Proenkephalin Processing for Biosynthesis of Enkephalin Peptide Neutrotransmitter

描述（由申请人提供）：脑啡肽神经递质是内源性阿片肽神经递质，通过蛋白水解加工由脑啡肽原（PE）合成。由于成熟的加工脑啡肽神经肽（不是PE）作为肽神经递质具有生物活性，参与疼痛缓解和脑功能，蛋白水解加工对脑啡肽的神经递质活性至关重要。最近的研究结果表明，分泌囊泡组织蛋白酶L在处理PE活性脑啡肽的关键作用。为了了解PE的蛋白水解加工机制，本项目的目标将是研究组织蛋白酶L加工过程中PE切割位点的构象特征，并结合定义PE-组织蛋白酶L复合物的相互作用结构域。这一目标将评估PE切割位点的构象特征参与组织蛋白酶L的蛋白水解加工的假设。该项目将实现三个具体目标。第一个目标将评估PE裂解位点相对accessibility的水性环境中DXMS研究暴露于水性溶剂，使用重组PE在氢氘交换质谱（DXMS）的研究进行pH值条件下，代表那些内分泌囊泡PE加工发生。第二个目标将定义组织蛋白酶L介导的PE和高分子量PE衍生中间体的切割位点，并将这些切割位点与DXMS研究的构象特征进行比较。第三个目标是通过DXMS和直接化学交联研究PE-组织蛋白酶L相互作用，以了解复合物的构象特征。该项目将获得有关PE裂解位点的构象特征的知识，相对于accessibility到水环境中，PE裂解产物产生的组织蛋白酶L，和PE-组织蛋白酶L复合物的相互作用域。研究结果将为脑啡肽阿片肽神经递质生物合成的蛋白水解机制提供新的知识。

DESCRIPTION (provided by applicant): Enkephalin peptide neurotransmitters are endogenous opioid peptide neurotransmitters that are synthesized from proenkephalin (PE) by proteolytic processing. Because the mature processed enkephalin neuropeptide (not PE) is biologically active as a peptide neurotransmitter, participating in pain relief and brain functions, proteolytic processing is crucial for neurotransmitter activities of enkephalins. Recent findings have indicated a key role for secretory vesicle cathepsin L in processing PE to active enkephalin peptides. To understand the PE proteolytic processing mechanism, the goal of this project will be to investigate the conformational features of PE cleavage sites during processing by cathepsin L, combined with defining interacting domains of PE-cathepsin L complexes . This goal will assess the hypothesis that conformational features of PE cleavage sites participate in the proteolytic processing by cathepsin L. This project will be achieved in three specific aims. The first aim will assess the relative accessibilities of PE cleavage sites to the aqueous environment in DXMS studies exposure to aqueous solvent, using recombinant PE in hydrogen deuterium exchange-mass spectrometry (DXMS) studies conducted under pH conditions representing those within secretory vesicles where PE processing occurs. The second aim will define the cathepsin L-mediated cleavage sites of PE and HMW PE-derived intermediates, and compare these cleavage sites with their conformational features from DXMS investigations. The third aim will study PE-cathepsin L interactions by DXMS and direct chemical cross-linking studies to understand conformational features of the complex. This project will have gained knowledge concerning the conformational features of PE cleavage sites with respect to accessibilities to the aqueous environment, PE cleavage products generated by cathepsin L, and interacting domains of PE-cathepsin L complexes. Results will provide new knowledge of the proteolytic mechanisms utilized for biosynthesis of active enkephalin opioid peptide neurotransmitters.