Neuropeptidase Function

神经肽是一组不同的小肽，在中枢神经系统中作为神经递质和神经调节剂，在外周作为神经激素。众所周知，这些信号分子的水平受到许多中枢活性物质的调节，并在许多疾病状态下受到影响--包括精神病、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来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，这既是因为底物的特异性还不清楚，也是因为关于底物和抑制剂结合的结构信息很少，可以用于改进治疗。这项拟议研究的目标是以内肽酶24.16(神经溶素)作为模型系统来了解神经肽酶的特异性和功能。这种酶是一种锌金属肽酶，可以使神经肽神经降压素失活，也可能参与其他神经活性多肽的失活。内肽酶24.16具有许多神经肽酶所共有的两个不同寻常的性质：它只切割短的寡肽，它识别一组高度多样化的切割位点。这些特殊性质的基础将通过结合高分辨率结构测定和酶的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，它显示了一个阻止蛋白质进入的深层活性通道。我们将检验这样的假设，即底物的长度进一步受到活性位点通道的性质的限制，并且催化区域的高度可塑性适应了不同的序列。提出了四个具体的目标：1)确定一系列多肽和抑制剂的晶体结构，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识别重要的识别特征；4)通过突变来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，并提供详细的抑制剂结合的观点，为未来的治疗开发奠定基础。

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.