CONTROL OF PEPTIDE HORMONE BIOSYNTHESIS BY PC2 AND 7B2

生物活性多肽激素的产生 神经内分泌细胞需要一系列复杂的生物合成、 成熟赛事和交通赛事。最关键的早期步骤之一 大前体多肽的蛋白水解性加工 枯草杆菌素样蛋白水解酶，即前激素转换酶(PC)。 关于个人计算机行动的许多关键问题仍然存在 回答；特别是，调节活动的机制 人们对个人电脑知之甚少。我们和其他人最近已经表明， 神经内分泌蛋白7B2代表PC2和 对其生物合成产生了深远的影响。羧基末端 该蛋白的多肽(7B2 CT多肽)是一种有效的PC2和 ProPC2的激活，而该蛋白的氨基末端部分 (21 KDa 7B2)矛盾地促进细胞内转运和 ProPC2活化为成熟的PC2。这项提议的主要目标是 阐明CT抑制终止的机制 以及21 kDa蛋白的作用机制。 促进proPC2的激活。具体目标包括以下几点：1)使用 反义和代谢标记实验精确定义 7B2在ProPC2成熟过程中的细胞作用 二者相互作用的结构-功能关系 分子；2)在体外使用纯化的重组分子 结合实验确定亲和力和最佳结合 7B2和PC2的各种形式相互作用的条件。我们会 还要检验7B2在折叠和/或折叠过程中是必需的假设 ProPC2激活。 综上所述，本提案中描述的实验应该提供 关于细胞生物学和生物化学的重要信息 ProPC2/PC2与其调控蛋白7B2的相互作用长期的 这些实验的目标是了解细胞内的关键调控机制 生物活性多肽激素的生产。最终这就是 理解可能为治疗的调节提供新的途径 多肽激素水平。

The generation of biologically active polypeptide hormones by neuroendocrine cells entails a complex series of biosynthetic, maturational, and transport events. One of the most crucial early steps is proteolytic processing of the large precursor polypeptide by subtilisin-like proteolytic enzymes, the prohormone convertases (PCs). Many critical questions regarding the action of the PCs remain to be answered; in particular, the mechanisms which regulate the activity of the PCs are poorly understood. We and others have recently shown that the neuroendocrine protein 7B2 represents a binding partner for PC2 and exerts a profound influence on its biosynthesis. The carboxyl terminal peptide of this protein (7B2 CT peptide) is a potent inhibitor of PC2 and of proPC2 activation, while the amino-terminal portion of this protein (21 kDa 7B2) paradoxically facilitates the intracellular transport and the activation of proPC2 to mature PC2. Major goals of this proposal are to elucidate both the mechanism for termination of inhibition of the CT peptide as well as the mechanism by which the 21 kDa protein acts to promote proPC2 activation. Specific aims include the following: 1) to use antisense and metabolic labelling experiments to precisely define the cellular roles of 7B2 in the maturation of proPC2, and to provide structure-function relationships for the interaction of these two molecules; and 2) to use purified recombinant molecules in in vitro binding experiments to determine the affinities and optimum binding conditions of the various forms of 7B2 and PC2 for each other. We will also test the hypothesis that 7B2 is required for folding and/or during proPC2 activation. Taken together, the experiments described in this proposal should provide significant information on the cell biology and biochemistry of the interaction of proPC2/PC2 with its regulatory protein, 7B2. The long-term goal of these experiments is to understand key regulatory mechanisms in the production of bioactive peptide hormones. Ultimately this understanding may provide new avenues for the therapeutic modulation of peptide hormone levels.