Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments

Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of cross-domain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.Large protein machines consist of long amino acid chains, often exceeding many hundreds or even over a thousand residues in length. Although the in vitro folding of small and medium-sized proteins is relatively well understood (1–5), very limited information exists about the complete folding process of such large proteins (6). In general, larger proteins often exhibit a multitude of intermediate and aggregation-prone misfolded states (4, 7). Recently, it has been shown that in multidomain proteins with homologous domains, cross-repeat intermediates can greatly slow down productive folding (8) but little is known about how size effects influence the folding of very large (>500 residues) nonhomologous multidomain proteins.Methods providing dynamic as well as structural information are rare, and many bulk methods often do not provide enough resolution to deal with the multitude of states expected for complex systems such as the aforementioned large protein complexes. Single-molecule force spectroscopy offers kinetic, energetic as well as coarse primary structural information combined with the possibility of actively manipulating systems, making it ideally suited for studying the folding of large proteins (5, 9–12).In this paper, we study the folding and assembly of the large chaperone machinery heat shock protein 90 from yeast (Hsp90), a protein that needs to fold and self-assemble before it can function as a chaperone in the cell. Hsp90 consists of three domains, the N-terminal domain (N domain, 211 residues), the middle domain (M domain, 266 residues), and the C-terminal domain (C domain, 172 residues). In eukaryotic Hsp90, the N and M domains are connected by a long (62 residues) charged linker that can bind transiently to the N domain (13). In addition, the C domain is partly unstructured (residues 678–709). Hsp90 protomers form biologically active dimers through helix pairs in the C domains (residues 640–672) (14). Early equilibrium bulk studies suggest that the unfolding and refolding of isolated Hsp90 is mostly reversible and an unspecified intermediate is populated (15).     

快速起搏诱发心室颤动过程中的倍周期分岔和浑沌

目的：探讨室心动过速（VT）向心室颤动（VF）的转化是否表现为浑沌动力学特征。方法：用快速速起和搏法造成犬在体心脏驱动性VT并诱发VF；用非线性分析方法研究VT向VF转化时，心肌电生理学参数的震荡特点，结果：在VT向VF的转化过程中，激动周期（CL），冲动传导速度（CV）和R波振幅出现了交替节律，倍周期分岔，半周期等非线性震荡并最终陷入浑沌，结论：驱动性VT向VF的转化遵循非线性途径，因而VF是心肌激动陷入浑沌所致。     

大鼠激素性股骨头坏死脂代谢基因的作用通路

目的了解脂代谢有关基因表达谱和通路的改变与激素性股骨头坏死发病的关系。方法采用脂多糖(12S 10μg/kg)与甲泼尼龙[20 mg/(kg·d)]连续4 d肌肉注射制作大鼠股骨头坏死动物模型,取模型组(M)和对照组(C)大鼠股骨头进行组织形态计量学分析;测定血清总胆固醇(TC)含量;采用微阵列技术,测定和分析2组大鼠股骨头基因表达。结果 (1)股骨头组织形态学检测显示模型组大鼠发生激素性股骨头坏死,与对照组比较空骨陷窝率明显升高(P0.05);骨小梁体积、骨矿化速率、毛细血管面积均明显降低(P0.01)。(2)模型组大鼠血清总胆固醇含量明显高于正常组(P0.001)。(3)基因表达谱芯片检测结果显示,模型组有111个较对照组差异倍数大于2.0的表达基因。经MAS系统分析后发现有18个显著性变化的通路。(4)模型组大鼠6个与脂肪酸代谢通路(FAM)上调表达的关键基因、2个与脂肪酸在线粒体延长通路的基因(FAEM)、有3个与脂肪细胞分裂素信号通路上调表达的基因(ACK)。结论脂代谢有关基因的表达上调在大鼠激素性股骨头坏死发病机制中起重要作用。     

羊瘙痒因子263K经多种途径感染仓鼠的脑外组织PrP~(Sc)分子和沉积特点研究

目的　比较研究实验仓鼠经不同途径感染羊瘙痒病 (Scrapie)毒株 2 6 3K的终末期病鼠脑外组织内PrPSc分子和沉积特点。方法　以Scrapie 2 6 3K毒株经颅内、腹腔、心内、肌肉注射及灌胃等途径接种金黄地鼠 ,在终末期取脑、脊髓、脾、肾、舌、肌肉、小肠回盲部和胃组织 ,用HE染色观察病理变化 ,免疫组化法检测PrPSc的组织沉积特点 ,Westernblot检测PrPSc分子特征。结果　五种感染方式均可引起动物发病 ,在脑和脊髓组织中观察到典型的病理改变 ;PrPSc免疫组化检测显示外周途径感染动物脊髓白质内有大量沿纤维走行的PrPSc沉积 ,灰质前后角内围绕空泡点状或网状沉积 ,而颅内感染主要在中央管附近和后角出现大量的点状PrPSc沉积 ;脾脏、肾、小肠回盲部、胃组织中均观察到点状PrPSc的沉积 ;WesternBlot检测发现不同感染途径动物脊髓提取物均出现可抵抗蛋白酶消化的PrPSc条带 ,与脑提取物中PrPSc电泳性状完全一致 ;外周组织仅在脾脏检测到抵抗蛋白酶消化的PrPSc,但与正常对照比较 ,各种组织中PrP的总量明显增高 ,同时呈现与中枢神经组织不同的PrP电泳特征。结论　在TSE感染发病过程中 ,多种组织细胞成分可能参与了TSE感染因子的向中枢神经系统传递过程 ,PrP蛋白在中枢神经组织和其他外周组织细胞中的翻译后修饰及     

应用普查法和捕获再捕获法对难于接触的女性性工作者开展规模估计研究

目的探索在难于接触的女性性工作者中开展规模估计的简便可行的方法,为评价项目效果提供依据。方法固定人群采用普查法,流动人群采用捕获再捕获法。结果用普查法估计固定人群规模为445人(409~480人),用捕获再捕获法估计流动人群规模为586人,95%CI:515~656人;18个调查点目标人群规模995~1 066人。结论根据规模估计的目的、目标人群类别及特征,结合现有资料和工作基础选择规模估计方法,简便可行。调查中,各利益相关群体积极参与和发挥作用至关重要。     

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE₂) plays pleiotropic roles at fetal-maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12-16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE₂ signaling in endometrial functions and establishment of pregnancy in ruminants.