School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
Animal House Facility & Radiation Signaling Section, Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, 400085, India.
Neuromolecular Med. 2022 Dec;24(4):452-468. doi: 10.1007/s12017-022-08711-4. Epub 2022 May 3.
GNE myopathy is an inherited neuromuscular disorder caused by mutations in GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase) gene catalyzing the sialic acid biosynthesis pathway. The characteristic features include muscle weakness in upper and lower extremities, skeletal muscle wasting, and rimmed vacuole formation. More than 200 GNE mutations in either epimerase or kinase domain have been reported worldwide. In Indian subcontinent, several GNE mutations have been recently identified with unknown functional correlation. Alternate role of GNE in various cellular processes such as cell adhesion, migration, apoptosis, protein aggregation, and cytoskeletal organization have been proposed in recent studies. We aim to understand and compare the effect of various GNE mutations from Indian origin on regulation of the cytoskeletal network. In particular, F-actin dynamics was determined quantitatively by determining F/G-actin ratios in immunoblots for specific proteins. The extent of F-actin polymerization was visualized by immunostaining with Phalloidin using confocal microscopy. The proteins regulating F-actin dynamics such as RhoA, cofilin, Arp2, and alpha-actinin were studied in various GNE mutants. The altered level of cytoskeletal organization network proteins affected cell migration of GNE mutant proteins as measured by wound healing assay. The functional comparison of GNE mutations will help in better understanding of the genotypic severity of the disease in the Indian population. Our study offers a potential for identification of therapeutic molecules regulating actin dynamics in GNE specific mutations.
GNE 肌病是一种遗传性神经肌肉疾病，由 GNE（UDP-N-乙酰氨基葡萄糖 2-差向异构酶/N-乙酰甘露糖胺激酶）基因突变引起，该基因突变可导致唾液酸生物合成途径。其特征性表现为四肢肌肉无力、骨骼肌萎缩和边缘空泡形成。全世界已经报道了超过 200 种 GNE 突变，位于差向异构酶或激酶结构域。在印度次大陆，最近已经鉴定出几种具有未知功能相关性的 GNE 突变。最近的研究提出，GNE 在细胞黏附、迁移、凋亡、蛋白聚集和细胞骨架组织等多种细胞过程中具有替代作用。我们旨在了解和比较来自印度的各种 GNE 突变对细胞骨架网络调节的影响。特别是通过测定免疫印迹中特定蛋白的 F/G-肌动蛋白比来定量测定 F-肌动蛋白动力学。通过使用鬼笔环肽进行免疫染色并用共聚焦显微镜观察 F-肌动蛋白聚合程度。研究了各种 GNE 突变体中调节 F-肌动蛋白动力学的蛋白质，如 RhoA、胞质动力蛋白、Arp2 和α-辅肌动蛋白。细胞迁移的伤口愈合试验测量了细胞骨架组织网络蛋白水平的改变对 GNE 突变蛋白的影响。GNE 突变的功能比较有助于更好地了解印度人群中该疾病的基因型严重程度。我们的研究为鉴定调节 GNE 特异性突变中肌动蛋白动力学的治疗分子提供了可能性。
