In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine: Glyoxylate Aminotransferase
In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine: Glyoxylate Aminotransferase
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DOI：
10.1074/jbc.m803525200
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发表时间：
2008-11-07
影响因子：
4.8
通讯作者：
Tucker, Chandra L.
中科院分区：
文献类型：
--
作者：
Hopper, Erin D.;Pittman, Adrianne M. C.;Tucker, Chandra L.
原发性高草酸尿I型是一种严重的肾结石疾病，由蛋白质丙氨酸：glyoxylate aminotransferase （AGT）突变引起。许多患者的AGT突变不是单独有害的，而是与一个常见的小等位基因多态变异协同作用，损害蛋白质折叠、二聚化或定位。虽然研究表明小等位基因变异本身是不稳定的，但没有进行直接的稳定性研究。在本报告中，我们使用三种方法分析AGT的功能和稳定性。首先，我们描述了AGT的酵母互补生长试验，在该试验中，我们发现人类AGT可以替代酵母Agx1的功能，并且与人类疾病相关的突变显示酵母的生长减少。次要等位基因突变体的生长减少反映了蛋白质水平的降低，表明这些蛋白质在酵母中比野生型AGT更不稳定。我们进一步研究了AGT等位基因在体外的稳定性，使用了两种直接的方法，一种是基于质谱的技术（通过H/D交换率检测未纯化蛋白的稳定性），另一种是差示扫描荧光法。我们还研究了已知配体吡哆醛5′-磷酸和氨基乙酸对稳定性的影响。我们的工作确定了次要等位基因是不稳定的，吡哆醛5'-磷酸和氨基乙酸结合显著地稳定了这两个等位基因。据我们所知，这是第一次直接测量AGT变体和配体复合物的相对稳定性。由于先前的研究表明，稳定化合物（即药物伴侣）可能对治疗原发性高草酸尿有效，我们建议本文描述的方法可用于高通量筛选稳定AGT突变体的化合物。
Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine: glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5'-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5'-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.