一种新短肽COX52-69抑制糖诱导的胰岛素分泌的机制研究

COX52-69, a novel truncated peptide we discovered, can inhibit glucose-induced insulin secretion, suggesting its clinical value. Our previous experiment found that the peptide could combine with the membrane protein CHRM3 and CCKAR, upregulates the mRNA level of the subunit of protein kinase, PRKACA, and some other genes; activates the BK channel (Large-conductance, calcium-activated-K+ channel) and increases the BK channel current. Base on our previous finding, we hypothesize that COX52-69 binds to the membrane protein CHRM3 or CCKAR, and its signal is mediated by a subunit of protein kinase that contains PRKACA, furthermore, the binding increases the intracellular calcium ion concentration and promotes the opening of the BK channel, causing an outflux of intracellular K+, thereby accelerates the membrane repolarization and even to the extent of hyperpolarization, therefore, inhibits the secretion of insulin. The project will perfect the hypothesis in more details by using techniques such as the fluorescent polarization technology to determine whether truncated peptide COX52-69 binds with membrane protein CHRM3 or CCKAR, and its target molecules from the two membrane proteins listed above. Other techniques include the use of Co-IP and crispr / cas9 to identify the signal transduction pathway involving PAKACA, and the use of patch-clamp to confirm whether the aforementioned factors will lead to the opening of the BK channel, thereby inhibiting glucose-induced insulin secretion. This study helps to reveal a new pathway for the regulation of insulin secretion and lay the foundation for the clinical treatment of hyperinsulinemia and related diseases.

我们发现的新短肽COX52-69能够抑制糖诱导的胰岛素分泌，暗示其临床应用价值。前期实验发现该多肽可以与膜蛋白CHRM3以及CCKAR结合，上调蛋白激酶亚基PRKACA等基因的mRNA水平；激活BK通道，增加BK通道电流。据此我们提出假说：COX52-69可以与细胞膜上蛋白CHRM3或CCKAR结合，其信号通过含PRKACA亚基的蛋白激酶传导，增加细胞内钙浓度，促进BK通道的开放，致膜复极化加速乃至超极化，从而抑制胰岛素的分泌。该课题将更深入地探索该假说的细节，大体包括利用荧光偏振技术明确该多肽与CHRM3/CCKAR的结合，确定COX52-69调控的靶点分子；利用Co-IP及crispr/cas9等技术确定PAKACA参与的信号传导途径；利用膜片钳确定前述因素致BK通道开放，从而抑制糖诱导的胰岛素分泌。该研究有助于揭示一条胰岛素分泌调控的新途径，为治疗高胰岛素血症等实际临床应用奠定基础。

在基金的支持下，该课题取得了如下的进展和结果：.1. 完成了从线肽向环形肽的改造，也就是口服改造。并且完成了其功能测试以及药代动力学工作，实现了有功能的口服的多肽COX52-69， 这极大地提高了该多肽的成药性，口服给药将是更方便更易被接受的给药途径。.2.其次发现了该环肽具有新的功能，也就是在降低胰岛素的同时，还有一定的降低血糖的作用或者说至少并不升高血糖，这是很少有的现象，目前已经对其机制进行了初步定探索。.3.通过Crispr方法，我们knock-out了筛选出的CHRM3、PRKACA、KCNMA1以及Camk2b，并完成了knock-out后的动物实验和细胞和分子水平的实验，这些结果可以很好的解释该多肽抑制胰岛素分泌的机制，该工作已成文，虽多次投稿被拒，仍在完善努力中。.4. 目前在该基金的资助下发表了3篇标注该基金的SCI论文。.5. 获授权发明专利一项：修饰肽在制备抑制胰岛分泌胰岛素药物上的用途及修饰方法（ZL202110380196.1）. 该专利为该多肽的成药应用奠定了基础。.6. 培养多肽研究的毕业硕士研究生5名。