NEURAL POLYHORMONE PROCESSING MECHANISMS

越来越多的神经分泌肽被发现来自 从多激素前体，其在其序列内含有 一种以上分泌产物的结构。 这引发了根本性的 关于控制生产的生物化学机制的问题 这些肽和它们的路由到适当的亚细胞位置。 具体来说，这些功能在多大程度上取决于 前体，而不是加工酶的性质？ 这 这个问题将在加工顺序的实验中解决 导致海兔袋中产生神经分泌产卵激素（ELH） 细胞 该序列中的前体具有已知的一级结构， 通过蛋白水解过程产生ELH和其他分泌肽 序列已被详细描述。 此外，中间体 在该序列中，导致不同产品被路由到不同的 细胞内的位置。 有许多假定的切割位点 但实际使用的是哪一种还不知道。 此外，前体及其中间体可能是膜结合的， 但这些分子的区域介导这种关联，这可能是 重要的路由，还有待确定。 此外， 还知道前体和中间体是否被相同的或 不同的内切蛋白酶。 本提案中的实验旨在 回答这些问题 前体的部分序列分析， 中间体和产物将用于鉴定切割位点， 参考前体的已知结构。 肽的比较 在无细胞翻译系统中合成的前体的图谱， 来自完整细胞合成的前体的那些将识别 包含“信号”序列的区域。 脂溶性标记 光活化探针将产生膜区域的信息 协会 前体和中间体裂解的表征 袋细胞匀浆中的活性应揭示多重性的程度 加工内切蛋白酶和亚细胞分级分离技术 将用于确定其亚细胞分布。 这些 实验应该能让我们深入了解基本的细胞过程 控制神经肽和肽激素的产生。

A growing number of neurosecretory peptides are being found to be derived from a polyhormonal precursor which contains within its sequence the structures of more than one secretory product. This raises fundamental questions concerning the biochemical mechanisms controlling the production of these peptides and their routing to appropriate subcellular locations. Specifically, to what extent do these functions depend on the structure of the precursor, as opposed to properties of the processing enzymes? This question will be addressed in experiments on the processing sequence leading to the neurosecretory egg-laying hormone (ELH) in Aplysia bag cells. The precursor in this sequence is of known primary structure and gives rises to ELH and other secretory peptides by a proteolytic processing sequence which has been characterized in detail. Moreover, intermediates in this sequence which lead to different products are routed to different locations within the cell. There are a number of putative cleavage sites in the precursor, but which of these are actually used is not yet known. Moreover, the precursor and its intermediates are probably membrane-bound, but the regions of these molecules mediating this association, which may be important in routing, have yet to be determined. In addition, it is not yet known if the precursor and intermediates are cleaved by the same or different endoproteases. The experiments in this proposal are designed to answer these questions. Partial sequence analysis of the precursor, intermediates, and products will be used to identify cleavage sites by reference to the known structure of the precursor. Comparison of peptide maps of the precursor synthesized in a cell-free translation system to those from the precursor synthesized by intact cells will identify the region containing the "signal" sequence. Labelling with a lipid-soluble photoactivated probe will yield information on regions of membrane association. Characterization of precursor and intermediate cleaving activity in bag cell homogenates should reveal the degree of multiplicity of the processing endoproteases, and subcellular fractionation techniques will be used to determine their subcellular distribution. These experiments should yield insights into the fundamental cellular processes controlling the production of neuropeptides and peptide hormones.