瘢痕疙瘩中DJ-1介导PTEN亚硝基化调控PI3K/Akt/mTOR信号通路的作用及机制研究

The continuous activation of PI3K/Akt/mTOR signal transduction pathway is the important mechanism of keloid formation. Our preliminary work found that the decrease of PTEN's phosphatase activity mediate the activation of PI3K/Akt/mTOR signal transduction pathway. However, the mechanism still remains uncertain. Our further work found that the expression of PTEN in keloid showed no significant difference compared with normal skin, however, the expression of SNO-PTEN in keloid increase obviously and its activity of phosphatase decreases obviously. We screen out DJ-1 protein with proteomic technology. The expression of DJ-1 in keloid is higher than the normal skin but the expression of SNO-DJ-1 decease. The suppression of DJ-1 can decrease the level of SNO-PTEN and recover the phosphatase activity of PTEN. DJ-1 and PTEN are interactional protein. So we speculate that the DJ-1 suppress the activity of PTEN's phosphatase activity by S-nitrosylation in keloid, resulting in the continuous activation of PI3K/Akt/mTOR signal transduction pathway. This research will take technologies like specimen collection,, deletion mutantion , FRET and mass spectromotry to expound the mechanism of PTEN's S-nitrosylation regulating PI3K/Akt/mTOR signal transduction pathway by DJ-1 in keloid.

PI3K/Akt/mTOR与瘢痕疙瘩形成密切相关。我们前期工作发现PTEN磷酸酶活性下调，介导了PI3K/Akt/mTOR通路的激活。然而，PTEN磷酸酶活性下调的机制不清楚。进一步工作发现： PTEN的表达水平无明显变化，而亚硝基化PTEN（SNO-PTEN）水平上调，SNO-PTEN的磷酸酶活性明显降低；运用蛋白质组学技术筛选到DJ-1蛋白；DJ-1在瘢痕疙瘩中高表达，但亚硝基化DJ-1（SNO-DJ-1）水平下降，抑制DJ-1可下调SNO-PTEN水平，恢复PTEN磷酸酶活性；DJ-1与PTEN为相互作用蛋白。因此，我们推测：瘢痕疙瘩中，DJ-1通过转亚硝基化作用抑制PTEN的磷酸酶活性，介导PI3K/Akt/mTOR通路的激活。本课题将采用临床标本收集检测、缺失突变、FRET 、质谱分析等方法技术，阐明DJ-1介导PTEN亚硝基化调控PI3K/Akt/mTOR通路的具体机制。

瘢痕疙瘩（Keloid）系临床常见的病理性瘢痕，是继发于皮肤损伤的一种胶原过度沉积性疾病，是烧伤、整形外科和康复医学领域的难治性疾病。PI3K/Akt/mTOR与瘢痕疙瘩形成密切相关。负反馈调节信号分子PTEN，通过促进PIP3去磷酸生成PIP2，实现PI3K/Akt/mTOR信号通路的负性调节。 我们前期工作发现PTEN磷酸酶活性下调，介导了PI3K/Akt/mTOR通路的激活。然而，PTEN磷酸酶活性下调的机制不清楚。我们的研究证实：1.亚硝基化PTEN (SNO-PTEN)在瘢痕疙瘩中表达上调，而PTEN的磷酸酶活性明显下降。2.采用质粒构建与转染、点突变、质谱分析等方法，确定PTEN的亚硝基化作用位点在Cys136。3.明确了瘢痕疙瘩成纤维细胞中SNO-DJ-1（亚硝基化DJ-1）水平下降，而瘢痕疙瘩成纤维细胞中DJ-1和PTEN有相互作用，DJ-1与PTEN 作用的位点在76-136aa序列。4.证实了SNO-DJ-1转移NO基团给PTEN蛋白，SNO-DJ-1转移NO基团给PTEN蛋白的位点在Cys136，而不是Cys46。5.明确PTEN亚硝基化激活了PI3K/Akt/mTOR信号通路，促进了瘢痕疙瘩的发生发展。我们的研究证实了瘢痕疙瘩中DJ-1通过转亚硝基化作用抑制PTEN磷酸酶活性的可靠证据，阐明DJ-1介导PTEN亚硝基化调控PI3K/Akt/mTOR信号通路的新机制，为确立DJ-1作为瘢痕疙瘩治疗的新靶点提供更充分的科学依据。