Dhanvantari Savita, Shen Fu-Sheng, Adams Tiffany, Snell Christopher R, Zhang ChunFa, Mackin Robert B, Morris Stephen J, Loh Y Peng
Section on Cellular Neurobiology, National Institutes of Health, Bethesda, Maryland 20892-4480, USA.
Mol Endocrinol. 2003 Sep;17(9):1856-67. doi: 10.1210/me.2002-0380. Epub 2003 Jun 26.
In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.
在家族性高胰岛素原血症中，胰岛素原基因的特定突变与循环血浆中胰岛素原水平的显著升高有关。然而，这种疾病的分子和细胞基础仍未明确。在这里，我们研究了这些突变如何破坏将胰岛素原靶向到调节性分泌途径的分泌颗粒所需的分选信号，从而导致胰岛素原的不受调控释放。通过结合分子建模和定点诱变，我们确定了胰岛素原中对于正确分选到内分泌细胞分泌颗粒中所必需的结构分子基序。我们表明，膜羧肽酶E（CPE），先前被鉴定为一种激素原分选受体，对于胰岛素原分选至关重要。这通过在β细胞系中利用短干扰RNA介导的CPE耗竭以及用CPE的显性负性突变体进行转染得以证明。在家族性高胰岛素原血症中发现的突变胰岛素原无法与CPE结合，并且不能被有效分选。这些发现提供了证据，表明家族性高胰岛素原血症患者中血浆胰岛素原水平的升高是由CPE介导的突变胰岛素原向调节性分泌途径的分选破坏所导致的。
