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，由前脑啡肽（PE）通过蛋白水解加工合成。由于成熟加工的脑啡肽（不是PE）作为多肽神经递质具有生物活性，参与疼痛缓解和脑功能，因此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在将PE加工成活性脑啡肽的过程中起关键作用。为了了解PE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复合物的相互作用域。该目标将评估PE裂解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。该项目将在三个具体目标中实现。第一个目的是评估DXMS研究中PE裂解位点对水环境的相对可及性，暴露于水溶液，在pH条件下使用重组PE在氢氘交换质谱（DXMS）研究中进行，代表PE加工发生的分泌囊泡内的条件。第二个目标将定义组织蛋白酶l介导的PE和HMW PE衍生中间体的裂解位点，并将这些裂解位点与DXMS研究的构象特征进行比较。第三个目标将通过DXMS和直接化学交联研究来研究PE-cathepsin L的相互作用，以了解配合物的构象特征。该项目将获得有关PE裂解位点的构象特征的知识，包括对水环境的可及性，由组织蛋白酶L产生的PE裂解产物，以及PE-组织蛋白酶L复合物的相互作用域。研究结果将为活性脑啡肽类阿片肽神经递质的生物合成提供新的蛋白水解机制。

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.