Biosynthesis of Enkephalin Opioid Peptides

描述(申请人提供)：需要对前脑啡肽(PE)进行蛋白质分解处理，以产生调节止痛、行为和免疫细胞功能的活性脑啡肽阿片肽。因此，确定将PE转化为活性脑啡肽所需的多步蛋白分解途径是至关重要的。我们对PE加工的研究发现，在含有脑啡肽的嗜铬颗粒中，被称为‘前激素硫醇蛋白水解酶’(PTP)的半胱氨酸蛋白酶是主要的PE裂解活性。值得注意的是，这一持续进行的项目的最新进展利用活性部位亲和标记和质谱仪多肽显微测序来确定组织蛋白酶L的相关活性。组织蛋白酶L与脑啡肽共同定位于分泌小泡中，组织蛋白酶L在与天然PTP相同的切割位点上裂解含有脑啡肽的底物。值得注意的是，组织蛋白酶L基因敲除的小鼠大脑中脑啡肽水平降低。这些新的结果表明，分泌性囊泡组织蛋白L在脑啡肽的产生中起着关键作用。组织蛋白酶L和PTP的切割特性产生带有NH2末端碱性残基延伸的多肽中间体。这些发现表明，精氨酸/赖氨酸氨基肽酶是去除这些碱性残基所必需的。事实上，精氨酸/赖氨酸氨基肽酶活性与PE、脑啡肽和PTP/组织蛋白L共同定位于嗜铬细胞颗粒中。这些组织蛋白酶L和精氨酸/赖氨肽酶酶用于PE加工的新发现补充了我们早期的发现，即枯草杆菌菌素样PC1和PC2以及羧肽酶E/H参与了PE加工。这些发现为研究分泌囊泡组织蛋白L和精氨酸/赖氨酸氨基肽酶以及PC1和PC2在将PE加工成脑啡肽类阿片肽所需的蛋白分解途径中的作用奠定了基础。这一目标将通过四个具体目标来实现，它们将(1)通过(A)确定分泌囊泡组织蛋白L体外处理PE的相对效率和切割位置，与PC1和PC2相比，以及(B)在PC12细胞中酶与PE共表达时评估细胞PE处理，(2)评估组织蛋白酶L与脑啡肽和PC酶在嗜铬细胞、转基因PC12细胞以及大鼠脑和神经内分泌组织中的共存情况，(3)检测酶活性降低对(A)嗜铬细胞和接受反义酶表达的皮质神经元的PE处理的影响，以及用选择性化学抑制剂直接抑制组织蛋白酶L，以及(B)组织蛋白酶L基因敲除和PC2基因敲除小鼠中脑啡肽水平降低，以及(4)获得精氨酸/赖氨酸氨基肽酶产生脑啡肽的生化和分子分析。结果证实了分泌囊泡组织蛋白L和精氨酸/赖氨酸氨基肽酶这两种新的蛋白水解酶在脑啡肽类阿片肽生物合成中的作用。这些发现将提高我们对内源性阿片系统复杂性的认识。

DESCRIPTION (provided by applicant): Proteolytic processing of proenkephalin (PE) is required to produce active enkephalin opioid peptides that regulate analgesia, behavior, and immune cell function. It is, therefore, critical to define the multi-step proteolytic pathway required to convert PE into active enkephalin peptides. Our studies of PE processing have identified the cysteine protease termed 'prohormone thiol protease' (PTP) as the major PE cleaving activity in enkephalin-containing chromaffin granules. Notably, recent progress on this continuing project utilized active site affinity labeling and peptide microsequencing by mass spectrometry to identify the responsible PTP activity as cathepsin L. Cathepsin L is colocalized in secretory vesicles with enkephalin, and cathepsin L cleaves enkephalin-containing substrates at identical cleavage sites as native PTP. Significantly, cathepsin L knockout mice show reduced levels of enkephalin in brain. These new results implicate a key role for secretory vesicle cathepsin L in enkephalin peptide production. The cleavage specificities of cathepsin L and PTP generate peptide intermediates with NH2-terminal basic residue extensions. These findings indicate that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Indeed, Arg/Lys aminopeptidase activity is colocalized in chromaffin granules with PE, enkephalin, and PTP/cathepsin L. These new discoveries of cathepsin L and Arg/Lys aminopeptidase enyzmes for PE processing complement our earlier findings showing participation of the subtilisin-like PC1 and PC2 and carboxypeptidase E/H in processing PE. These findings provide the basis for the goal of this proposal that will investigate the roles of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, with PC1 and PC2, in the proteolytie pathway required for processing PE into enkephalin opioid peptides. This goal will be achieved in four specific aims, which will (1) evaluate PE processing by (a) determining the relative efficiency and cleavage sites for in vitro processing of PE by secretory vesicle cathepsin L, compared to PC1 and PC2, and (b) assessing cellular PE processing during enzyme coexpression with PE in PC12 cells, (2) assess the colocalization of cathepsin L with enkephalin and PC enzymes in chromaffin cells, transfected PC12 cells, and rat brain and neuroendocrine tissues, (3) examine the effects of reduced enzyme activity on PE processing in (a) chromaffin cells and cortical neurons subjected to antisense enzyme expression, as well as direct inhibition of cathepsin L with a selective chemical inhibitor, and in (b) cathepsin L knockout and PC2 knockout mice that show reduced enkephalin levels in brain, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for enkephalin production. Results can establish functional roles for two new protease components, secretory vesicle cathepsin L and Arg/Lys aminopeptidase, in the biosynthesis of enkephalin opioid peptides. These findings will enhance our knowledge of the complexity of the endogenous opioid system.