Abstract:
Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix. MMP-9 has been implicated in several diseases including neurodegeneration, arthritis, cardiovascular diseases, fibrosis and several types of cancer, resulting in a high demand for MMP-9 inhibitors for therapeutic purposes. For such drug design efforts, large amounts of MMP-9 are required. Yet, the catalytic domain of MMP-9 (MMP-9Cat) is an intrinsically unstable enzyme that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. We set our goal to design MMP-9Cat variant that is active but stable to auto-cleavage. For this purpose, we first identified potential auto-cleavage sites on MMP-9Cat using mass spectroscopy and then eliminated the auto-cleavage site by predicting mutations that minimize auto-cleavage potential without reducing enzyme stability. Four computationally designed MMP-9Cat variants were experimentally constructed and evaluated for auto-cleavage and enzyme activity. Our best variant, Des2, with 2 mutations, was as active as the wild-type enzyme but did not exhibit auto-cleavage after 7 days of incubation at 37°C. This MMP-9Cat variant, with an identical with MMP-9Cat WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9CAT stabilization could be applied to redesign other proteases to improve their stability for various biotechnological applications.
摘要:
基质金属蛋白酶-9(MMP-9)是重塑细胞外基质的内肽酶。MMP-9与几种疾病有关,包括神经变性,关节炎,心血管疾病,纤维化和几种类型的癌症,导致对用于治疗目的的MMP-9抑制剂的高需求。对于这样的药物设计努力,需要大量的MMP-9。然而,MMP-9的催化域(MMP-9Cat)是一种本质上不稳定的酶,往往在几分钟内自动切割,使其难以用于药物设计实验和其他生物物理研究。我们设定我们的目标是设计有活性但对自切割稳定的MMP-9Cat变体。为此,我们首先使用质谱鉴定了MMP-9Cat上潜在的自切割位点,然后通过预测突变消除了自切割位点,所述突变可在不降低酶稳定性的情况下最小化自切割潜能.实验构建四种计算设计的MMP-9Cat变体,并评估其自身切割和酶活性。我们最好的变种,Des2,有2个突变,具有与野生型酶一样的活性,但在37°C下孵育7天后没有表现出自动切割。这种MMP-9Cat变体,具有与MMP-9CatWT相同的活性位点,是靶向MMP-9的药物设计实验和酶结晶实验的理想候选者。所开发的MMP-9CAT稳定策略可用于重新设计其他蛋白酶,以提高其在各种生物技术应用中的稳定性。