Proenkephalin Processing for Biosynthesis of Enkephalin Peptide Neutrotransmitter

说明(申请人提供)：脑啡肽类神经递质是由前脑啡肽(PE)通过蛋白分解过程合成的内源性阿片肽类神经递质。由于成熟的脑啡肽(不是PE)作为一种多肽类神经递质具有生物活性，参与疼痛缓解和脑功能，因此蛋白水解性加工对脑啡肽类神经递质的活性至关重要。最近的研究表明，分泌囊泡组织蛋白L在将PE加工成活性脑啡肽过程中起着关键作用。为了了解PE的蛋白降解加工机制，本项目的目标是研究组织蛋白L在加工过程中PE裂解位点的构象特征，并确定PE-组织蛋白酶L复合体的相互作用结构域。这一目标将评估PE裂解位点的构象特征参与组织蛋白酶L的蛋白质降解过程的假设。该项目将在三个特定目标下实现。第一个目标是在DXMS研究中评估PE裂解位点对水环境的相对可达性，使用重组PE在氢氚交换-质谱仪(DXMS)研究中进行，该研究在pH条件下进行，代表发生PE加工的分泌囊中的那些。第二个目的是定义组织蛋白酶L介导的PE和高分子聚乙烯衍生中间体的裂解位点，并将这些裂解位点与DXMS研究中的构象特征进行比较。第三个目标是用DXMS和直接化学交联研究PE-组织蛋白L的相互作用，以了解该复合体的构象特征。这个项目将获得关于PE裂解位点的构象特征的知识，这些特征涉及到水环境的可访问性、组织蛋白酶L产生的PE裂解产物以及PE-组织蛋白酶L复合体的相互作用结构域。这些结果将为活性脑啡肽类神经递质的生物合成提供新的蛋白质分解机制的知识。

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.