HCdc14A在胰岛β细胞自噬中的作用和机制研究

The number and function declining of β cell is the key reason for diabetes. Autophagy can reduce β cell apoptosis caused by glycolipids toxicity and endoplasmic reticulum stress. HCdc14A (Human Cell division cycle gene 14A) is a double specificity serine/threonine phosphatase. We found that LC3-Ⅱ markedly increased in mice β TC3 Cells with HCdc14A over-expression, and HCdc14A also increased in serum deprivation induced autophagy, which indicating that HCdc14A is involved in the regulation of autophagy. ZIPK (Zipper-interacting protein kinase) is the necessary factor of serum deprivation induced autophagy in muscle smooth cells. We verified ZIPK interacted with HCdc14A. ZIPK can interact with HCdc14A N-terminal with phosphatase activity. The co-localization relied on the kinase activity of ZIPK. ZIPK can release HCdc14A N-terminal from the nucleus to the cytoplasm. Therefore, we propose that ZIPK regulates HCdc14A localization, stability and phosphatase activity, and thus controls autophagy. This study is to investigate the interaction of ZIPK and HCdc14A, and their roles in serum starvation and high sugar induced autophagy on β cell. This will strive to provide a new therapeutic target and to make contributions to elucidate the pathogenesis of diabetes.

β细胞的数量减少和功能减退是糖尿病发病的核心环节。HCdc14A是一种重要的双特异性丝/苏氨酸磷酸酶，我们在小鼠β-TC3细胞转染HCdc14A，发现LC3-Ⅱ的表达明显增多。饥饿诱导β-TC3细胞自噬，HCdc14A的表达也明显增多，提示HCdc14A参与自噬的调控过程。ZIPK是饥饿诱导平滑肌细胞自噬的必要调控因子。我们发现ZIPK与HCdc14A相互作用，ZIPK能与HCdc14A的N端（磷酸酶活性端）结合，其共定位依赖于ZIPK的激酶活性，ZIPK可以使HCdc14A的N端从细胞核释放到细胞浆内。因此，我们猜测ZIPK调控HCdc14A的定位、稳定性和磷酸酶活性，并由此调控自噬。本课题通过生物信息学预测ZIPK对HCdc14A的调控位点并探明调控机制，同时深入研究饥饿和高糖环境下，HCdc14A对β细胞自噬的调控。这将为干预糖尿病的发生发展提供重要的理论依据以及目标明确的靶蛋白。

糖尿病（DM）是全球常见的慢性疾病，也是严重的公共卫生问题。胰岛β细胞的耗竭在糖尿病的发展中起着至关重要的作用。细胞自噬作为一种自我修复的过程，有助于维持代谢稳态，并可以保护胰岛β细胞免于饥饿或高葡萄糖应激时的凋亡。 然而，胰岛β细胞中自噬过程的潜在调控网络尚未得到充分探索。本报告在细胞和分子水平对Hcdc14A在胰岛β细胞自噬中的作用和机制进行研究。首先，我们研究了在不同葡萄糖浓度刺激下，Hcdc14A过表达质粒或siRNA处理的胰腺β细胞的生理反应。结果提示高糖刺激下，Hcdc14A过表达可以刺激β-TC3细胞的生长速率，对细胞存活有利，导致G1/G0相停滞，诱导β-TC3细胞产生和分泌胰岛素。Hcdc14A siRNA则抑制β-TC3细胞的生长，引起β-TC3细胞凋亡，导致G2/M相停滞，减少β-TC3细胞产生和分泌胰岛素。并且，发现高糖刺激下，Hcdc14A过表达增强自噬，而Hcdc14A siRNA抑制自噬。接着，根据预测的磷酸化位点Ser484和Ser583构建了Hcdc14A的去磷酸化突变体。与野生型和Ser583突变体相比，Ser484突变体不影响AMPK或mTOR信号通路以及β-TC3细胞的自噬启动。免疫荧光分析表明，Ser484突变体使LC3II表达减少，这提示自噬激活取决于Hcdc14A的Ser484磷酸化。为了证实这一假设，我们用雷帕霉素或磷酸酶抑制剂进一步处理β-TC3细胞，观察到Ser484突变体损害了雷帕霉素介导的自噬。最后，用GFP免疫沉淀β-TC3细胞用于质谱分析，发现ZIPK是与Hcdc14A相互作用的潜在靶蛋白。但ZIPK无法与Ser484 突变体Hcdc14A结合。与只转染Hcdc14A野生型过表达质粒相比，ZIPK siRNA既不促进高糖刺激的β-TC3细胞的自噬，也不调节细胞周期阻滞。以上数据表明ZIPK通过调节Ser484位点的Hcdc14A磷酸化影响自噬。我们的结果为Hcdc14A在胰岛β细胞自噬中的作用提供新的见解，并提示Hcdc14A磷酸化在预防和治疗糖尿病方面的潜在治疗价值。