Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo (USP), Av. Prof. Lineu Prestes 748, 05508-000 Sao Paulo, Brazil; Institute of Clinical Biochemistry, Hannover Medical School (MHH), Carl-Neuberg-Straße, 1, 30625, Hannover, Germany.
Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo (USP), Av. Prof. Lineu Prestes 748, 05508-000 Sao Paulo, Brazil.
Free Radic Biol Med. 2019 Apr;134:394-405. doi: 10.1016/j.freeradbiomed.2019.01.023. Epub 2019 Jan 27.
Maintaining islet cell viability in vitro, although challenging, appears to be a strategy for improving the outcome of pancreatic islet transplantation. We have shown that prolactin (PRL) leads to beta-cell cytoprotection against apoptosis, an effect mediated by heat shock protein B1 (HSPB1). Since the role of HSPB1 in beta-cells is still unclear and the hormone concentration used is not compatible with clinical applications because of all the side effects displayed by the hormone in other tissues, we explored the molecular mechanisms by which HSPB1 mediates beta-cell cytoprotection. Lysates from PRL- and/or cytokine-treated MIN6 beta-cells were subjected to HSPB1 immunoprecipitation followed by identification through mass spectrometry. PRL-treated cells presented an enrichment of several proteins co-precipitating with HSPB1. Of note were oxidative stress resistance-, protein degradation- and carbohydrate metabolism-related proteins. Wild type, HSPB1 silenced or overexpressing MIN6 cells were exposed to menadione and hydrogen peroxide and analysed for several oxidative stress parameters. HSPB1 knockdown rendered cells more sensitive to oxidative stress and led to a reduced antioxidant capacity, while prolactin induced an HSPB1-mediated cytoprotection against oxidative stress. HSPB1 overexpression, however, led to opposite effects. PRL treatment, HSPB1 silencing or overexpression did not change the expression nor activities of antioxidant enzymes, it also did not lead to a modulation of total glutathione levels nor G6PD expression. However, HSPB1 levels are related to a modulation of GSH/GSSG ratio, G6PD activity and NADPH/NADP ratio. We have shown that HSPB1 is important for pro-survival effects against oxidative stress-induced beta-cell death. These results are in accordance with PRL-induced enrichment of HSPB1-interacting proteins related to protection against oxidative stress. Finally, our results outline the need of further studies investigating the importance of HSPB1 for beta-cell viability, since this could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway.
在体外维持胰岛细胞活力虽然具有挑战性，但似乎是改善胰腺胰岛移植效果的一种策略。我们已经表明，催乳素 (PRL) 导致β细胞抵抗细胞凋亡的细胞保护作用，这种作用是由热休克蛋白 B1 (HSPB1) 介导的。由于 HSPB1 在β细胞中的作用尚不清楚，并且由于该激素在其他组织中显示出的所有副作用，所用激素浓度与临床应用不兼容，因此我们探索了 HSPB1 介导β细胞细胞保护的分子机制。用 PRL 和/或细胞因子处理的 MIN6 β细胞的裂解物进行 HSPB1 免疫沉淀，然后通过质谱鉴定。用 PRL 处理的细胞中存在与 HSPB1 共沉淀的几种蛋白质的富集。值得注意的是与氧化应激抗性、蛋白质降解和碳水化合物代谢相关的蛋白质。野生型、HSPB1 沉默或过表达的 MIN6 细胞暴露于甲萘醌和过氧化氢中，并分析了几种氧化应激参数。HSPB1 敲低使细胞对氧化应激更敏感，并导致抗氧化能力降低，而催乳素诱导 HSPB1 介导的抗氧化应激保护作用。然而，HSPB1 的过表达导致相反的效果。PRL 处理、HSPB1 沉默或过表达既不会改变抗氧化酶的表达和活性，也不会导致总谷胱甘肽水平或 G6PD 表达的调节。然而，HSPB1 水平与 GSH/GSSG 比、G6PD 活性和 NADPH/NADP 比的调节有关。我们已经表明，HSPB1 对于抵抗氧化应激诱导的β细胞死亡的生存作用很重要。这些结果与 PRL 诱导的与抵抗氧化应激相关的 HSPB1 相互作用蛋白的富集一致。最后，我们的结果概述了需要进一步研究 HSPB1 对β细胞活力的重要性，因为这可能通过上调内源性保护途径来减轻β细胞死亡。
