Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, 570020, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad- 201002, India.
Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, 570020, India.
Biochimie. 2024 Oct;225:106-113. doi: 10.1016/j.biochi.2024.05.016. Epub 2024 May 19.
Follistatin like-1 (FSTL-1) is a secreted glycoprotein of mesenchymal in origin. In human skin, FSTL1 is upregulated in the epidermal keratinocytes upon acute injury and is required for the migration of keratinocytes. Failure to upregulate FSTL1 leads to the lack of keratinocyte migration and the non-healing nature of diabetic foot ulcer (DFU). FSTL1 undergoes extensive post-translational modification (PTM) at specific residues. Glycosylation at N144, N175 and N180, are the only experimentally demonstrated PTM in FSTL1, wherein, N180 and N144 glycosylations have been found to be critical for its function in cardiac tissue regeneration and pre-adipocyte differentiation, respectively. However, it is not known if PTMs other than glycosylation occurs in FSTL1 and how it impacts its pro-migratory function. Using in-silico analysis of mass spectrometric datasets, we found a novel PTM, namely, Serine 165 (S165) phosphorylation in FSTL1. To address the role of S165 phosphorylation in its pro-migratory function, a phosphorylation defective mutant of FSTL1 (S165A) was constructed by converting serine 165 to alanine and over expressed in 293T cells. S165A mutation did not affect the secretion of FSTL1 in vitro. However, S165A abolished the pro-migratory effect of FSTL1 in cultured keratinocytes likely via its inability to facilitate ERK signaling pathway. Interestingly, bacterially expressed recombinant FSTL1, trans-dominantly inhibited wound closure in keratinocytes highlighting the prime role of FSTL1 phosphorylation for its pro-migratory function. Further, under high glucose conditions, which inhibited scratchwound migration of keratinocytes, we noticed a significant decrease in S165 phosphorylation. Taken together, our results reveal a hitherto unreported role of FSTL1 phosphorylation PTM with profound implications in wound healing.
卵泡抑素样蛋白 1（Follistatin like-1，FSTL-1）是一种起源于间充质的分泌糖蛋白。在人类皮肤中，FSTL1 在急性损伤时在上皮角质形成细胞中上调，并需要角质形成细胞的迁移。FSTL1 上调失败会导致角质形成细胞迁移缺失和糖尿病足溃疡（DFU）的非愈合性质。FSTL1 在特定残基上经历广泛的翻译后修饰（PTM）。N144、N175 和 N180 的糖基化是 FSTL1 中唯一经过实验证实的 PTM，其中，N180 和 N144 的糖基化已被发现对其在心脏组织再生和前脂肪细胞分化中的功能至关重要。然而，目前尚不清楚 FSTL1 是否发生除糖基化以外的 PTM 及其对促迁移功能的影响。通过对质谱数据集的计算机分析，我们发现了 FSTL1 中的一种新的翻译后修饰，即丝氨酸 165（S165）磷酸化。为了研究 S165 磷酸化在其促迁移功能中的作用，我们构建了 FSTL1 的磷酸化缺陷突变体（S165A），即将丝氨酸 165 突变为丙氨酸，并在 293T 细胞中过表达。S165A 突变不影响 FSTL1 的体外分泌。然而，S165A 突变消除了 FSTL1 在培养角质形成细胞中的促迁移作用，可能是因为其无法促进 ERK 信号通路。有趣的是，细菌表达的重组 FSTL1 可显著抑制角质形成细胞的伤口闭合，突出了 FSTL1 磷酸化在其促迁移功能中的主要作用。此外，在高葡萄糖条件下，可抑制角质形成细胞的划痕迁移，我们注意到 S165 磷酸化显著减少。总之，我们的研究结果揭示了 FSTL1 磷酸化 PTM 的一个前所未有的作用，这对伤口愈合具有深远的意义。
