Bodvard Kristofer, Mohlin Johanna, Knecht Wolfgang
Molecular Pharmacology - Target Production, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
Protein Expr Purif. 2007 Feb;51(2):308-19. doi: 10.1016/j.pep.2006.07.015. Epub 2006 Aug 1.
Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the highly purified enzyme. Featuring a new chromogenic substrate (Acetyl-Arg-Arg-Leu-Leu-p-nitroanilide) we show that the enzymatic activity of S1P is not calcium dependent, but can be modulated by a variety of mono- and divalent cations. S1P displayed pronounced positive cooperativity with a substrate derived from the viral coat glycoprotein of the lassa virus. The screening of a limited number of protease inhibitors showed that S1P was not inhibited by specific inhibitors of other proprotein convertases or by Pefabloc SC (4-(2-aminoethyl) benzene sulphonyl fluoride, AEBSF). We found 3,4-dichloroisocoumarin (DCI) to be a potent slow binding inhibitor of human S1P, with a K(iapp) = 6.8 microM, thus representing a new small molecule inhibitor of S1P. These findings show that S1P differs significantly from other proprotein convertases with respect to kinetics, co-factor requirement and inhibition.
人1型位点蛋白酶（S1P，酶学委员会编号S08.8063），也被广泛称为枯草杆菌蛋白酶/克新同工酶1（SKI-1），是一种与膜结合的、与枯草杆菌蛋白酶相关的丝氨酸蛋白酶，属于一组九种哺乳动物前体蛋白转化酶。在这些蛋白酶中，S1P表现出独特的底物特异性，偏好切割非碱性氨基酸之后的位点。S1P在控制膜、细胞和血液中胆固醇含量的蛋白水解途径中起关键作用。S1P还参与拉沙病毒、淋巴细胞性脉络丛脑膜炎病毒和克里米亚刚果出血热病毒的病毒包膜糖蛋白的激活。我们使用杆状病毒表达载体系统表达了重组人S1P，并对高度纯化的酶进行了表征。使用一种新的生色底物（乙酰基-精氨酸-精氨酸-亮氨酸-亮氨酸-对硝基苯胺），我们发现S1P的酶活性不依赖于钙，但可被多种单价和二价阳离子调节。S1P与源自拉沙病毒病毒包膜糖蛋白的底物表现出明显的正协同性。对有限数量的蛋白酶抑制剂进行筛选表明，S1P不受其他前体蛋白转化酶的特异性抑制剂或Pefabloc SC（4-(2-氨基乙基)苯磺酰氟，AEBSF）的抑制。我们发现3,4-二氯异香豆素（DCI）是一种有效的人S1P慢结合抑制剂，其表观抑制常数K(iapp) = 6.8 microM，因此代表了一种新的S1P小分子抑制剂。这些发现表明，S1P在动力学、辅因子需求和抑制方面与其他前体蛋白转化酶有显著差异。
