分子伴侣、蛋白质聚集和大分子拥挤环境对蛋白质折叠的影响

 摘要

 3－磷酸甘油醛脱氢酶折叠中间体与分子伴侣GroEL的“半位结合”模式

     “分子伴侣”的发现和鉴定改变了蛋白质折叠研究的经典概念，开辟了崭新的蛋白质生物合成研究的时代，为解译第 二遗传密码并最终全面阐明分子生物学中心法则奠定了基础。分子伴侣功能的研究是蛋白质折叠研究领域中最活跃的前沿，其中分子伴侣与其帮助的靶蛋白之间的识别、相互作用以及解离又是分子伴侣作用机制研究最根本的内容。李剑研究了当前最受注意的分子伴侣GroEL与同源四聚体的3－磷酸甘油醛脱氢酶（GAPDH）、单肽链的溶菌酶以及GroEL的辅助分子伴侣GroES的相互作用模式。通过分析（GroEL－靶蛋白折叠中间体）复合物的成分，以及抑制变性靶蛋白复性过程中的聚集等细致的实验，他发现不同的靶蛋白与GroEL的结合有不同的模式。

       一个GroEL分子能够结合两个亚基的GAPDH折叠中间体而形成稳定的复合物，该复合物不能再结合其他的GAPDH折叠中间体，却能再结合一个溶菌酶折叠中间体或者一个GroES分子。该结果表明二体的GAPDH折叠中间体是结合在GroEL分子的一端，表现为“半位结合”。对溶菌酶，GroEL既能在一端结合一分子的溶菌酶折叠中间体又能在两端各结合一个中间体而形成1：1 和1：2的“全位结合”复合物。由一分子GroEL和一分子GroES形成的复合物还能够再结合一个溶菌酶中间体或一个二体的GAPDH中间体而形成一个稳定的三元复合物。此外，结合了一分子溶菌酶或是两个亚基的GAPDH的GroEL，分别为（GroEL-溶菌酶1）(GroEL-GAPDH2)，只能在另一端再结合一分子GroES而形成反式三元复合物。根据GroEL同GAPDH折叠中间体结合的化学计量比以及包含有（GroEL－GAPDH2）的三元复合物的形成规律，推断在GroEL帮助GAPDH复性过程中是仅在其分子一端结合一个二聚化的GAPDH中间体。该工作为四聚体GAPDH的折叠和组装的复性机制以及分子伴侣GroEL帮助其复性的作用机制贡献了新的丰富的信息。

       聚集的蛋白加速但不增加3－磷酸甘油醛脱氢酶的聚集 — 蛋白聚集的特异性

       近年来认识到蛋白质构象病的共同机制是蛋白质发生聚集。最近一二年又认识到所有的蛋白质都可能在某些条件下发生聚集，而蛋白质的聚集对细胞往往是有害的，甚至引起疾病。因此蛋白质聚集的固有性质和其危害性成为蛋白质折叠研究的另一侧重热点。李剑通过蛋白聚集体对3－磷酸甘油醛脱氢酶（GAPDH）去折叠和重折叠过程中聚集的影响研究蛋白质聚集的规律性。GAPDH在去折叠和重折叠过程中发生的聚集呈现S型曲线。蛋白聚集体的存在加速GAPDH的聚集，但不增加聚集的量也不影响GAPDH最终的复性效率。说明蛋白聚集体作为种子通过疏水相互作用只诱导那些注定要聚集的GAPDH折叠中间体聚集，而不改变GAPDH原来的折叠途径中正确重折叠蛋白和错误折叠而发生聚集的分配比例。此外，两种不同的蛋白在同一系统中共同复性时不相互干扰。这些发现有助于我们了解细胞如何保证一种蛋白在折叠时不受其他蛋白聚集体影响的机制。

       只有还原脱钙的牛乳清白蛋白能够被蛋白聚集体诱导而发生聚集

       牛乳清白蛋白（LA）是一种钙结合蛋白，其不同状态的构象已为很好鉴定，因此是蛋白质折叠研究的好材料。还原脱钙的牛乳清白蛋白（r-LA）在25℃中性还原性的缓冲液中是可溶性蛋白，以“前熔球态”存在。但是随着不同种类蛋白聚集体的加入，r-LA便发生聚集。但蛋白聚集体不能诱导天然的LA和只脱钙的处于“熔球态”的LA发生聚集。分子伴侣蛋白二硫键异构酶或者化学分子伴侣聚乙二醇抑制这种诱导聚集的发生。天然蛋白、不聚集的折叠中间体以及可溶性的聚集体都不会诱导LA的聚集。经鉴定r-LA和蛋白聚集体之间的相互作用本质上是疏水相互作用。以前的工作表明分子伴侣识别r-LA，李剑的工作揭示了处于“前熔球态”的牛乳清白蛋白不仅是分子伴侣而且也是蛋白聚集体的识别对象，说明生物大分子之间相互作用的构象基础。

       大分子拥挤对6-磷酸葡萄糖脱氢酶和蛋白质二硫键异构酶复性的影响

       蛋白质体外折叠的研究已经为人们认识细胞内新生肽链的折叠提供了大量的信息。为了避免伴随蛋白质去折叠和折叠过程中的聚集干扰研究，或为了通过基因工程得到可溶性蛋白，研究通常是在简单组成的缓冲液体系中进行，所用蛋白的浓度一般较低。可是细胞内的实际情况完全不是如此。所有的细胞中都存在着大量的蛋白质、核酸、多糖等各种生物大分子，总浓度高达50－400 g/l，它们大约占用细胞容积的20－30%，因此任何一种大分子都处于一个充满其他大分子的拥挤环境中。对源于排斥容积效应的拥挤理论分析表明它对所有大分子之间的反应在热力学和动力学上都有很大的影响。可是以往人们在体外研究生物大分子的性质和相互作用时几乎都忽略了这样一个细胞大分子拥挤的实际环境。最近几年建议把大分子拥挤与pH、离子强度和溶液组成等一样作为常规因素来研究生物大分子的呼声很高，然而“拥挤”对蛋白质折叠影响的实验研究目前还是处于初步阶段。李剑及时开展了向复性体系添加高浓度的多糖，聚乙二醇和蛋白拥挤试剂以模拟细胞内环境，研究大分子拥挤对变性6-磷酸葡萄糖脱氢酶和蛋白质二硫键异构酶复性的影响。

       李剑发现，随着蛋白复性时浓度的升高，两种蛋白复性过程中的聚集明显增加，复性产率降低。在高浓度的拥挤试剂存在时，选定浓度的蛋白的复性产率保持不变但速率降低。特别是当拥挤试剂存在时，6-磷酸葡萄糖脱氢酶的复性动力学由一相一级反应变为由快慢两相构成的两相一级反应，而且其慢相的比例随拥挤试剂浓度的增加而增大。分子伴侣GroEL不仅能够大大提高6-磷酸葡萄糖脱氢酶的复性产率，而且能够使它的复性动力学从两相逆转回到一相，从而加速整个复性的进程。对比其他实验室的工作，李的结果揭示了大分子拥挤环境对蛋白质折叠的热力学和动力学影响的复杂性和多样性。

      金属离子对金属硫蛋白参与氧化还原反应的调控

      金属硫蛋白（MT）是一种非常特殊的含高达30％半胱氨酸，却没有二硫键和芳香氨基酸的小分子蛋白。所有半胱氨酸在序列中保守，并结合各种金属离子，一个分子可结合7个二价离子或是11－18个一价离子。近年来MT的发现者Vallee报导了细胞内的氧化还原电位调控MT中锌离子释放和结合的新的性质。我们提出了一个与此观点相反的观点，认为细胞内大量的MT是否可能通过锌离子的结合和释放反过来调节其巯基的反应性，从而调控整个细胞内的氧化还原电位呢。以往虽然也有人曾经设想过，但一直没有实验证据。在该实验室有关蛋白二硫键异构酶（PDI）催化性质研究的基础上，李剑与张森一起发现MT像谷胱甘肽、二巯基苏糖醇一样，也能影响PDI催化的巯基磺酸化的核糖核酸酶A (RNase A)形成天然核糖核酸酶A。利用这一反应他们发现MT在金属螯合剂EDTA存在时，能够提高RNase A的复性产率；但EDTA与谷胱甘肽或二巯基苏糖醇共存对RNase A复性无影响。表明EDTA确能通过鏊合锌离子使MT代替谷胱甘肽、二巯基苏糖醇充当体系中的还原剂。进一步又证明EDTA确能通过鏊合锌离子而增加MT的巯基同巯基试剂5,5’-dithiobis-2-nitrobenzoic acid 的反应性。实验结果表明MT确实可以通过锌离子的结合和释放来调节其巯基的反应性，从而构成细胞内巯基库的一部分来调控细胞内的氧化还原电位。

      Effects of Molecular Chaperones, Protein Aggregation and Macromolecular Crowding on Protein Folding

   “Half of the sites” binding of d-glyceraldehyde-3-phosphate dehydrogenase folding intermediate with GroEL. Two D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) folding intermediate subunits bind with GroEL to form a stable complex, which can no longer bind with additional GAPDH intermediate subunits, but does bind with one more lysozyme folding intermediate or one GroES molecule, suggesting that the two GAPDH subunits bind at one end of the GroEL molecule displaying a “half of the sites” binding profile. For lysozyme, GroEL binds with either one or two folding intermediates to form a stable 1:1 or 1:2 complex with one substrate on each end of the GroEL double ring for the latter. The 1:1 complex of (GroEL-GroES) binds with one lysozyme or one dimeric GAPDH folding intermediate to form a stable ternary complex. Both complexes of (GroEL-lysozyme1) and (GroEL-GAPDH2) bind with one GroES molecule only at the other end of the GroEL molecule forming a trans ternary complex. According to the stoichiometry of GroEL binding with the GAPDH folding intermediate and the formation of ternary complexes containing GroEL-GAPDH2, it is suggested that the folding intermediate of GAPDH binds, very likely in the dimeric form, with GroEL at one end only.

       Aggregated proteins accelerate but do not increase the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation. The effect of protein aggregates on the aggregation of GAPDH during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH, but did not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insights into a mechanism by which cells prevent protein folding against interference from aggregation of other proteins.

      Only the reduced conformer of a-lactalbumin is inducible to aggregation by protein aggregates. Reduced apo-a-lactalbumin (r-LA) in pre-molten globule state is soluble in neutral and reduced buffer at 25 oC but becomes aggregated when aggregates of various proteins are added. However, protein aggregates do not induce the aggregation of apo-a-lactalbumin in the molten globule state. The presence of molecular chaperone, protein disulfide isomerase (PDI), or the “chemical chaperone”, polyethyleneglycol, inhibits the induced aggregation. Native proteins, aggregation-free folding intermediates and soluble aggregates do not induce the aggregation. The interaction between r-LA and protein aggregates is hydrophobic in nature. These finding suggest that pre-molten globule state of LA is the target not only for chaperones but also for protein aggregates.

       Effects of macromolecular crowding on the refolding of glucose-6-phosphate dehydrogenase and protein disulfide isomerase. Intracellular environment is actually crowded or confined. The effects of high concentrations of polysaccharide, polyethelene glycol, and protein- crowding agents on the refolding of glucose-6-phosphate Dehydrogenase (G6PDH) and protein disulfide isomerase have been examined. By increasing concentration during refolding, the reactivation yields of the two proteins decreased with the formation of soluble aggregates. In the presence of high concentrations of crowding agents the reactivation yields remain constant but with decreased refolding rates. The refolding of G6PDH changed from monophasic to biphasic first-order reactions in the presence of crowding agents, and the amplitude of the new slow phase increases with increasing concentrations of crowding agents. The molecular chaperone GroEL assists the refolding of G6PDH with marked increased reactivation yield and reverses the refolding kinetics from biphase back to monophase and accelerates the refolding process. The results display the complexity and the diversity of effects of macromolecular crowding on both the thermodynamics and kinetics of protein folding.

       Metal regulation of metallothionein participation in redox reactions. Like glutathione or dithiothreitol, metallothionein effects the formation of pancreatic ribonuclease A from its S-sulfonated derivative catalyzed by protein disulfide isomerase. EDTA increases the yield of ribonuclease A activity recovery with metallothionein but does not affect the reaction with glutathione or dithiothreitol. EDTA also increases the reactivity of thiol groups in metallothionein with 5,5’-dithiobis-(2-nitrobenzoic acid) by chelation of zinc ions. It is suggested that the thiol groups in metallothionein form a part of the pool of cellular thiols in the regulation of cellular redox reactions and their availability is modulated by zinc chelation.