Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 ± 7 s, much longer than it remained bound to the ribosomes (t_½ ∼ 10–14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.     

Plant Vascular Biology and Agriculture

The evolution of animal and plant vascular systems played a pivotal role in the advancement from simple to complex organisms, through the provision of a delivery system for the distribution     

不同术式的经内镜下行乳头括约肌切开术（EST）对肠胆反流的影响

目的：目前认为,十二指肠胆道反流是引起胆道反复感染,进而导致胆道结石再发和胆道狭窄的原因之一。近年来应用以内镜下逆行胆胰管造影术（endoscopicretrogradecholangiopancreatography,ERCP）为基础的微创治疗胆总管结束的技术开展颇为广泛。它主要包括ERCP、Oddi括约肌切开术（endoscopicsphincterotomy,EST）、十二指肠乳头球囊扩张术（endoscopicpapillarybal—Iondilation,EPBD）、胆管结石碎石取石术、胆总管支架植入术和鼻胆管引流术六大技术。本文主要研究了采用不同术式的EST,即EST中切口和EST小切口＋EPBD术,在术后早期对患者十二指肠胆道反流的影响。方法：63例胆总管结石患者,男30例,女33例,予行经内镜下逆行的胆胰管造影（ERCP）后分别采用不同术式EST,术后安放胆总管引流管。术后l周留取胆汁采用口服核素和测定胆汁中的胃蛋白酶I、II的浓度,对十二指肠胆道反流进行定量和定性的测定。结果：EST中切口术组、EST小切口＋球囊扩张（EPBD）组分别与无EST组相比,年龄和性别无统计学意义（P=0．07,P=0．416）。行EST中切开和小切开＋球囊扩张患者胆汁中的锝计数明显高于无EST组,且这两组不同术式的患者锝计数存在显著的统计学差异（P〈0．05）。行EST中切口者、EST小切口＋球囊扩张术者胆汁中的PGII质量浓度明显低于无EST组（P〈0．05）,但是EST中切口者和EST小切口＋球囊扩张术后两组间胆汁中PGII的质量浓度无统计学差异。结论：行EST中切口取胆总管结石的患者在手术早期较易发生十二指肠胆道的反流。因此,建议对于胆总管结石患者尽量选择行EST小切口＋球裳扩张术（EPBD）的手术方式。     

苎麻光合生理生态特性研究

以大田栽培的苎麻植株为材料,用TPS-2便携式光合作用测定系统测定自然条件下生长的苎麻叶片的光合气体交换参数,以及光响应曲线和CO2响应曲线,并通过回归和相关法分析探讨净光合速率与主要生理、生态因子间的关系.结果表明:(1)苎麻叶片的净光合速率(Pn)日变化曲线呈现双峰型,2个光合峰高度接近,其净光合速率具有典型的午休"现象;蒸腾速率(Tr)日变化曲线呈现单峰型,其走势与气孔导度(Gs)日变化一致.(2)苎麻叶片光合作用的光饱和点(LSP)为1 568.5μmol?m-2?s-1,光补偿点(LCP)为54.18μmol?m-2?s-1,表观量子效率(AQY)为0.025 8 mol?mol-1;而其CO2补偿点(CCP)、饱和点(CSP)和羧化效率(CE)分别为49.25、1 746.9μmol?mol-1和0.045;因此,苎麻属于喜光性阳生植物,且对强光有一定的耐受能力.(3)苎麻叶片Pn日变化的主要限制因子是胞间CO2浓度(Ci),主要决定生理因子是气孔导度(Gs).     

E. coli chaperones DnaK,Hsp33 and Spy inhibit bacterial functional amyloid assembly

Amyloid formation is an ordered aggregation process, where β-sheet rich polymers are assembled from unstructured or partially folded monomers. We examined how two Escherichia coli cytosolic chaperones, DnaK and Hsp33, and a more recently characterized periplasmic chaperone, Spy, modulate the aggregation of a functional amyloid protein, CsgA. We found that DnaK, the Hsp70 homolog in E. coli, and Hsp33, a redox-regulated holdase, potently inhibited CsgA amyloidogenesis. The Hsp33 anti-amyloidogenesis activity was oxidation dependent, as oxidized Hsp33 was significantly more efficient than reduced Hsp33 at preventing CsgA aggregation. When soluble CsgA was seeded with preformed amyloid fibers, neither Hsp33 nor DnaK were able to efficiently prevent soluble CsgA from adopting the amyloid conformation. Moreover, both DnaK and Hsp33 increased the time that CsgA was reactive with the amyloid oligomer conformation-specific A11 antibody. Since CsgA must also pass through the periplasm during secretion, we assessed the ability of the periplasmic chaperone Spy to inhibit CsgA polymerization. Like DnaK and Hsp33, Spy also inhibited CsgA polymerization in vitro. Overexpression of Spy resulted in increased chaperone activity in periplasmic extracts and in reduced curli biogenesis in vivo. We propose that DnaK, Hsp33 and Spy exert their effects during the nucleation stages of CsgA fibrillation. Thus, both housekeeping and stress induced cytosolic and periplasmic chaperones may be involved in discouraging premature CsgA interactions during curli biogenesis.Key words: chaperone, curli, functional amyloid, CsgA, DnaK, Hsp33, Spy     

核心组蛋白的融合表达和纯化

目的:克隆核心组蛋白H2A、H2B、H3和H4的基因,表达并纯化组蛋白与谷胱甘肽S-转移酶(GST)的融合蛋白。方法:用PCR方法从乳腺文库中扩增核心组蛋白H2A、H2B、H3和H4的编码序列,分别将其以正确相位与pGEX-KG载体中的GST编码序列融合,得到重组质粒pGST-H2A、pGST-H2B、pGST-H3和pGST-H4,分别转化大肠杆菌BL21,表达融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4;用谷胱甘肽-Sepharose 4B亲和纯化融合蛋白;用Western印迹检测融合蛋白的表达及纯化。结果:分别构建了核心组蛋白H2A、H2B、H3和H4的融合表达载体;Western印迹检测表明,融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4获得表达及纯化。结论:表达并纯化了H2A、H2B、H3和H4的融合蛋白,为进一步研究核心组蛋白的功能奠定了基础。     

Quantitative proteomics reveals that Hsp90 inhibition preferentially targets kinases and the DNA damage response

Despite the increasing importance of heat shock protein 90 (Hsp90) inhibitors as chemotherapeutic agents in diseases such as cancer, their global effects on the proteome remain largely unknown. Here we use high resolution, quantitative mass spectrometry to map protein expression changes associated with the application of the Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG). In depth data obtained from five replicate SILAC experiments enabled accurate quantification of about 6,000 proteins in HeLa cells. As expected, we observed activation of a heat shock response with induced expression of molecular chaperones, which refold misfolded proteins, and proteases, which degrade irreparably damaged polypeptides. Despite the broad range of known Hsp90 substrates, bioinformatics analysis revealed that particular protein classes were preferentially affected. These prominently included proteins involved in the DNA damage response, as well as protein kinases and especially tyrosine kinases. We followed up on this observation with a quantitative phosphoproteomic analysis of about 4,000 sites, which revealed that Hsp90 inhibition leads to much more down- than up-regulation of the phosphoproteome (34% down versus 6% up). This study defines the cellular response to Hsp90 inhibition at the proteome level and sheds light on the mechanisms by which it can be used to target cancer cells.