FS36作为新型人源Hsp90抑制剂的发现（英文）

热休克蛋白90(Hsp90)通过对几百种蛋白质底物(客户蛋白质)进行合理的折叠、成熟其构象并且激活,在肿瘤细胞的生长和繁殖中发挥重要作用.因此,Hsp90成为非常有吸引力、有前途的抗肿瘤药物靶点,并且超过20种抑制剂已经进入临床实验阶段.我们在这里设计并合成了一个小分子抑制剂:FS36.收集了Hsp90~N-FS36复合物晶体结构的X射线衍射实验数据.高分辨率X射线晶体结构表明,FS36在ATP结合位点上与Hsp90~N相互作用,并且FS36可能替代核苷酸与Hsp90~N结合.FS36和Hsp90~N的复合物晶体结构和相互作用为后期设计和优化新型抗肿瘤药物奠定基础.     

热休克蛋白90的N端与ATP类似物的晶体结构揭示其功能调控

分子伴侣热休克蛋白90(Hsp90)对于许多涉及细胞周期调控、信号转导以及细胞生长调控蛋白质的折叠、成熟及稳定是必需的．Hsp90的N端结构高度保守,包含一个ATP结合口袋并具有ATP酶活性,Hsp90的功能依赖于ATP与Hsp90结合后诱导的构象重排及之后的ATP水解．为了深入研究ATP与Hsp90结合后N端的结构及其功能状态,使用悬滴法共结晶了Hsp90的N端与ATP类似物AMPPNP及ATPγS的复合物,并利用分子置换法对其结构进行了解析．两个复合物晶体结构都捕获到了核苷酸的电子密度,尤其是γ-磷酸的电子密度,从而观察到γ-磷酸与蛋白质之间的相互作用．ATPγS中γ-磷酸的捕获证实了之前报道的结构中没有捕获到γ-磷酸是其处于无序状态而非被水解．单体状态下的人源Hsp90^N- AMPPNP与处于二聚体化的酵母Hsp90-AMPPNP结构对比可见S1和ATP lid的位置有明显区别,结构分析表明,E18-K100和N40-D127之间形成的氢键相互作用,在一定程度上阻碍了S1和ATP lid的摆动,很可能阻止了二聚体的形成．     

人二氢乳清酸脱氢酶蛋白的表达、纯化及其与新型抑制剂的晶体结构

人二氢乳清酸脱氢酶（human dihydroorotate dehydrogenase, hDHODH）是催化嘧啶从头合成途径的一个关键酶。近年来,多种研究表明,抑制该酶可缓解类风湿性关节炎的症状。但该酶的抑制剂甚少,寻找该酶的高效抑制剂具有重要意义。本研究利用PCR技术扩增hDHODH基因,构建重组质粒pET-19b-hDHODH,并在大肠杆菌(Escherichia coli, E.coli ) BL21(DE3)中表达,获得可溶性蛋白质。用Ni2+-NTA亲和层析柱对蛋白质进行纯化,获得较高（90%）纯度的hDHODH蛋白,将蛋白质与抑制剂3-(5-乙硫基)-1H-1, 2, 4-三氮唑-3-)苯甲酸和底物DHO混合孵育。用Hampton试剂盒初筛晶体并用棋盘法进行优化,获得晶形完美、衍射能力很强的hDHODH蛋白复合物单晶。用X射线衍射晶体,用CCP4、Coot软件解析结构,获得hDHODH蛋白复合物晶体结构。从解析的结构中可以看出,抑制剂与蛋白质的吻合度非常高,且抑制剂通过亲水的羧基端与蛋白质356位和147位的酪氨酸形成氢键网络。抑制剂的5元环与蛋白质359位的亮氨酸和360位的苏氨酸相互作用,使抑制剂与蛋白质牢固结合。该复合物晶体结构的顺利解析,将为开发新型特异性抗类风湿性关节炎药物提供重要基础。     

综述: 大肠杆菌RNA分子伴侣Hfq与DsrA和rpoS作用机制的研究进展

RNA分子伴侣Hfq是细菌重要的转录后调节因子,它能够帮助非编码small RNA(sRNA)与目标mRNA配对．mRNA rpoS编码的稳态sigma因子σ^S是大肠杆菌中应激响应的核心调控因子．在低温下,Hfq蛋白对于sRNA DsrA介导的mRNA rpoS的翻译激活是必需的．然而,Hfq使用何种机制来促进sRNA和mRNA配对一直有两种并不互斥的模型存在：Hfq远侧和近侧两个表面同时结合sRNA和mRNA,使得两条RNA相互靠近,便于形成碱基配对;Hfq可能结合一条或者两条RNA,改变它们的二级结构或者三级结构,从而促进sRNA-mRNA配对的实现．最近的研究报道,成功在体外捕捉到了sRNA-Hfq-mRNA三元复合物,测定了AU₆A-Hfq-A₇三元复合物的晶体结构,并且在大肠杆菌体内证实了三元复合物的形成对于Hfq帮助sRNA DsrA激活mRNA rpoS的翻译是重要的．本文以sRNA DsrA和mRNA rpoS为例综述了蛋白质Hfq与RNA的结合特性,同时也讨论了sRNA-Hfq-mRNA三元复合物的存在对于研究sRNA介导的调控机制的一些启示．     

川西平原小流域不同水体N2O排放特征及驱动因素

氧化亚氮（N₂O）是一种潜在的、强大的温室气体,应该根据京都议定书规定开展监测和削减。河流、水库、鱼塘和沟渠等受人类影响的小流域水生生态系统是氮素生物地球化学循环的活跃区域,更是N₂O重要的源和汇。然而,同一流域不同水体N₂O的排放特征差异及其驱动因素尚不清楚。因此,选择川西平原西河流域作为研究区,于2016年6月到2017年5月连续监测不同水体水气界面的N₂O排放强度,并结合聚类分析解析N₂O排放特征的驱动因素。结果显示,不同水体的N₂O年排放通量差异显著,沟渠的N₂O年排放通量最高（（52.68±36.09）μg m^-2 h^-1）,城市段河流和鱼塘次之（（34.16±23.97）μg m^-2 h^-1和（29.03±31.41）μg m^-2 h^-1）,乡镇段和农区段河流再次（（8.32±28.60）μg m^-2 h^-1和（8.52±9.43）μg m^-2 h^-1）,水库最低（（-16.45±29.76）μg m^-2 h^-1）。除水库表现为N₂O的汇,其他水体均表现为N₂O的排放源。另外,不同水体N₂O排放的季节特征差异显著,农区段河流和农业沟渠表现为夏天最高,冬春最低（P<0.05）,而其他水体均表现为冬春显著高于夏秋（P<0.05）。根据N₂O排放季节特征及其驱动因素可将西河流域水体分为四类：第一类农业类水体的N₂O排放季节特征受气象因素和农业活动的联合驱动;第二类城乡类河流和第三类鱼塘分别受控于人类活动和养殖活动,与降雨温度等气象指标关系较弱;第四类水库主要受控于气象因素。并且,第一类农业类水体已成为大气N₂O排放的重要源,农业氮素管控是区域控制N₂O排放的重点。     

黄土高原旱塬区陇东苜蓿草地N2O释放动态及微生物驱动因子的研究

在甘肃庆阳黄土高原,采用静态箱-气相色谱法测定了3年龄陇东苜蓿草地不同物候期的N²O释放通量,采用最大或然数法(MPN)测定了土壤0～30 cm硝化细菌和反硝化细菌数量,探讨旱区紫花苜蓿草地N²O释放规律与其土壤微生物的关系。结果表明:（1）3年龄陇东苜蓿草地年内N²O释放通量在-0.099～0.085 mg·m^-2·h^-1之间;N²O释放通量在头茬花期和越冬前再生期达到高峰,而二茬再生期和越冬期则处于N²O吸收期,成为N²O贮存库。（2）陇东苜蓿草地亚硝酸细菌、硝酸细菌和反硝化细菌数量在不同物候期和土壤层次间存在差异。（3）3年龄陇东苜蓿草地N²O释放通量与土壤水分含量、土壤pH、硝酸细菌和反硝化细菌数量间呈显著正相关线性关系。（4）庆阳黄土高原旱区陇东苜蓿草地是较弱的农业N²O释放源,远低于同类研究和国际释放平均水平。     

黄河上游灌区稻田N2O排放特征

黄河上游灌区稻田高产区过量施肥现象十分突出,氮肥过量施用引起土壤氮素盈余,导致N₂O排放量增大,由此引起的温室效应引起广泛关注。采用静态箱-气相色谱法研究黄河上游灌区稻田不同施肥处理下N₂O排放特征。试验设置5个施肥处理,包括常规氮肥300 kg/hm²下单施尿素和有机肥配施2个处理,分别用N300和N300-OM代表;优化氮肥240 kg/hm²下单施尿素和有机肥配施2个处理,分别用N240和N240-OM代表;对照不施氮肥用N0代表。试验结果得出,灌区水稻生长季稻田土壤N₂O排放主要集中在水稻分蘖前及水稻生长的中后期,稻田氮肥施用、灌水及土壤温度的变化对N₂O排放通量影响较大,不同处理水稻各生育阶段N₂O累积排放量与稻田土壤耕层NO^-₃-N含量动态变化显著相关。稻田N₂O排放不是黄河上游灌区稻田氮素损失的主要途径,但灌区稻田N₂O排放的增温潜势较大;稻田氮肥过量施用会显著增加N₂O排放量,在相同氮素水平下,有机肥配施会显著增加稻田土壤N₂O的排放量(P＜0.01)。优化施氮能有效减少灌区稻田水稻生长季N₂O排放量。稻田不同处理的水稻整个生长季土壤N₂O排放总量为2.69-3.87 kg/hm²,肥料氮通过N₂O排放损失的百分率仅为0.43%-0.64%。在灌区习惯灌水和高氮肥300 kg/hm²时,N300-OM处理的稻田N₂O排放量达3.87 kg/hm²,在100 a时间尺度上的全球增温潜势(GWPs)为20.76×10⁷ kg CO₂/hm²;优化施氮240 kg/hm²水平下,N240和N240-OM处理的N₂O累计排放量较N300-OM处理,分别降低了1.18 kg/hm²和0.57 kg/hm²,在100 a尺度上每年由稻田N₂O排放引起的GWPs分别降低了6.33×10⁷ kg CO₂/hm²和3.06×10⁷ kg CO₂/hm²。     

TPR蛋白对Hsp90功能的调节

热激蛋白Hsp90是一类在进化中形成的高度保守的且可参与多种细胞功能的特异分子伴侣。TPR蛋白通常存在于Hsp90的多蛋白质复合物中,它对Hsp90的功能的多样性起着至关重要的作用,同时Hsp90可能为TPR蛋白提供“泊位”,允许不同的TPR蛋白在Hsp90分子伴侣底物附近有序而特异结合,从而使Hsp90在细胞内环境中以特定的方式完成其各种细胞功能。了解TPR蛋白与Hsp90的相互作用机制为阐明细胞内Hsp90的功能多样性和特异性奠定了基础。     

氮添加对马尾松人工林土壤团聚体氮矿化及土壤酶活性的影响

土壤氮库是生态系统氮素重要的源和汇。以三峡库区马尾松（Pinus massoniana）人工林为研究对象,从团聚体视角出发分析土壤养分和酶活性对氮添加的响应规律,以及相应的变化对氮矿化的影响,为预测该地区在大气氮沉降持续增加的背景下土壤氮动态提供参考。设置4种量的氮添加处理（N₀：0 kg N hm^-2 a^-1;N₃₀：30 kg N hm^-2 a^-1;N₆₀：60 kg N hm^-2 a^-1;N₉₀：90 kg N hm^-2 a^-1）,将土壤按粒径分为>2000 μm （大团聚体）、250-2000 μm （小团聚体）和<250 μm （微团聚体）3个组分的团聚体,观察团聚体氮矿化特征。结果表明：（1）与对照相比,N₃₀和N₆₀处理提高了有机质（SOM）含量,但土壤SOM和全氮（TN）含量在N₉₀下开始出现下降;氮添加降低了土壤速效磷（aP）含量,在小团聚体中表现最为显著。除微团聚体中的POD和NAG以外,其余3种酶的活性均在N₃₀和N₆₀处理之下被提高。（2）土壤平均净硝化速率整体高于土壤平均净氨化速率;大团聚体和小团聚体中净氨化速率在氮添加处理后显著降低,大团聚体净硝化速率低于其他两个粒径;土壤净氮转化速率在N₉₀处理下最高。（3）土壤养分和无机氮含量与土壤酸性磷酸酶（AP）、N-乙酰-β-D-葡糖苷酶（NAG）、过氧化物酶（POD）、硝酸还原酶（NR）和脲酶（UE）的活性呈显著相关,酶活性变化是多因子综合作用的结果;RDA分析显示,UE与土壤净氨化速率存在显著正相关,NAG和POD是与净氮转化速率分别存在显著正相关和显著负相关的关键土壤酶。综上所述,硝化作用是土壤净氮转化的主要贡献者,微团聚体在土壤氮矿化中发挥主要作用,NAG和POD是改变土壤净氮转化的主要生物酶。此外,氮添加会引起土壤氮素的流失,引起土壤的磷限制,并对土壤养分循环产生显著影响。     

城市化地区典型水体的N2O排放通量特征——以南京市为例

内陆淡水水体是大气中N₂O的重要排放源,然而目前对于内陆典型城市水体N₂O排放通量的监测数据依然匮乏,典型城市水体的N₂O排放特征及驱动因素尚不清楚。本研究选取了南京市江北新区的典型水体,包括湖库、河流、养殖池塘和景观池塘,在2020年5月-2021年4月利用漂浮箱法连续监测了不同水体类型的水-气界面N₂O排放特征,并通过测定水环境特征,探究驱动水体N₂O排放通量的关键因素。研究结果表明,典型城市水体整体均表现为N₂O排放源,河流和养殖池塘的日平均排放通量最大,分别为（503±1236）μg m^-2 d^-1和（508±797）μg m^-2 d^-1,其次为景观池塘（（179±989）μg m^-2 d^-1）,而湖库的N₂O排放通量最小,仅表现为微弱的N₂O排放源（（54±212）μg m^-2 d^-1）。水体的N₂O排放呈现季节性差异,河流和养殖池塘夏季的N₂O排放通量显著高于其他季节（P<0.01）。水体全年N₂O排放数据与水体温度和溶解氧含量（DO）呈显著相关。而在温度较高的5月份-9月份（>20℃）,氮输入成为影响N₂O排放通量的关键因素（P<0.01）,因此控制城市水体的氮输入尤其是在水温较高的夏季是减少N₂O排放的有利措施。此外,由于水文化学条件差异等因素,小型封闭水体包括养殖池塘和景观池塘的N₂O排放通量差异较大,未来应加强监测不同水体的水文化学特征和N₂O的时空排放特征,探讨影响小型封闭水体水-气界面N₂O排放通量的具体驱动因素。此研究为城市区域N₂O排放的精准核算提供了数据支撑,为N₂O排放模型的修正提供了科学依据。     

Anti-NSCLC activity in vitro of Hsp90N inhibitor KW-2478 and complex crystal structure determination of Hsp90N-KW-2478

KW-2478 is a promising anti-cancer lead compound targeting to the molecular chaperone heat shock protein 90 ^N (Hsp90^N). Absence of complex crystal structure of Hsp90^N-KW-2478, however, hampered further structure optimization of KW-2478 and understanding on the molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90^N-KW-2478 was determined by X-ray diffraction (XRD, resolution limit: 1.59 Å; PDB ID: 6LT8) and their molecular interaction was analyzed in detail, which suggested that KW-2478 perfectly bound in the N-terminal ATP-binding pocket of Hsp90 to disable its molecular chaperone function, therefore suppressed or killed cancer cells. The results from thermal shift assay (TSA, ΔTm, 18.82 ± 0.51 °C) and isothermal titration calorimetry (ITC, K_d, 7.30 ± 2.20 nM) suggested that there is an intense binding force and favorable thermodynamic changes during the process of KW-2478 binding with Hsp90^N. Additionally, KW-2478 exhibited favorable anti-NSCLC activity in vitro, as it inhibited cell proliferation (IC₅₀, 8.16 μM for A549; 14.29 μM for H1975) and migration, induced cell cycle arrest and promoted apoptosis. Thirty-six novel KW-2478 derivatives were designed, based on the complex crystal structure and molecular interaction analysis of Hsp90^N-KW-2478 complex. Among them, twenty-two derivatives exhibited increased binding force with Hsp90^N evaluated by molecular docking assay. The results would provide new guidance for anti-NSCLC new drug development based on the lead compound KW-2478.     

Synthesis and in vitro antiproliferative activity of C5-benzyl substituted 2-amino-pyrrolo[2,3-d]pyrimidines as potent Hsp90 inhibitors

A novel series of heat shock protein 90 (Hsp90) inhibitors was identified by X-ray crystal analysis of complex structures at solvent-exposed exit pocket C. The 2-amino-pyrrolo[2,3-d]pyrimidine derivatives, 7-deazapurines substituted with a benzyl moiety at C5, showed potent Hsp90 inhibition and broad-spectrum antiproliferative activity against NCI-60 cancer cell lines. The most potent compound, 6a, inhibited Hsp90 with an IC₅₀ of 36 nM and showed a submicromolar mean GI₅₀ value against NCI-60 cell lines. The interaction of 6a at the ATP-binding pocket of Hsp90 was confirmed by X-ray crystallography and Western blot analysis.     

Lead generation of heat shock protein 90 inhibitors by a combination of fragment-based approach, virtual screening, and structure-based drug design

Heat shock protein 90 (Hsp90) is a molecular chaperone which regulates maturation and stabilization of its substrate proteins, known as client proteins. Many client proteins of Hsp90 are involved in tumor progression and survival and therefore Hsp90 can be a good target for developing anticancer drugs. With the aim of efficiently identifying a new class of orally available inhibitors of the ATP binding site of this protein, we conducted fragment screening and virtual screening in parallel against Hsp90. This approach quickly identified 2-aminotriazine and 2-aminopyrimidine derivatives as specific ligands to Hsp90 with high ligand efficiency. In silico evaluation of the 3D X-ray Hsp90 complex structures of the identified hits allowed us to promptly design CH5015765, which showed high affinity for Hsp90 and antitumor activity in human cancer xenograft mouse models.     

The Ig VH complementarity-determining region 3-containing Rb9 peptide,inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the V_HCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca²⁺ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.     

1H, 15N and 13C backbone resonance assignments of the TPR1 and TPR2A domains of mouse STI1

Hop/STI1 (Hsp-organizing protein/stress-induced-phosphoprotein 1) is a molecular co-chaperone, which coordinates Hsp70 and Hsp90 activity during client protein folding through interactions with its TPR1 and TPR2A domains. Hsp90 substrates include a diverse set of proteins, many of which have been implicated in tumorigenesis. Over-expression of Hsp90 in cancer cells stabilizes mutant oncoproteins promoting cancer cell survival. Disruption of Hsp90 and its co-chaperone machinery has become a promising strategy for the treatment of cancer. STI1 has also been described as a neurotrophic signaling molecule through its interactions with the prion protein (PrP^C). Here, we report the ¹H, ¹³C and ¹⁵N backbone assignments of the TPR1 and TPR2A domains of mouse STI1, which interact with Hsp70 and Hsp90, respectively. ¹H-¹⁵N HSQC spectra of TPR2A domain in the presence of a peptide encoding the C-terminal Hsp90 binding site revealed significant chemical shift changes indicating complex formation. These results will facilitate the screening of potential molecules that inhibit STI1 complex formation with Hsp70 and/or Hsp90 for the treatment of cancer and detailed structural studies of the STI1-PrP^C complex.     

Hsp90 inhibitor 17-AAG sensitizes Bcl-2 inhibitor (-)-gossypol by suppressing ERK-mediated protective autophagy and Mcl-1 accumulation in hepatocellular carcinoma cells

Natural BH3-memitic (-)-gossypol shows promising antitumor efficacy in several kinds of cancer. However, our previous studies have demonstrated that protective autophagy decreases the drug sensitivities of Bcl-2 inhibitors in hepatocellular carcinoma (HCC) cells. In the present study, we are the first to report that Hsp90 inhibitor 17-AAG enhanced (-)-gossypol-induced apoptosis via suppressing (-)-gossypol-triggered protective autophagy and Mcl-1 accumulation. The suppression effect of 17-AAG on autophagy was mediated by inhibiting ERK-mediated Bcl-2 phosphorylation while was not related to Beclin1 or LC3 protein instability. Meanwhile, 17-AAG downregulated (-)-gossypol-triggered Mcl-1 accumulation by suppressing Mcl-1^Thr163 phosphorylation and promoting protein degradation. Collectively, our study indicates that Hsp90 plays an important role in tumor maintenance and inhibition of Hsp90 may become a new strategy for sensitizing Bcl-2-targeted chemotherapies in HCC cells.     

Luteolin Induces Carcinoma Cell Apoptosis through Binding Hsp90 to Suppress Constitutive Activation of STAT3

Background

Abnormal activity of STAT3 is associated with a number of human malignancies. Hsp90 plays a central role in stabilizing newly synthesized proteins and participates in maintaining the functional competency of a number of signaling transducers involved in cell growth, survival and oncogenesis, such as STAT3. Hsp90 interacts with STAT3 and stabilizes Tyr-phosphorylated STAT3. It has been reported that luteolin possesses anticancer activity through degradation of Tyr⁷⁰⁵-phosphorylated STAT3.

Methodology/Principal Findings

We found that overexpression of Hsp90 inhibited luteolin-induced degradation of Tyr⁷⁰⁵-phosphorylated STAT3 and luteolin also reduced the levels of some other Hsp90 interacting proteins. Results from co-immunoprecipitation and immunoblot analysis demonstrated that luteolin prevented the association between Hsp90 and STAT3 and induced both Tyr⁷⁰⁵- and Ser⁷²⁷-phosphorylated STAT3 degradation through proteasome-dependent pathway. The molecular modeling analysis with CHARMm–Discovery Studio 2.1(DS 2.1) indicated that luteolin could bind to the ATP-binding pocket of Hsp90. SPR technology-based binding assay confirmed the association between luteolin and Hsp90. ATP-sepharose binding assay displayed that luteolin inhibited Hsp90-ATP binding.

Conclusions/Significance

Luteolin promoted the degradation of Tyr⁷⁰⁵- and Ser⁷²⁷-phosphorylated STAT3 through interacting with Hsp90 and induced apoptosis of cancer cells. This study indicated that luteolin may act as a potent HSP90 inhibitor in antitumor strategies.     

Sulforaphane inhibits pancreatic cancer through disrupting Hsp90–p50Cdc37 complex and direct interactions with amino acids residues of Hsp90

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl) butane)], an isothiocyanate derived from cruciferous vegetables, has been shown to possess potent chemopreventive activity. We analyzed the effect of sulforaphane on the proliferation of pancreatic cancer cells. Sulforaphane inhibited pancreatic cancer cell growth in vitro with IC₅₀s of around 10–15 μM and induced apoptosis. In pancreatic cancer xenograft mouse model, administration of sulforaphane showed remarkable inhibition of tumor growth without apparent toxicity noticed. We found that sulforaphane induced the degradation of heat shock protein 90 (Hsp90) client proteins and blocked the interaction of Hsp90 with its cochaperone p50^Cdc37 in pancreatic cancer cells. Using nuclear magnetic resonance spectroscopy (NMR) with an isoleucine-specific labeling strategy, we overcame the protein size limit of conventional NMR and studied the interaction of sulforaphane with full-length Hsp90 dimer (170 kDa) in solution. NMR revealed multiple chemical shifts in sheet 2 and the adjacent loop in Hsp90 N-terminal domain after incubation of Hsp90 with sulforaphane. Liquid chromatography coupled to mass spectrometry further mapped a short peptide in this region that was tagged with sulforaphane. These data suggest a new mechanism of sulforaphane that disrupts protein–protein interaction in Hsp90 complex for its chemopreventive activity.     

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K_d values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 × 10^− 4 min^− 1 μM^− 1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.     

Characterization of Celastrol to Inhibit Hsp90 and Cdc37 Interaction

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotide-free Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.