Department of Microbiology and Molecular Genetics, University of Texas McGovern Medical School at Houston, Houston, TX 77030.
MD Anderson UT Health Graduate School of Biomedical Sciences, Houston, TX 77030.
Mol Biol Cell. 2019 Mar 1;30(5):554-565. doi: 10.1091/mbc.E18-10-0616. Epub 2019 Jan 2.
The eukaryotic cytosolic proteome is vulnerable to changes in proteostatic and redox balance caused by temperature, pH, oxidants, and xenobiotics. Cysteine-containing proteins are especially at risk, as the thiol side chain is subject to oxidation, adduction, and chelation by thiol-reactive compounds. The thiol-chelating heavy metal cadmium is a highly toxic environmental pollutant demonstrated to induce the heat shock response and recruit protein chaperones to sites of presumed protein aggregation in the budding yeast Saccharomyces cerevisiae. However, endogenous targets of cadmium toxicity responsible for these outcomes are largely unknown. Using fluorescent protein fusion to cytosolic proteins with known redox-active cysteines, we identified the yeast glycolytic enzyme triose phosphate isomerase as being aggregation-prone in response to cadmium and to glucose depletion in chronologically aging cultures. Cadmium-induced aggregation was limited to newly synthesized Tpi1 that was recruited to foci containing the disaggregase Hsp104 and the peroxiredoxin chaperone Tsa1. Misfolding of nascent Tpi1 in response to both cadmium and glucose-depletion stress required both cysteines, implying that thiol status in this protein directly influences folding. We also demonstrate that cadmium proteotoxicity is conserved between yeast and human cells, as HEK293 and HCT116 cell lines exhibit recruitment of the protein chaperone Hsp70 to visible foci. Moreover, human TPI, mutations in which cause a glycolytic deficiency syndrome, also forms aggregates in response to cadmium treatment, suggesting that this conserved enzyme is folding-labile and may be a useful endogenous model for investigating thiol-specific proteotoxicity.
真核细胞质的蛋白质组容易受到温度、pH 值、氧化剂和外源化学物质引起的蛋白质稳态和氧化还原平衡变化的影响。含有半胱氨酸的蛋白质尤其处于危险之中，因为巯基侧链容易受到氧化、加成和与巯基反应化合物的螯合。含硫重金属镉是一种高度毒性的环境污染物，已被证明可诱导热休克反应，并在出芽酵母酿酒酵母中募集蛋白伴侣到假定的蛋白聚集部位。然而，导致这些结果的镉毒性的内源性靶标在很大程度上是未知的。我们使用荧光蛋白融合到具有已知氧化还原活性半胱氨酸的细胞质蛋白，鉴定出酵母糖酵解酶磷酸丙糖异构酶对镉和葡萄糖耗尽的chronologically 老化培养物具有聚集倾向。镉诱导的聚集仅限于新合成的 Tpi1，该蛋白被募集到含有解聚酶 Hsp104 和过氧化物还原酶伴侣 Tsa1 的焦点中。新合成的 Tpi1 对镉和葡萄糖耗尽应激的错误折叠需要两个半胱氨酸，这意味着该蛋白中的巯基状态直接影响折叠。我们还证明了镉蛋白毒性在酵母和人类细胞之间是保守的，因为 HEK293 和 HCT116 细胞系显示蛋白伴侣 Hsp70 募集到可见焦点。此外，引起糖酵解缺陷综合征的人类 TPI 突变也会在镉处理下形成聚集体，这表明这种保守的酶易折叠不稳定，可能是研究巯基特异性蛋白毒性的有用内源性模型。
