Stability, Folding and Formation of Deleterious Proteins

总结：人α-1-蛋白酶抑制剂（A1-PI）是一种丝氨酸蛋白酶抑制剂，在体内抑制嗜中性粒细胞弹性蛋白酶的活性，在25 ℃下，在1.5 M Gu中经历胍-HCl（Gu）诱导的具有中间状态的双相解折叠。该中间状态似乎短暂稳定（超过1-2小时的时间），主要是形成单体和二聚体的中间体，进一步更缓慢地分解。 Marszal博士通过用缓冲液稀释来折叠这些较小的中间体，并观察到在折叠物质的混合物中，折叠的二聚体而不是折叠的单体进一步聚合。 她已通过MALDI-TOF测量证实该物质为二聚体，并显示其为二硫键连接（通过每个单体中的单个巯基）。 杜博士已经证实了这一点。80%的A1-PI单体具有游离巯基，并且二聚体没有表现出抑制活性。 重要的是，Marszal博士已经证明，纯化二聚体聚合产生的物质似乎是其倍数，即没有形成三聚体。 此外，具有与A1-PI的RCL相同的序列的反应中心环（RCL）肽（其通常靶向嗜中性粒细胞弹性蛋白酶的活性位点）与单体结合，但不与二聚体结合，从而防止单体中间体的聚合，但不防止折叠二聚体的聚合。 先前已经报道了肽对单体聚合的抑制作用，并部分用于支持聚合模型，该模型涉及将一个A1-PI分子的RCL插入相邻分子上β-折叠的中心链之间，从而形成线性链（即，用于聚合的环-折叠模型）。 电子显微镜数据表明，从纯化的二聚体形成的聚合物是线性珠状结构，类似于从单体（通过在高温下加热）形成的那些和从来自具有分泌不良的突变体A1-PI的患者的肝细胞中的包涵体分离的那些。 二硫键连接的二聚体的结构限制和其形成线性聚合物的能力难以与所提出的环片模型相协调。 我们提出，形成的二硫键连接的二聚体可能涉及RCL埋葬，而不是插入，和二聚体的聚合物可能形成的β-折叠暴露在二聚体的外表面上的相互作用。 这里提出的模型，涉及二硫键连接的二聚体作为聚合物的最小重复单元（即基本单元），是一种替代机制，因为在某些反应条件下，不同的聚合机制可能会竞争。 在1.5 M Gu中形成的中间物质的明显瞬时稳定分布主要由单体和二硫键连接的二聚体物质组成。 二硫键连接的二聚体物质的形成基本上是不可逆的，因此，提出了为什么不是所有单体中间体在瞬时稳定期间反应形成二聚体中间体的问题。 Du博士折叠了这种明显稳定的物质分布，并分离了未反应的A1-PI单体。 通过用纯化单体在1.5 M Gu中重复实验，他证明获得了相同的瞬时稳定分布，因此A1-PI的微观异质性不是所有单体物质在1-2 h内在1.5 M Gu中未能形成二硫键连接二聚体的原因。 他通过跟踪在1.5M Gu的半胱氨酸封闭和未封闭的A1-PI单体中聚合4周的时间过程来进行该研究，并且事实上，对于两种样品，发现最终几乎所有的单体中间体随着二聚体和更高聚合物物质的产生而消失。 因此，明显的瞬态稳定分布是由于连续相、较快相和慢得多的相的动力学而发生的。 未封闭的A1-PI形成非二硫键连接的二聚体，这也进一步增加了巯基;因此，高度聚合的未封闭单体（仅含80%游离巯基）的SE-HPLC和非还原SDS凝胶结果的比较将允许独立估计游离巯基的最低水平。 非二硫键连接的二聚体的结构及其聚合机制仍有待确定。 从2000年9月开始，Dr. Du开始了一项计划，以确定CBER A1-PI参比标准品1号的效价和稳定性。 然而，经过大量努力熟悉相关试验后，发现参比标准品1对HIV呈阳性（NAT法），尽管该标准品是在1988年从经第一代HIV检测筛选的血浆中商业生产的。 我们的目标是重新开发新的参考标准或参与世卫组织合作计划。

Summary: Human alpha-1-proteinase inhibitor (A1-PI), which is a serine protease inhibitor and which in vivo inhibits the activity of neutrophil elastase, undergoes guanidine-HCl (Gu) induced biphasic unfolding with an intermediate state in 1.5 M Gu at 25 deg C. The intermediate state appears transiently stable (over a 1-2 h period) primarily with the formation of monomeric and dimeric intermediates that further polymerize more slowly. Dr. Marszal has folded these smaller intermediates by dilution with buffer and observed that in the mixture of folded species, folded dimer, but not folded monomer, further polymerizes. She has confirmed that this species is dimer by MALDI-TOF measurements and shown that it is disulfide linked (through the single sulfhydryl in each monomer). Dr. Du has established that ca. 80% of the A1-PI monomer has free sulfhydryl and that the dimer exhibits no inhibitory activity. Importantly, Dr. Marszal has demonstrated that the species generated by polymerization of the purified dimer appear to be multiples of it, i.e. no trimer is formed. Furthermore, a reactive center loop (RCL) peptide, which has the same sequence at that of the RCL of A1-PI, which normally targets the active site of neutrophil elastase, binds to the monomer, but not to the dimer, thereby preventing the polymerization of the monomeric intermediate but not of the folded dimer. The inhibition of polymerization of monomer by the peptide has been previously reported and used, in part, to support a model of polymerization involving the insertion of the RCL of one A1-PI molecule between the central strands of a beta-sheet on an adjacent molecule thereby forming a linear chain (i.e. a loop-sheet model for polymerization). Electron micrograph data demonstrate that polymers formed from purified dimer are linear bead-like structures that are similar to those formed from monomer (by heating at elevated temperature) and those isolated from inclusion bodies in hepatocytes from patients with a mutant A1-PI that is poorly secreted. The structural constraints of the disulfide linked dimer and its ability to form linear polymers are difficult to reconcile with the proposed loop-sheet model. We propose that formation of the disulfide linked dimer may involve RCL burial, not insertion, and that polymers of the dimer may form by interaction of beta-sheets exposed on the outer surfaces of the dimer. The model proposed here, involving disulfide linked dimer as the smallest repeating unit (i.e. elementary unit) of the polymer, is an alternate mechanism since under certain reaction conditions, different mechanisms of polymerization may compete. The apparently transiently stable distribution of intermediate species that forms in 1.5 M Gu primarily consists of monomeric and disulfide linked dimeric species. The formation of the disulfide linked dimer species is essentially irreversible and, thus, raises the issue as to why not all of the monomeric intermediate reacts to form dimeric intermediate during the transiently stable period. Dr. Du folded this apparently stable distribution of species and isolated the unreacted A1-PI monomer. By repeating the experiment in 1.5 M Gu with the purified monomer, he demonstrated that the same transiently stable distribution was obtained and thus that microheterogeneity of the A1-PI was not responsible for the failure of all of the monomeric species to form disulfide linked dimer in 1.5 M Gu over 1-2 h. He pursued this investigation by following the time course of polymerization in 1.5 M Gu of cysteine blocked and unblocked A1-PI monomer over period of 4 weeks and found, in fact, for both samples, that eventually almost all the monomeric intermediate disappears with production of dimeric and higher polymeric species. Thus, the apparently transient stable distribution occurs as a result of the kinetics of sequential phases, a faster phase and a much slower phase. Unblocked A1-PI forms non-disulfide linked dimers, which also further polymerize; thus, a comparison of SE-HPLC and non-reducing SDS gel results for the highly polymerized unblocked monomer, which has only 80% free sulfhydryl, will allow an independent estimation of the minimum level of free sulfhydryl. The structure of the non-disulfide linked dimer and its mechanism of polymerization remain to be determined. Beginning is September 2000, Dr. Du began a program to establish the potency and stability of the CBER A1-PI Ref. Standard No. 1. However, after substantial effort in familiarizing himself with the relevant assays, Ref. Standard No. 1 was found to be positive for HIV by NAT although it had been produced commercially in 1988 from plasma that had been screened by a first generation test for HIV. Our goal is to either develop a new reference standard de novo or participate in a WHO collaborative program.