分子内分子伴侣机制的研究进展

在原核生物、真核生物及病毒中,一些蛋白质的折叠不符合Anfinsen原则,即依靠自身的氨基酸序列是不够的,还需一段被称为分子内分子伴侣(IMC)的肽段来协助折叠．根据机制不同,IMC可分为两类：第一类IMC引导成熟肽折叠为具有空间结构的蛋白质;第二类IMC协助成熟肽的多聚化而使其获得生物学功能．IMC能提供比分子伴侣更契合的结构,更有效地引导成熟肽折叠,是一种更优的折叠策略．研究IMC分子机制,不仅能够确定IMC上哪些残基的协同作用引导成熟肽折叠,而且可通过改变或修饰其侧链来改造成熟肽,拓展传统的蛋白质工程．     

前导肽介导的蛋白折叠机制及其对脂肪酶影响的研究进展

大多数蛋白质的形成过程主要由合成前体蛋白和合成功能蛋白两个步骤组成。在这个过程中,前导肽能够辅助蛋白质折叠或抑制它的活性。前导肽作为脂肪酶结构中重要的一段多肽链,通常作为分子内分子伴侣来辅助脂肪酶的折叠,同时该序列上包括糖基化位点在内的一些特殊位点,对酶的活性、极端环境稳定性、甲醇耐受性和底物特异性等性质具有重要影响。研究前导肽介导的蛋白折叠机制,以及前导肽对脂肪酶的作用和影响,可以实现通过改变前导肽来调控脂肪酶成熟肽性能的目的,进一步拓展蛋白质工程的研究。     

新生肽链折叠的新概念

本文介绍近几年来才形成的“新生肽折叠”的新概念,即在核糖体上合成的多肽链成熟为具有特定空间结构和全部生物活力的功能蛋白质的全过程往往不是自发进行的,需 是需要细胞内已经存在的其他蛋白质,分子伴侣和折叠酶的帮助才能完成,文中还简述了分子伴侣和折叠的研究现状。     

分子伴侣及其在蛋白质折叠中的作用研究进展

蛋白质折叠是一个复杂的、动态的过程,蛋白质的折叠不是自发的,需要其他物质的帮助.了解分子伴侣在蛋白质折叠过程中的的作用,有助于进一步研究蛋白质折叠机制.本文介绍了分子伴侣及其分类,重点综述了各类分子伴侣在蛋白质折叠中的机制,并提出了研究分子伴侣在蛋白质折叠中的作用的重要意义.     

分子伴侣在蛋白质折叠中的作用

分子伴侣主要由三个高度保守的蛋白质家族组成,这三个家族的成员广泛分布于原核和真核细胞中。TCP1复合物是真核细胞细胞溶质内的伴侣蛋白。分子伴侣在蛋白质折叠过程中防止多肽链形成聚集物或无活性结构,提高正确折叠率。本文重点讨论Stress-70家族蛋白质和伴侣蛋白协助蛋白质折叠过程中的协同性以及伴侣蛋白GroEL和GroES的作用机理。     

具有分子伴侣功能的抗体

蛋白质的折叠问题一直是生物学研究的前沿之一，蛋白质稳态平衡的破坏与衰老及很多神经退行性疾病的发病机理密切相关，而蛋白质的正确折叠与蛋白质稳态在很大程度上取决于分子伴侣参与构建的复杂网络。许多研究表明，抗体可以作为分子伴侣促进蛋白质的正确折叠，并阻止蛋白质的异常聚集，抗体所具有的严格底物特异性使其具备了治疗特定蛋白质折叠病、帮助包涵体复性等应用潜力。本文简要介绍了分子伴侣的研究进展，详细阐述了具有分子伴侣功能的抗体及单链抗体的研究进展，最后重点讨论了可抑制蛋白质聚集的抗体的研究近况。     

分子伴侣的多重功能

分子伴侣(molecular chaperone)在原核生物和真核生物的细胞中广泛存在.分子伴侣可稳定未折叠或部分折叠的多肽,并防止不适当的多肽链内或链间相互作用;有些分子伴侣也可与天然构象的蛋白质相互作用以促使寡聚态蛋白质发生结构重排.基于分子伴侣能识别并调节细胞内多肽的折叠,因此它们还具有介导线粒体蛋白跨膜转运,调控信息传导通路和转录、复制,以及参与微管形成与修复等功能.     

分子伴侣研究概况

分子伴侣主要是在进化上高度保守的热休克蛋白的几个家族。从细菌到哺乳动物,分子伴侣对体内蛋白质的折叠、运输和组装都起到非常重要的作用。本文简要地概述了分子伴侣的组成、它们在蛋白质折叠中的作用以及它们在生物工程下游处理过程中的应用情况。     

GroEL的耗能分子伴侣机制

双环结构Gro EL及其辅分子伴侣Gro ES是目前研究得最深入的分子伴侣.然而,Gro EL/Gro ES帮助蛋白质折叠的一些关键理化机制,尤其是水解ATP,Gro EL发生构象改变,能否主动调节蛋白质错误折叠中间体的构象,以促进错误折叠中间体的复性,仍然存在争议.结合本研究组近年的工作,作者着力介绍Gro EL促进蛋白质折叠的主动解折叠机制.     

主要组织相容性复合体Ⅰ类分子抗原肽

被主要组织相容性复合体（MHC）Ⅰ类分子呈递在细胞表面的抗原肽大部分来源于细胞内新合成蛋白质的降解产物,抗原肽直接体现细胞内功能蛋白质的部分变化,蛋白酶体、氨肽酶和抗原转运体（TAP）参与调控抗原肽的生成。在MHC的组装、折叠过程中,抗原肽促进各亚基的结合和折叠进程;而在起始细胞的免疫应答过程中,抗原肽不仅诱导T细胞抗原受体的特异结合,更为重要的是延长MHC同T细胞抗原受体特异结合的作用时间。     

Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding

Applications of sensitive new technologies, in particular, two-dimensional NMR spectroscopy, have allowed detection of folded structures in short peptide fragments of proteins in aqueous solution under conditions where native proteins fold. These structures are in rapid dynamic exchange with unfolded states. These observations provide evidence in support of models for protein folding which postulate localized regions of folded structure as initiation sites for the folding process. Since these initiation processes are expected to be rapid, such models are consistent with kinetic evidence that the rate-determining steps of protein folding occur late in the process and probably involve rearrangement of incorrectly folded intermediates.     

Contribution of glutamatergic signaling to nitrosative stress-induced protein misfolding in normal brain aging and neurodegenerative diseases

Glutamatergic hyperactivity, associated with Ca2+ influx and consequent production of nitric oxide (NO), is potentially involved in both normal brain aging and age-related neurodegenerative disorders. Many neurodegenerative diseases are characterized by conformational changes in proteins that result in their misfolding and aggregation. Normal protein degradation by the ubiquitin-proteasome system can prevent accumulation of aberrantly folded proteins. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. In particular, molecular chaperones - such as protein disulfide isomerase, glucose regulated protein 78, and heat shock proteins - can provide neuroprotection from misfolded proteins by facilitating proper folding and thus preventing aggregation. Here, we present evidence for the hypothesis that NO contributes to normal brain aging and degenerative conditions by S-nitrosylating specific chaperones that would otherwise prevent accumulation of misfolded proteins.     

GroEL-Mediated Protein Folding: Making the Impossible,Possible

ABSTRACT

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

Interdependent folding of the N- and C-terminal domains defines the cooperative folding of alpha-lytic protease

Alpha-lytic protease (alphaLP) serves as an important model in achieving a quantitative and physical understanding of protein folding reactions. Synthesized as a pro-protease, alphaLP belongs to an interesting class of proteins that require pro regions to facilitate their proper folding. alphaLP's pro region (Pro) acts as a potent folding catalyst for the protease, accelerating alphaLP folding to its native conformation nearly 10(10)-fold. Structural and mutational studies suggested that Pro's considerable foldase activity is directed toward structuring the alphaLP C-terminal domain (CalphaLP), a seemingly folding-impaired domain, which is believed to contribute significantly to the high-energy folding and unfolding transition states of alphaLP. Pro-mediated nucleation of alphaLP folding within CalphaLP was hypothesized to subsequently enable the alphaLP N-terminal domain (NalphaLP) to dock and fold, completing the formation of native protease. In this paper, we find that ternary folding reactions of Pro and noncovalent NalphaLP and CalphaLP domains are unaffected by the order in which the components are added or by the relative concentrations of the alphaLP domains, indicating that neither discrete CalphaLP structuring nor docking of the two alphaLP domains is involved in the folding transition state. Instead, the rate-limiting step of these folding reactions appears to be a slow and concerted rearrangement of the NalphaLP and CalphaLP domains to form active protease. This cooperative and interdependent folding of both protease domains defines the large alphaLP folding barrier and is an apparent extension of the highly cooperative alphaLP unfolding transition that imparts the protease with remarkable kinetic stability and functional longevity.     

A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co‐incubation with a GST‐cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine‐tagged recombinant protein in a batch process. The addition of the GST‐cleanser protein in the presence of ATP‐Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.     

Mimicking the action of GroEL in molecular dynamics simulations: application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results support the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.     

Mimicking the action of folding chaperones in molecular dynamics simulations: Application to the refinement of homology-based protein structures

A novel method for the refinement of misfolded protein structures is proposed in which the properties of the solvent environment are oscillated in order to mimic some aspects of the role of molecular chaperones play in protein folding in vivo. Specifically, the hydrophobicity of the solvent is cycled by repetitively altering the partial charges on solvent molecules (water) during a molecular dynamics simulation. During periods when the hydrophobicity of the solvent is increased, intramolecular hydrogen bonding and secondary structure formation are promoted. During periods of increased solvent polarity, poorly packed regions of secondary structures are destabilized, promoting structural rearrangement. By cycling between these two extremes, the aim is to minimize the formation of long-lived intermediates. The approach has been applied to the refinement of structural models of three proteins generated by using the ROSETTA procedure for ab initio structure prediction. A significant improvement in the deviation of the model structures from the corresponding experimental structures was observed. Although preliminary, the results indicate computationally mimicking some functions of molecular chaperones in molecular dynamics simulations can promote the correct formation of secondary structure and thus be of general use in protein folding simulations and in the refinement of structural models of small- to medium-size proteins.     

Translation‐coupled protein folding assay using a protease to monitor the folding status

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large‐scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli‐based cell‐free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation‐prone fractions. We translated ～90 E. coli proteins in the protease‐inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large‐scale analysis.     

Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation-screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non-native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain- or loss-of-function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease-causing misfolded proteins. Rescue of misfolded, "trafficking-defective", but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or "pharmacoperones") are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native-type-like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin-releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.     

The complete peptide dictionary--a meta-proteomics resource

Recent developments in MS-based proteomics have increased the emphasis on peptides as a primary observable. While peptides are identified by tandem mass spectra, the link between peptide and protein remains implicit given the bottom-up nature of the experiment in which proteins are enzymatically digested prior to sequencing. It is therefore useful to provide a fast lookup from peptide to protein in order to systematically establish the broadest possible protein basis for the observed peptides. Here, we describe Pep2Pro, a fast web-service providing protein lookup by peptides covering the entire protein space comprising ～10 million UniRef100 sequences. We demonstrate the usefulness of the service by reanalyzing peptides from two recent meta-proteomic data sets and identifying taxon-specific peptides, thereby implicating individual species as being present in these complex samples. The Pep2Pro web service can be accessed at http://www.pep2pro.org.