STRUCTURE/FUNCTION OF CARBOXYPEPTIDASE D

许多神经内分泌肽和蛋白质是由前体通过一系列内肽酶和羧肽酶反应产生的。羧基肽酶E （CPE）最初被认为是大多数神经内分泌肽生成所必需的唯一的羧基肽酶。最近我们发现，由于基因编码区域的点突变，肥胖/肥胖小鼠缺乏CPE活性。这些老鼠体重超标，而雄性老鼠血糖过高。虽然在这些小鼠中，羧基肽酶步骤的肽加工减少了，但尽管完全没有功能性CPE，它并没有被消除。在寻找具有cpe样性质的新型酶的过程中，我们最近发现了羧基肽酶D （CPD）。根据我们过去三年的研究，我们假设CPD有助于神经内分泌肽的加工以及通过分泌途径的蛋白质。在目前的应用中，我们将继续测试这一假设，并探索CPD在细胞中执行其他功能的可能性。在Specific Aim 1中，我们将比较CPD的三个羧基肽酶样结构域的酶学性质。由于CPD的第三个羧基肽酶样结构域似乎没有酶活性，我们将探索该结构域的其他潜在功能。在目标2中，我们将研究在细胞和各种组织中发现的CPD形式的分子基础。之前，我们已经发现了多种形式的CPD的证据。在Aim 3中，我们将通过在缺乏CPE的细胞系中过表达这种酶来解决CPD的生理功能，然后测试升高的CPD是否可以进一步补偿CPE的缺乏。此外，我们将使用各种策略来降低培养细胞中的CPD（抑制剂，反义RNA）水平，然后检查对肽/蛋白质加工和分选的影响。总之，这种协调一致的方法将提供关于CPD在神经内分泌肽产生中的作用的重要信息。

Many neuroendocrine peptides and proteins are produced from precursors by a series of endopeptidase and carboxypeptidase reactions. Carboxypeptidase E (CPE) was initially thought to be the only carboxypeptidase required for the generation of most neuroendocrine peptides. Recently we found that fat/fat mice lack CPE activity due to a point mutation in the coding region of the gene. These mice are overweight, and males are hyperglycemic. Although peptide processing by the carboxypeptidase step is reduced in these mice, it is not eliminated despite the complete absence of functional CPE. In a search for novel enzymes with CPE-like properties, we recently discovered carboxypeptidase D (CPD). Based on our studies over the past three years, we hypothesize that CPD contributes to the processing of neuroendocrine peptides as well as proteins that transit the secretory pathway. In the present application, we will continue to test this hypothesis, as well as to explore the possibility that CPD performs additional functions in the cell. In Specific Aim 1, we will compare the enzymatic properties of the three carboxypeptidase-like domains of CPD. Since the third carboxypeptidase- like domain of CPD does not appear to be enzymatically active, we will explore other potential functions for this domain. In Aim 2, we will examine the molecular basis for the forms of CPD found in the cell, and in various tissues. Previously, we have found evidence for multiple forms of CPD. In Aim 3, we will address the physiological function(s) of CPD by overexpressing this enzyme in cell lines that are deficient in CPE, and then testing whether elevated CPD can further compensate for the deficiency of CPE. In addition, we will use various strategies to reduce levels of CPD (inhibitors, antisense RNA) in cultured cells and then examine the effect on peptide/protein processing and sorting. Together, this concerted approach will provide important information regarding the role of CPD in the production of neuroendocrine peptides.