Neuropeptidase Function

神经肽是多种小肽的集合，在中枢神经系统中作为神经递质和神经调节剂，在外围神经系统中作为神经激素。这些信号分子的水平被许多中枢活性因子调节，并在许多疾病状态中受到影响，包括精神障碍、阿尔茨海默病和高血压。神经肽被一组被称为神经肽酶的蛋白水解酶灭活。这些酶的治疗调节可以通过改变其神经肽底物的水平形成有效治疗的基础，因此它们是重要的药物靶点。然而，这些努力受到阻碍，一方面是因为底物特异性不被理解，另一方面是因为底物和抑制剂结合的结构信息很少，这些信息可以用于改进治疗方法。本研究的目的是利用内肽酶24.16（神经溶解素）作为模型系统，了解神经肽酶的特异性和功能。这种酶，一种锌金属肽酶，使神经肽神经紧张素失活，也可能参与其他神经活性肽的失活。内啡肽酶24.16具有许多神经肽酶共有的两个不寻常的特性：它只切割短的寡肽，并且它识别高度多样化的切割位点。这些不寻常性质的基础将通过结合酶的高分辨率结构测定和功能研究的方法来探索。我们已经确定了内肽酶24.16的晶体结构，它显示出一个深层的活性通道，阻止蛋白质进入。我们将测试底物的长度进一步受到活性位点通道性质限制的假设，以及催化区域高度可塑性容纳不同序列的假设。提出了四个具体目标：1)用一系列肽和抑制剂来确定晶体结构，从而揭示底物结合的细节；2)通过底物特征的系统变化来表征特异性的功能方面；3)通过表征密切相关的内肽酶来确定识别的重要特征；4)通过诱变调节和改变酶的特异性来测试和完善我们的识别模型。这项工作将明确神经肽失活的一般机制，并提供抑制剂结合的详细视图，为未来的治疗发展奠定基础。

Neuropeptides are a diverse assembly of small peptides that serve as neurotransmitters and neuromodulators in the central nervous system and as neurohormones in the periphery. The levels of these signaling molecules are known to be modulated by many centrally active agents and affected in a number of disease states-including psychotic disorders, Alzheimer's disease, and hypertension. Neuropeptides are inactivated by a group of proteolytic enzymes known as neuropeptidases. Therapeutic modulation of these enzymes can form the basis of effective treatments by altering the levels of their neuropeptide substrates, and they are therefore important drug targets. These efforts are hampered, however, both because substrate specificity is not understood and because there is a little structural information on the binding of substrates and inhibitors, which could be used for improving therapeutics. The goal of the proposed study is an understanding of specificity and function in neuropeptidases using endopeptidase 24.16 (neurolysin) as a model system. This enzyme, a zinc metallopeptidase, inactivates the neuropeptide neurotensin and may also participate in the inactivation of other neuroactive peptides. Endopeptidase 24.16 has two unusual properties shared by a number of neuropeptidases: it cleaves only short oligopeptides, and it recognizes a highly diverse set of cleavage sites. The basis for these unusual properties will be explored by an approach combining high- resolution structure determination and functional studies of the enzyme. We have determined the crystal structure of endopeptidase 24.16, which shows a deep active channel that prevents access by proteins. We will test the hypotheses that the length of substrates is further limited by the nature of the active site channel and that diverse sequences are accommodated by a high degree of plasticity in the catalytic region. Four specific aims are proposed: 1) to determine crystal structures with a series of peptides and inhibitors that will reveal the details of substrate binding, 2) to characterize the functional aspects of specificity through systematic variation of substrate features, 3) to identify important features of recognition by characterizing a closely related endopeptidase, 4) to test and refine our models of recognition by modulating and altering the specificity of the enzyme through mutagenesis. This work will define the general mechanisms of neuropeptide inactivation and provide a detailed view of inhibitor binding, laying the foundation for future therapy development.