Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Italy.
Department of Experimental Medicine, University of Perugia, Italy.
FEBS J. 2017 Nov;284(21):3739-3764. doi: 10.1111/febs.14269. Epub 2017 Nov 1.
Protein aggregate formation is the basis of several misfolding diseases, including those displaying loss-of-function pathogenesis. Although aggregation is often attributed to the population of intermediates exposing hydrophobic surfaces, the contribution of electrostatic forces has recently gained attention. Here, we combined computational and in vitro studies to investigate the aggregation process of human peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme involved in glyoxylate detoxification. We demonstrated that AGT is susceptible to electrostatic aggregation due to its peculiar surface charge anisotropy and that PLP binding counteracts the self-association process. The two polymorphic mutations P11L and I340M exert opposite effects. The P11L substitution enhances the aggregation tendency, probably by increasing surface charge anisotropy, while I340M plays a stabilizing role. In light of these results, we examined the effects of the most common missense mutations leading to primary hyperoxaluria type I (PH1), a rare genetic disorder associated with abnormal calcium oxalate precipitation in the urinary tract. All of them endow AGT with a strong electrostatic aggregation propensity. Moreover, we predicted that pathogenic mutations of surface residues could alter charge distribution, thus inducing aggregation under physiological conditions. A global model describing the AGT aggregation process is provided. Overall, the results indicate that the contribution of electrostatic interactions in determining the fate of proteins and the effect of amino acid substitutions should not be underestimated and provide the basis for the development of new therapeutic strategies for PH1 aimed at increasing AGT stability.
蛋白质聚集体的形成是几种错误折叠疾病的基础，包括那些表现出功能丧失发病机制的疾病。尽管聚集通常归因于暴露疏水面的中间物群体，但静电相互作用的贡献最近引起了关注。在这里，我们结合计算和体外研究来研究人类过氧化物酶体丙氨酸：乙醛酸氨基转移酶（AGT）的聚集过程，AGT 是一种依赖吡哆醛 5'-磷酸（PLP）的酶，参与乙醛酸解毒。我们证明 AGT 由于其特殊的表面电荷各向异性而易受到静电聚集的影响，并且 PLP 结合会抵消自缔合过程。两种多态性突变 P11L 和 I340M 产生相反的效果。P11L 取代增强了聚集倾向，可能是通过增加表面电荷各向异性，而 I340M 则起到稳定作用。鉴于这些结果，我们研究了导致原发性高草酸尿症 1 型（PH1）的最常见错义突变的影响，PH1 是一种罕见的遗传疾病，与泌尿道中异常草酸钙沉淀有关。它们都赋予 AGT 强烈的静电聚集倾向。此外，我们预测表面残基的致病突变可能会改变电荷分布，从而在生理条件下诱导聚集。提供了描述 AGT 聚集过程的整体模型。总的来说，这些结果表明静电相互作用在决定蛋白质命运中的贡献以及氨基酸取代的影响不应被低估，并为开发旨在增加 AGT 稳定性的 PH1 新治疗策略提供了基础。
