Department of Nutrition, University of California Davis, Davis, CA, USA.
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan.
Biochim Biophys Acta Mol Cell Res. 2024 Jan;1871(1):119590. doi: 10.1016/j.bbamcr.2023.119590. Epub 2023 Sep 18.
Glomerular podocytes are instrumental for the barrier function of the kidney, and podocyte injury contributes to proteinuria and the deterioration of renal function. Protein tyrosine phosphatase 1B (PTP1B) is an established metabolic regulator, and the inactivation of this phosphatase mitigates podocyte injury. However, there is a paucity of data regarding the substrates that mediate PTP1B actions in podocytes. This study aims to uncover novel substrates of PTP1B in podocytes and validate a leading candidate. To this end, using substrate-trapping and mass spectroscopy, we identified putative substrates of this phosphatase and investigated the actin cross-linking cytoskeletal protein alpha-actinin4. PTP1B and alpha-actinin4 co-localized in murine and human glomeruli and transiently transfected E11 podocyte cells. Additionally, podocyte PTP1B deficiency in vivo and culture was associated with elevated tyrosine phosphorylation of alpha-actinin4. Conversely, reconstitution of the knockdown cells with PTP1B attenuated alpha-actinin4 tyrosine phosphorylation. We demonstrated co-association between alpha-actinin4 and the PTP1B substrate-trapping mutant, which was enhanced upon insulin stimulation and disrupted by vanadate, consistent with an enzyme-substrate interaction. Moreover, we identified alpha-actinin4 tandem tyrosine residues 486/487 as mediators of its interaction with PTP1B. Furthermore, knockdown studies in E11 cells suggest that PTP1B and alpha-actinin4 are modulators of podocyte motility. These observations indicate that PTP1B and alpha-actinin4 are likely interacting partners in a signaling node that modulates podocyte function. Targeting PTP1B and plausibly this one of its substrates may represent a new therapeutic approach for podocyte injury that warrants additional investigation.
肾小球足细胞对于肾脏的屏障功能至关重要，而足细胞损伤导致蛋白尿和肾功能恶化。蛋白酪氨酸磷酸酶 1B（PTP1B）是一种已被确定的代谢调节剂，该磷酸酶失活可减轻足细胞损伤。然而，关于介导 PTP1B 在足细胞中作用的底物的研究数据却很少。本研究旨在揭示足细胞中 PTP1B 的新底物，并验证一个主要候选物。为此，我们使用底物捕获和质谱技术，鉴定了该磷酸酶的潜在底物，并研究了肌动蛋白交联细胞骨架蛋白α-辅肌动蛋白 4。PTP1B 和α-辅肌动蛋白 4 在鼠和人肾小球中共同定位，并瞬时转染 E11 足细胞。此外，体内和培养条件下的足细胞 PTP1B 缺乏与α-辅肌动蛋白 4 的酪氨酸磷酸化升高有关。相反，用 PTP1B 重建敲低细胞可减弱α-辅肌动蛋白 4 的酪氨酸磷酸化。我们证明了α-辅肌动蛋白 4 与 PTP1B 底物捕获突变体的共同关联，这种关联在胰岛素刺激下增强，在钒酸钠作用下被破坏，这与酶-底物相互作用一致。此外，我们鉴定出α-辅肌动蛋白 4 串联酪氨酸残基 486/487 是其与 PTP1B 相互作用的介导物。此外，E11 细胞的敲低研究表明，PTP1B 和α-辅肌动蛋白 4 是足细胞运动的调节剂。这些观察结果表明，PTP1B 和α-辅肌动蛋白 4 可能是调节足细胞功能的信号节点中的相互作用伙伴。靶向 PTP1B 并可能靶向其一个底物可能代表一种新的治疗足细胞损伤的方法，值得进一步研究。
