Sluzala Zachary B, Shan Yang, Elghazi Lynda, Cárdenas Emilio L, Hamati Angelina, Garner Amanda L, Fort Patrice E
Department of Ophthalmology & Visual Sciences, The University of Michigan, Ann Arbor, MI 48109, USA.
Interdepartmental Program in Medicinal Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA.
Cells. 2024 Dec 4;13(23):2000. doi: 10.3390/cells13232000.
HSPB4 and HSPB5 (α-crystallins) have shown increasing promise as neuroprotective agents, demonstrating several anti-apoptotic and protective roles in disorders such as multiple sclerosis and diabetic retinopathy. HSPs are highly regulated by post-translational modification, including deamidation, glycosylation, and phosphorylation. Among them, T148 phosphorylation has been shown to regulate the structural and functional characteristics of HSPB4 and underlie, in part, its neuroprotective capacity. We recently demonstrated that this phosphorylation is reduced in retinal tissues from patients with diabetic retinopathy, raising the question of its regulation during diseases. The kinase(s) responsible for regulating this phosphorylation, however, have yet to be identified. To this end, we employed a multi-tier strategy utilizing in vitro kinome profiling, bioinformatics, and chemoproteomics to predict and discover the kinases capable of phosphorylating T148. Several kinases were identified as being capable of specifically phosphorylating T148 in vitro, and further analysis highlighted mTORC2 as a particularly strong candidate. Altogether, our data demonstrate that the HSPB4-mTORC2 interaction is multi-faceted. Our data support the role of mTORC2 as a specific kinase phosphorylating HSPB4 at T148, but also provide evidence that the HSPB4 chaperone function further strengthens the interaction. This study addresses a critical gap in our understanding of the regulatory underpinnings of T148 phosphorylation-mediated neuroprotection.
HSPB4和HSPB5（α-晶状体蛋白）作为神经保护剂已显示出越来越大的前景，在诸如多发性硬化症和糖尿病性视网膜病变等疾病中发挥了多种抗凋亡和保护作用。热休克蛋白（HSPs）受到翻译后修饰的高度调控，包括脱酰胺作用、糖基化和磷酸化。其中，T148磷酸化已被证明可调节HSPB4的结构和功能特性，并在一定程度上构成其神经保护能力的基础。我们最近证明，糖尿病性视网膜病变患者视网膜组织中的这种磷酸化水平降低，这就引发了其在疾病过程中调控机制的问题。然而，负责调节这种磷酸化的激酶尚未被确定。为此，我们采用了一种多层次策略，利用体外激酶组分析、生物信息学和化学蛋白质组学来预测和发现能够磷酸化T148的激酶。几种激酶被确定能够在体外特异性磷酸化T148，进一步分析突出显示mTORC2是一个特别有力的候选者。总之，我们的数据表明HSPB4与mTORC2的相互作用是多方面的。我们的数据支持mTORC2作为在T148位点磷酸化HSPB4的特异性激酶的作用，但也提供了证据表明HSPB4的分子伴侣功能进一步加强了这种相互作用。这项研究填补了我们在理解T148磷酸化介导的神经保护的调控基础方面的一个关键空白。
