天冬酰胺内肽酶的结构、自活化及催化机制研究

Mammalian asparaginyl endopeptidase (AEP) is a cysteine endopeptidase cleaving specifically after asparagine that itself undergoes partly autocatalytic proteolytic maturation upon reaching acidic compartments. AEP has emerged as an important enzyme in antigen and autoantigen processing. Overexpression of AEP in tumors is significant for invasion/metastasis. AEP has been implicated in TLR9 processing.TLR9 recognize unmethylated CpG DNA and play an essential role in innate and adaptive immunity.It is still not clear how AEP get self-activation, catalyze the substrate and how it is processed to 36kDa. We will study the molecular mechanism of AEP autoactivation with decrease of pH by structural biology and mechanism of AEP catalyzing substrate by analyzing the crystal sructure of AEP in inactive form, active form and complex of AEP with inhibtor and substrate analogue. We have already solved the crystal structure of AEP in inactive form, analyzed the mutants of partial cleavage sites and active sites and found that the process of AEP self-activation is reversible. We will analyze in which circumstance AEP involve in the process of TLR9 processing. These achievements will build reliable foundation for drug discovery on tumor invasion/metastasis.

AEP是一种溶酶体半胱氨酸内肽酶，随pH降低自活化后，能特异性水解天冬酰胺残基羧基端肽键，是参与抗原及自身抗原加工的一个重要酶。AEP在肿瘤细胞中高表达，对于肿瘤的侵袭和迁移具有重要作用。AEP还参与TLR9的加工，而TLR9能够识别非甲基化CpG DNA，在起始和适应免疫中发挥作用。目前关于AEP自活化、催化底物的分子机理以及AEP是如何被加工为36kDa的过程均不清楚。本课题以AEP为研究对象，结构生物学为主要研究手段，通过获得AEP非活化、活化状态及其分别与抑制剂、底物类似物复合物的精细三维结构，结合酶活性分析来阐明AEP自活化及催化底物的分子机制。同时分析AEP如何参与TLR9的加工。我们已经获得了AEP非活化状态的晶体结构，并且首次发现AEP自活化过程是可逆的。本项目的实施将为开发以AEP为靶标、能够抑制肿瘤侵袭和迁移的药物打下坚实的结构基础。

天冬酰胺内肽酶(asparaginyl endopeptidase, AEP)是一种溶酶体半胱氨酸内肽酶，能够特异性水解天冬酰胺残基羧基端肽键，也能够对天冬氨酸残基羧基端肽键进行水解。AEP在溶酶体中活化时，需切除N端及C端的两个多肽，产生46kDa的活性形式，并进一步被加工为36kDa。本课题解析了非活化及活化状态AEP晶体结构。结构比对发现，AEP关键催化残基及催化机制与caspases相似。突变及酶活分析显示，N44, R46, H150, E189, C191, S217/S218 和 D233组成活性中心。AEP活化时，自身将帽子结构切掉，暴露活性位点。我们发现，部分活化的AEP（SDS-PAGE分析表明，AEP的 N及C端处于被切开状态）通过改变pH可以变回非活化状态。这一结果在获得部分活化AEP晶体结构时得到验证。AEP的活性可以被cystatin C抑制，在制备AEP与cystatin C复合物时，发现cystatin C是作为AEP的底物，通过底物竞争机制来调节AEP活性。我们还发现AEP具有连接酶活性。