors but also activate viral proteins and bacterial toxins during infections. The short-term treatment with PC inhibitors in acute viral or bacterial infectious diseases could be a promising therapeutic strategy. However, the selectivity profile of most PC inhibitors is limited as they inhibit several PCs in a similar range, e.g. in the case of furin and PC5. The development of selective PC inhibitors would be of great interest for therapeutically purposes and for the further characterization of the role of PCs in various diseases. So far, the recognition motif of other kexin-like PCs than furin has been less investigated and remains poorly understood. In case of PC5 only limited data are available on its specificity and properties. Therefore, the aim of this proposal is to further characterize PC5 as antiviral target regarding its specificity, structure as well as biological and chemical properties to identify differences to other PCs.For the determination of its selectivity profile and further characterization two different strategies will be applied. On the one hand already available PC inhibitors and substrates will be screened for selectivity in enzyme kinetic measurements. On the other hand experimental crystal structures of PC5 should be solved to enable a rational, structure-based design of more selective drugs. Therefore, PC5 will be expressed in human embryonic kidney cells and purified using a three-step chromatography procedure. An enzyme kinetic assay will be established and optimized for the determination of inhibition constants of available PC inhibitors. In parallel, the production of highly purified enzyme will be scaled up for the structure determination of PC5 using crystallographic methods.Furin and PC5 activate the hemagglutinin (HA) of highly pathogenic avian influenza virus H5 and H7 strains at a mulitbasic cleavage site. In co-expression experiments it will be investigated, whether PC5 can also activate HA containing di- or tribasic sequence motifs. These results will be compared to the cleavage analysis of short synthetic HA-derived substrates. Furthermore, the role of PC5 in the activation of HA under endogenous conditions has to be clarified using small-interfering RNAs or peptide-conjugated phosphordiamidate morpholino oligomers. In additional co-expression experiments the effect of available inhibitors on the PC5-mediated HA cleavage will be tested.The combination of these results will enable the development of more selective PC5 inhibitors with a low in vitro toxicity suitable for the inhibition of influenza HA-activation. In future, potential drug candidates have to be examined regarding their pharmacokinetic and pharmacodynamic properties as well as their toxicity and therapeutic effects on diseases in vivo.

前蛋白转化酶（proprotein convertases，PC）家族由9个成员组成，其催化结构域高度保守。PC参与激素或生长因子的几种前体蛋白的加工，但也在感染期间激活病毒蛋白和细菌毒素。在急性病毒或细菌感染性疾病中，使用PC抑制剂进行短期治疗可能是一种有前途的治疗策略。然而，大多数PC抑制剂的选择性特征是有限的，因为它们在类似的范围内抑制几种PC，例如在弗林蛋白酶和PC 5的情况下。选择性PC抑制剂的发展将是极大的兴趣，用于治疗目的和PC在各种疾病中的作用的进一步表征。到目前为止，识别基序的其他kexin样的PC比弗林蛋白酶已经较少的研究，仍然知之甚少。在PC5的情况下，关于其特异性和特性的数据有限。因此，本研究的目的是进一步表征PC5作为抗病毒靶点的特异性、结构以及生物学和化学性质，以确定其与其他PCs的差异。一方面，将筛选已经可用的PC抑制剂和底物在酶动力学测量中的选择性。另一方面，应该解决PC5的实验晶体结构，以实现更有选择性的药物的合理的，基于结构的设计。因此，PC5将在人胚肾细胞中表达，并使用三步色谱法进行纯化。将建立并优化酶动力学测定法，以测定可用PC抑制剂的抑制常数。与此同时，高纯度的酶的生产将被放大用于使用晶体学方法确定PC5的结构。弗林蛋白酶和PC5在多碱基切割位点激活高致病性禽流感病毒H5和H7株的血凝素（HA）。在共表达实验中，将研究PC 5是否也可以激活含有二碱基或三碱基序列基序的HA。将这些结果与短合成HA衍生底物的裂解分析进行比较。此外，PC 5在内源性条件下激活HA的作用必须使用小干扰RNA或肽缀合的磷酰二胺吗啉代寡聚物来澄清。这些结果的组合将使得能够开发具有更高选择性的PC5抑制剂，其具有低的体外毒性，适用于抑制流感HA活化。在未来，潜在的候选药物必须检查其药代动力学和药效学特性以及它们的毒性和对体内疾病的治疗效果。