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Biochemical characterization of Ty1 retrotransposon protease

机译：TY1转烷蛋白酶蛋白酶的生化特征

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Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae . The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e . g . the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae , untagged and His 6 -tagged forms of Ty1 protease were expressed in E . coli . After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.
机译：TY1是芽胺酵母酿酒酵母中的许多转座子之一。 TY1的生命周期显示了逆转录病毒的许多相似之处，即G 。最初合成的多蛋白前体通过蛋白酶经历蛋白水解加工。由于GAG-POL Polyprotein在复制循环中的有限蛋白分解的关键作用，逆转录病毒蛋白酶已成为目前的抗逆转录病毒疗法的重要靶标，因此它们属于最良好研究的酶。逆转录病毒和逆转录病毒样蛋白酶的比较分析可以有助于探讨关键的相似性和差异，这可能有助于了解抗蛋白酶抑制剂的抵抗，但有关逆转录病毒样的结构和生化特性的可用信息，以及尤其是回复转瘤蛋白酶是有限的。为了研究酿酒酵母酿酒酵母的TY1回复蛋白酶蛋白酶的主要特征，在e中表达了未标记的和他的6种-Graged形式的Ty1蛋白酶。大肠杆菌。在重组蛋白纯化后，使用合成的寡肽和荧光重组蛋白质基质进行活性测量，其代表野生型和蛋白酶的天然发生的裂解位点的野生型和改性形式。我们调查了酶活性对不同反应条件（pH，温度，离子强度和尿素浓度）的依赖性，以及所研究的基材的确定酶动力学参数。还测试了10种不同的蛋白酶抑制剂的抑制潜力。通过针对人免疫缺陷病毒1型蛋白酶设计的抑制剂不抑制TY1蛋白酶，并被批准为抗逆转录病毒治疗剂。基于同源性建模，提出了同源二聚体TY1蛋白酶的季结构，并且该结构用于支持实验结果的解释，并将一些结构和生化特性与其他逆转录病毒蛋白酶的解释相关。

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