Stability, Folding and Formation of Deleterious Proteins

摘要：人α -1蛋白酶抑制剂（A1-PI）是一种丝氨酸蛋白酶抑制剂，在体内抑制中性粒细胞弹性酶的活性，在25℃下，胍- hcl （Gu）诱导双相展开，中间状态在1.5 M Gu下出现短暂稳定（1-2小时），主要形成单体和二聚体中间体，进一步聚合更慢。Marszal博士用缓冲液稀释了这些较小的中间体，并观察到在折叠物种的混合物中，折叠的二聚体，而不是折叠的单体，进一步聚合。她通过MALDI-TOF测量证实了该物种是二聚体，并表明它是二硫连接的（通过每个单体中的单个巯基）。杜博士已经确定，大约80%的A1-PI单体具有游离巯基，二聚体没有表现出抑制活性。重要的是，Marszal博士已经证明，由纯化二聚体聚合产生的物种似乎是它的倍数，即没有形成三聚体。此外，一个与A1-PI的RCL序列相同的反应性中心环（RCL）肽通常靶向中性粒细胞弹性酶的活性位点，它与单体结合，而不是与二聚体结合，从而阻止了单体中间体的聚合，而不是折叠二聚体的聚合。先前已经报道过这种肽对单体聚合的抑制作用，并在一定程度上用于支持一种聚合模型，该模型涉及将一个A1-PI分子的RCL插入相邻分子的β -片的中心链之间，从而形成一条线性链（即聚合的环片模型）。电子显微镜数据表明，纯化二聚体形成的聚合物是线性的珠状结构，类似于单体（在高温下加热）形成的结构，也类似于从分泌不良的A1-PI突变患者肝细胞包体体中分离出来的结构。二硫键二聚体的结构限制及其形成线性聚合物的能力很难与所提出的环片模型相协调。我们提出二硫键二聚体的形成可能涉及RCL埋藏，而不是插入，二聚体的聚合物可能通过暴露在二聚体外表面的β片的相互作用形成。这里提出的模型，涉及二硫键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中形成的中间物种的短暂稳定分布主要由单体和二硫键连接的二聚体物种组成。二硫键二聚体的形成本质上是不可逆的，因此，提出了一个问题，即为什么不是所有的单体中间体反应形成二聚体中间体在瞬态稳定时期。杜博士折叠了这种明显稳定的物种分布，并分离出未反应的A1-PI单体。通过重复实验1.5顾纯化单体,他证明了同样的暂时性的稳定分布,从而得到微观不均一性A1-PI没有责任的失败有关的所有的单体的物种形成二硫化二聚体在1 - 2 h。1.5顾他追求这个调查后聚合的时间进程1.5顾半胱氨酸阻塞和疏通A1-PI单体在4周内,发现,事实上,对于这两种样品，最终几乎所有的单体中间体都随着二聚体和更高的聚合体的产生而消失。因此，明显的瞬态稳定分布是连续相、快速相和慢得多的相动力学的结果。未阻断的A1-PI形成非二硫键二聚体，也会进一步聚合；因此，比较SE-HPLC和非还原SDS凝胶对高度聚合的无阻断单体的结果，只有80%的游离巯基，将允许独立估计游离巯基的最低水平。非二硫键二聚体的结构及其聚合机理有待进一步研究。从2000年9月开始，杜博士开始了一项建立CBER A1-PI参考标准1号的效力和稳定性的计划。然而，经过大量的努力，熟悉了相关的检测方法后，参考标准1号被NAT检测为HIV阳性，尽管参考标准1号是在1988年从第一代HIV检测筛选过的血浆中生产出来的。我们的目标是要么重新制定一个新的参考标准，要么参与世卫组织的一个合作规划。

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.