GSK-3调控GAPDH嵌入线粒体的作用和机制

GAPDH plays an important role in neurodegeneration and neuronal apoptosis process. The present study demonstrates that siRNA knockdown of GAPDH protected neuron from apoptosis induced by activity deprivation. But GAPDH was neither upregulated nor translocated to nucleus or mitochondria during apoptosis. Furthermore, we found that GAPDH inserted into mitochondria during neuronal apoptosis, and the "insertion" was crucial for the pro-apoptotic function of GAPDH. Inhibition of pro-apoptotic kinase GSK-3 could block GAPDH insert into mitochondria; the in vitro kinase assay showed that GSK-3α/β could directly phosphorylate GAPDH and the Thr235 of GAPDH was a putative phosphorylation site of GSK-3. Based on the above findings, we put forward the hypothesis: GSK-3 mediates GAPDH insertion into mitochondria and neuronal apoptosis through phosphorylation of GAPDH at Thr235. In this project, we will use phosphorylation site mutation and in vitro kinase analysis to obtain the reliable evidence that GSK-3 phosphorylates of GAPDH at Thr235 to promote its insertion into mitochondria. This project will reveal the mechanism underlying GSK-3 regulation of GAPDH insertion into mitochondria which results in mitochondria dysfunction and provide the scientific basis for the establishment of GAPDH as a new treatment target of neurodegenerative diseases.

GAPDH在神经退行性病变和神经元凋亡过程中发挥重要作用。我们发现： siRNA沉默GAPDH能保护性干预神经元凋亡的发生，但是GAPDH的促凋亡功能不依赖于表达上调和核或者线粒体转位。进一步实验表明，神经元凋亡时GAPDH嵌入（insertion）到线粒体膜上，提示这一"嵌入"与GAPDH的促凋亡作用密切相关。抑制GSK-3抑制了GAPDH嵌入线粒体；激酶反应实验显示GSK-3能直接磷酸化GAPDH；序列分析显示GAPDH的Thr235是GSK-3的潜在磷酸化位点。由此，我们提出假设：GSK-3磷酸化GAPDH的Thr235介导其嵌入线粒体并触发神经元凋亡。本项目拟采用位点突变和激酶分析等手段，旨在获得GSK-3磷酸化GAPDH Thr235介导其嵌入线粒体的可靠证据，揭示GAPDH的嵌入对线粒体损伤的作用及其机制，为确立GAPDH作为神经退行性疾病治疗新靶点提供科学依据。

研究发现GAPDH参与调控神经元凋亡，观察到凋亡神经元线粒体中定位于线粒体内膜的GAPDH显著增多。进一步研究证实GSK-3通过磷酸化GAPDH Thr235来介导GAPDH转位进入线粒体内膜并介导细胞凋亡。同时，排除了GAPDH 通过形成Cyto C 通道促进Cyto C释放来介导细胞凋亡的可能性。有报道表明，GAPDH可以被亚硝基化，并把亚硝基转运传递给相互作用蛋白从而调控其功能。由此，我们提出进一步的假设：磷酸化GAPDH 的转位到线粒体内膜并通过亚硝基化阻断氧化呼吸链的功能并触发神经元凋亡。本项目需要进一步肯定GAPDH的亚硝基化，并寻找到其氧化呼吸链的相互作用蛋白，从而最终阐明GAPDH促神经元凋亡的机制。. 此外，在肿瘤细胞中我们也发现GAPDH具有与神经元细胞内类似的作用，这说明在细胞凋亡时GAPDH转位进入线粒体内膜具有较普遍的意义。同时，GAPDH还具有调控凋亡和细胞自噬切换的功能，这一作用不仅对退行性神经系统疾病的研究有提示作用，其对肿瘤细胞耐药性、上皮间质化和转移的研究也具有一定价值。因此，我们将深入探讨GAPDH调控细胞死亡和自噬的具体机制。