A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.     

Hsp90 as a capacitor of phenotypic variation

Heat-shock protein 90 (Hsp90) chaperones the maturation of many regulatory proteins and, in the fruitfly Drosophila melanogaster, buffers genetic variation in morphogenetic pathways. Levels and patterns of genetic variation differ greatly between obligatorily outbreeding species such as fruitflies and self-fertilizing species such as the plant Arabidopsis thaliana. Also, plant development is more plastic, being coupled to environmental cues. Here we report that, in Arabidopsis accessions and recombinant inbred lines, reducing Hsp90 function produces an array of morphological phenotypes, which are dependent on underlying genetic variation. The strength and breadth of Hsp90's effects on the buffering and release of genetic variation suggests it may have an impact on evolutionary processes. We also show that Hsp90 influences morphogenetic responses to environmental cues and buffers normal development from destabilizing effects of stochastic processes. Manipulating Hsp90's buffering capacity offers a tool for harnessing cryptic genetic variation and for elucidating the interplay between genotypes, environments and stochastic events in the determination of phenotype.     

Hsp90抑制剂17-AAG通过EGFR,IGFR途径抑制乳腺癌细胞的增殖

 17-AAG通过抑制Hsp90的功能而抑制多种肿瘤细胞的生长增殖,用不同浓度17-AAG作用于乳腺癌细胞株MDA-MB-231和BT474,用SRB法检测细胞相对存活数,计算半数抑制药物浓度IC₅₀;并用Westernblotting检测Hsp90的"顾客"蛋白EGFR,IGFR-1以及G2/M细胞周期蛋白cdc2的表达,研究17-AAG抑制乳腺癌细胞的增殖,促进细胞凋亡的途径.结果显示,1μmol/L的17-AAG已能完全抑制MDA-MB-231和BT474细胞增殖,2种细胞的IC₅₀分别为0.0587,0.0576μmol/L;17-AAG作用的乳腺癌细胞EGFR,IGFR-1表达明显下调,此作用呈剂量依赖性.这些研究结果表明17-AGG可显著抑制乳腺癌细胞MDA-MB-231和BT474的生长增殖,此作用与17-AGG抑制细胞生长因子受体途径有关.     

基于Hsp90 ATPase活性的抑制剂筛选模型研究

热休克蛋白90(Heat shock protein,Hsp90)作为分子伴侣,参与调控肿瘤细胞多条信号通路,对肿瘤细胞生长有重要作用.以pGEX-4T-1为载体,构建了Hsp90表达载体于宿主大肠杆菌中表达.通过孔雀绿磷钼酸铵-无机磷检测法,测定异源表达的Hsp90 ATPase活性.实验得出Hsp90对ATP的米氏常数(Km)为369.1 μmol/L,在Hsp90和ATP浓度分别为0.18,1 mmol/L时转换效率常数(kcat)为1.6 min-1,最大反应速率(Vmax)为1.0 μmol/(L·min).基于该方法,建立Hsp90 ATPase活性抑制剂筛选模型,以Geldanamycin和Radicicol为阳性对照,发现Rifamycin、Rugulosin及MycoE也有较强的Hsp90 ATPase抑制活性.该方法可用于筛选抗肿瘤候选化合物的研究,同时也可为具有ATPase活性蛋白的抑制剂筛选提供参考.     

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.     

Hsp90抑制剂木犀草苷对非小细胞肺癌的体外抑制活性及作用机理研究

本文利用荧光偏振法(FP)构建分子伴侣Hsp90的筛选平台,筛选得到黄酮类化合物木犀草苷,并用热稳定实验验证了它与蛋白的结合作用;评价了木犀草苷对14株癌细胞的体外增殖抑制作用,发现木犀草苷对非小细胞肺癌有良好的选择性抑制活性;利用细胞克隆形成、western blot以及流式细胞检测技术进一步深入研究其可能的作用机制,发现木犀草苷能剂量依赖性地抑制非小细胞肺癌的增殖;下调Hsp90的客户蛋白Raf-1、HER2的表达;将H460细胞阻滞在G2/M期;诱导细胞凋亡,并且引起Bax的表达上调。实验结果表明,黄酮类化合物木犀草苷可以通过抑制Hsp90的活性进而降解肿瘤细胞内的客户蛋白,诱导H460细胞G2/M期阻滞,上调Bax表达诱导细胞凋亡来发挥抗肿瘤活性。     

持续干旱下4个柳树无性系生理生态适应性研究

为比较柳树无性系对干旱胁迫生理生态适应性的差异,通过盆栽试验研究了持续干旱胁迫下4个柳树无性系一年生扦插苗渗透调节?保护酶活性等指标的动态变化?研究结果表明,随着干旱胁迫时间的增加,电导率?丙二醛逐渐增加;4个无性系渗透调节物质脯氨酸?可溶性糖和保护酶系统过氧化物酶?超氧化物歧化酶在胁迫过程中整体呈上升的趋势,随着胁迫程度的加强,抗旱性弱的无性系最先表现出下降的趋势?利用主成分分析所有生理生态指标,对4个无性系抗旱性进行综合评价?     

水分胁迫下高羊茅幼苗的生理适应性

采用不同浓度PEG6000模拟水分胁迫处理6种高羊茅幼苗,测定了胁迫72 h后叶片叶绿素含量、相对电导率、丙二醛含量、超氧化物歧化酶和过氧化物酶活性等抗旱指标,以综合评价6种高羊茅的抗旱能力。综合分析可知,6种高羊茅的苗期抗旱能力最强的是阳光宝贝。     

Development of multifunctional lightweight cellular metals through interdisciplinary efforts

In spite of her phenomenal economic achievement since the historically imperative implementation of the ＂open door＂ policy in 1978, China as a whole remains in the middle stage of industrialization, faced with fresh challenges from energy resources and environment protection. The issue becomes even more acute in view of the current economic and financial crisis worldwide,     

Continuous on-line adaptation of the rotor time constant in FOC induction machine system

Rotor time constant is an important parameter for the indirect lleld oraentateO control of mauc- tion motor. Incorrect rotor tittle constant value will cause the flux observer generating a wrong angu- lar orientation of the rotor field. A new approach serves for rotor time constant on-line adaptation by setting the stator current to be zero for a short period. A smooth eorrector is designed to prevent ab- normal detection result from making adaptation. Impact of zero current duration on detection error and rotor speed is analyzed by experiments.     

Regulation of cellular response to oncogenic and oxidative stress by Seladin-1

Expression of multiple oncogenes and inactivation of tumour suppressors is required to transform primary mammalian cells into cancer cells. Activated Ha-RasV12 (Ras) is usually associated with cancer, but it also produces paradoxical premature senescence in primary cells by inducing reactive oxygen species followed by accumulation of tumour suppressors p53 and p16(INK4a) (ref. 4). Here we identify, using a direct genetic screen, Seladin-1 (also known as Dhcr24) as a key mediator of Ras-induced senescence. Following oncogenic and oxidative stress, Seladin-1 binds p53 amino terminus and displaces E3 ubiquitin ligase Mdm2 from p53, thus resulting in p53 accumulation. Additionally, Seladin-1 associates with Mdm2 independently of p53, potentially affecting other Mdm2 targets. Ablation of Seladin-1 causes the bypass of Ras-induced senescence in rodent and human fibroblasts, and allows Ras to transform these cells. Wild-type Seladin-1, but not mutants that disrupt its association with either p53 or Mdm2, suppresses the transformed phenotype. The same mutants are also inactive in directing p53-dependent oxidative stress response. These results show an unanticipated role for Seladin-1, previously implicated in Alzheimer's disease and cholesterol metabolism, in integrating cellular response to oncogenic and oxidative stress.     

杜氏盐藻hsp9O基因的克隆及胁迫条件下表达量的变化

利用RACE方法首次从杜氏盐藻中获得hsp90基因全长序列,并将其提交至GeneBank(Accession No:JQ735968).进一步研究表明,在热激与盐胁迫条件下杜氏盐藻hsp90基因的转录水平及蛋白表达水平出现明显上调,暗示该基因可能是杜氏盐藻细胞中与逆境响应相关的调控元件.     

Dynamic modeling of cellular response to DNA damage based on p53 stress response networks

Under acute perturbations from the outside, cells can trigger self-defensive mechanisms to fight against genome stress. To investigate the cellular response to continuous ion radiation (IR), a dynamic model for p53 stress response networks at the cellular level is proposed. The model can successfully be used to simulate the dynamic processes of double-strand breaks (DSBs) generation and their repair, switch-like ataxia telangiectasia mutated (ATM) activation, oscillations occurring in the p53-MDM2 feedback loop, as well as toxins elimination triggered by p53 stress response networks. Especially, the model can predict the plausible outcomes of cellular response under different IR dose regimes.     

渗透胁迫对苗期不同品种春小麦叶片叶绿素荧光动力学的影响

利用OS1—FL调制式叶绿素荧光分析仪对渗透胁迫下的幼苗期春小麦叶片叶绿素荧光动力学参数的变化进行了研究。结果表明,渗透胁迫下4个品种春小麦叶片的可变荧光(F_v)、小麦叶片光系统Ⅱ(PSⅡ),原初光能转换效率(F_v/F_m)和PSⅡ潜在活性(F_v/F_o)均降低,光量子产额(Yield)减少,光合机构受到不同程度的损伤(F_m/F_o),以上各值经方差分析可知,在品种间、处理浓度、处理天数及以上各因素二二互作或三因素间互作等方面的结果皆达到显著或极显著水平。     

基于Crowbar的DFIG低电压穿越控制方法研究

针对双馈风力发电机(DFIG)在电网故障期间面临的脱网问题,研究基于Crowbar电路的低电压穿越控制方法。首先,在分析电网电压跌落过程中DFIG暂态特性的基础上,对Crowbar电路进行阻值整定,得到最优的Crowbar阻值范围;其次,根据电网电压跌落状况下DFIG的暂态响应特性,提出了相应的Crowbar电路投切控制方法;最后,在MATLAB/Simulink上对双馈风电系统低电压穿越问题进行仿真研究。仿真结果证明了采取Crowbar电路的必要性与所提出的控制策略的可行性。     

Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins

Flooding of soils results in acute oxygen deprivation (anoxia) of plant roots during winter in temperate latitudes, or after irrigation, and is a major problem for agriculture. One early response of plants to anoxia and other environmental stresses is downregulation of water uptake due to inhibition of the water permeability (hydraulic conductivity) of roots (Lp(r)). Root water uptake is mediated largely by water channel proteins (aquaporins) of the plasma membrane intrinsic protein (PIP) subgroup. These aquaporins may mediate stress-induced inhibition of Lp(r) but the mechanisms involved are unknown. Here we delineate the whole-root and cell bases for inhibition of water uptake by anoxia and link them to cytosol acidosis. We also uncover a molecular mechanism for aquaporin gating by cytosolic pH. Because it is conserved in all PIPs, this mechanism provides a basis for explaining the inhibition of Lp(r) by anoxia and possibly other stresses. More generally, our work opens new routes to explore pH-dependent cell signalling processes leading to regulation of water transport in plant tissues or in animal epithelia.