Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1
Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1
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DOI：
10.1042/bcj20190507
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发表时间：
2019-12-01
影响因子：
4.1
通讯作者：
Cellini, Barbara
中科院分区：
文献类型：
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作者：
Dindo, Mirco;Grottelli, Silvia;Cellini, Barbara
过氧化体丙氨酸：乙醛酸氨基转移酶(AGT)负责人体肝脏中乙醛酸的解毒作用，以5‘-磷酸吡哆醛(PLP)为辅酶。AGT的缺陷导致了原发性高草酸尿症I型(PH1)，这是一种罕见的疾病，其特征是乙醛蓄积导致草酸钙结石沉积在尿路。大多数错义突变都会导致AGT错误折叠，就像G41R一样，它会导致聚集和蛋白降解。我们研究了野生型AGT和致病的G41R变异体与用于治疗耐多药结核病的天然产物D-环丝氨酸及其合成对映体L-环丝氨酸的相互作用。与以前报道的其他PLP酶不同，这两种配体都是AGT可逆抑制剂，其抑制常数在微摩尔范围内。LCS经历半转氨基反应产生酮胺中间体，表现为典型的竞争性抑制物，而DCs则表现出依赖时间的结合，主要产生酮胺中间体。使用哺乳动物细胞模型，我们发现DC，而不是LCS，能够促进G41R变体的正确折叠，这表明它作为一种可溶性蛋白质比活性和表达增加。这一效应还转化为在用DC处理时表达该变体的细胞的乙醛解毒能力增强。总体而言，我们的研究结果表明，DCs可以发挥药理伴侣的作用，因此提出了一种基于药物重新定位方法的针对PH1的新的干预路线。在最大程度上，这一策略也可以应用于其他导致AGT错误折叠的致病突变。
Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5'-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with D-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer L-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.