芹黄素、白杨黄素和木犀草素选择性抑制肿瘤细胞胰凝乳蛋白酶样和胰蛋白酶样蛋白酶体催化活性

<正>泛素-蛋白酶体途径在调节细胞凋亡和细胞周期中起重要作用。蛋白酶体功能由3种主要的催化活性介导,即胰凝乳蛋白酶样(CT-L)、胰蛋白酶样(T-L)和肽基谷氨酰肽水解样(PGPH)活性。近期研究显示,蛋白酶体抑制剂具抗肿瘤活性并已用于肿瘤(如     

蛋白酶体抑制剂YSY01A不依赖NF-κB通路抑制乳腺癌细胞增殖及诱导凋亡研究

目的研究YSY01A对乳腺癌的增殖抑制作用及其抗癌机制。方法荧光底物法检测蛋白酶体活性;蛋白酶体荧光探针法研究YSY01A与蛋白酶体β催化亚基结合;Annexin V-FITC、JC-1探针检测YSY01A对细胞凋亡和线粒体膜电位的影响;用蛋白免疫印迹法检测细胞内的蛋白表达。结果YSY01A抑制细胞内外蛋白酶体的活性,其中对CT-L的抑制作用最强,对T-L的作用比PS341强3-4倍;MCF-7对YSY01A最敏感,且比PS341敏感性高;YSY01A以时间依赖性诱导细胞凋亡,与对照组比较,差异均有显著性(P<0.05)。YSY01A增加ubiquitin、P-IκBα、wt-p53、Bax、endonuclearse G蛋白表达,减少IκBα、Bcl-xL、XIAP水平,不影响NF-κB表达。结论 YSY01A是新蛋白酶体抑制剂,具有较强的抑制MCF-7细胞增殖的作用,抗癌机制与诱导细胞凋亡有关。     

顺铂对蛋白酶体抑制作用的研究

目的 研究顺铂对蛋白酶体的抑制作用。方法 采用蛋白酶体特异性底物S-LLVY-AMC、B-LRR-AMC及ZLLE-AMC观察了顺铂对纯化的人20S蛋白酶体及CCD-1103 KidTr细胞内蛋白酶体活力的影响，同时分析了受蛋白酶体降解的p53蛋白在蛋白质及mRNA水平的表达。结果顺铂对纯化的人20S蛋白酶体具有明显的抑制作用，对3种酶活力抑制作用的半数抑制浓度(IC50)分别为32．17(S-LLVY-AMC)、37．96(Z-LLE-AMC)和37．8lμg／ml(Z-LLE-AMC)。在30和60μg/ml顺铂作用下，CCD-1103 KidTr细胞内蛋白酶体活力也受到显著抑制。在30μg／ml顺铂作用下，p53 mRNA表达在3、6、12和24h未见明显改变，而p53蛋白质水平在3、6h明显增高。结论 顺铂对蛋白酶体活力有抑制作用，但对蛋白酶体3种酶活力的抑制没有特异性。顺铂作用CCD-1103 KidTr细胞后p53蛋白质表达增高，可能与蛋白酶体抑制有关。     

蛋白酶体抑制剂筛选模型的建立

蛋白酶体对肿瘤生长相关蛋白的降解起十分重要的调控作用,能够通过多种机制抑制肿瘤生长和扩散,已成为一个新的抗癌靶点[1].特异性蛋白酶体抑制剂PS-341在体外和小鼠肿瘤模型中表现出明显的抗肿瘤活性,目前已在进行Ⅱ～Ⅲ期临床试验[1].寻找特异的蛋白酶体抑制剂,关键是建立特异、简便的筛选模型.我们根据荧光底物Suc-Leu-Leu-Val-Tyr-AMC在SDS活化的蛋白酶体作用下发生水解,释放出具有荧光的AMC(7-氨基-4-甲基香豆素)的原理,建立了蛋白酶体抑制剂的筛选模型.本文就该模型的建立作一介绍.     

复智散通过细胞周期依赖性蛋白激酶5通路减轻皮层神经元Tau蛋白过度磷酸化

目的探讨复方中药复智散(FZS)能否通过抑制细胞周期依赖性蛋白激酶5(CDK5)通路,减轻Aβ_(25-351)诱导的新生鼠皮层神经元Tau蛋白过度磷酸化。方法选用24 h内新生Wistar大鼠,分离纯化皮层神经元,进行体外培养。皮层神经元在体外培养7 d后,应用20μmol·L-1Aβ_(25-351)作用于皮层神经元24 h。药物治疗组则应用FZS(20 mg·L~(-1))、CDK5抑制剂Roscovitine(15μmol·L~(-1))、钙蛋白酶(calpain)制剂Calpeptin(20μmol·L~(-1))预处理24 h,然后用20μmol·L~(-1)Aβ_(25-351)作用24 h。用Western blot检测Tau蛋白Ser396、Ser202和Thr231位点磷酸化水平和CDK5的激活蛋白p25/p35的蛋白水平;荧光酶标仪测定荧光强度来反映calpain活性;免疫沉淀法检测CDK5激酶活性。结果 20μmol·L~(-1)Aβ_(25-351)作用于皮层神经元24 h后,Tau蛋白在Ser396、Ser202、Thr231位点磷酸化水平增加,CDK5激酶活性升高,CDK5激活蛋白p25水平升高,calpain活性升高。20 mg·L-1FZS治疗组则明显抑制了Aβ_(25-351)导致的Tau蛋白在Ser396、Ser202、Thr231位点磷酸化水平增加,抑制了CDK5激酶活性、p25蛋白水平以及calpain活性升高。CDK5抑制剂roscovitine和calpain抑制剂calpeptin作为阳性对照药物也显示了抑制Tau蛋白过度磷酸化的作用。结论复智散可能通过calpain-p25/CDK5通路抑制Aβ_(25-351)导致的皮层神经元Tau蛋白过度磷酸化。     

磺胺类黄酮衍生物20S蛋白酶体抑制剂的设计、合成与活性评价

本文通过计算机辅助设计,设计并合成了一系列磺胺类黄酮衍生物作为非共价20S蛋白酶体抑制剂,并对其生物活性进行了测试。与先导化合物相比（β5亚基的IC50值为14.0μM）,化合物仅表现出局部的改善,但仍可作为一类潜在的20S蛋白酶体抑制剂。     

四氢异喹啉-噁二唑类非共价型蛋白酶体抑制剂研究初探

目的探索四氢异喹啉-噁二唑类非共价型蛋白酶体抑制剂的可行性。方法基于拼合原理,将新型非共价型蛋白酶体抑制剂PI-1833(5)的1,2,4-噁二唑靶头与本研究组发现的四氢异喹啉脲拟肽骨架相拼合,设计了四氢异喹啉-噁二唑类非共价型蛋白酶体抑制剂,合成了N-((3-苯基-1,2,4-噁二唑-5-基)甲基)-3,4-二氢异喹啉-2(1H)-甲酰异丙胺(8),并采用MTT法评价其对人胃癌细胞MGC-803的生长抑制活性;利用分子对接的方法分析化合物与20S蛋白酶体的结合状态。结果与结论化合物8在10μmol·L~(-1)浓度下没有显示出抗肿瘤活性;通过比较化合物5和8与20S蛋白酶体的对接结果,发现化合物8不产生活性的主要原因可能是噁二唑和四氢异喹啉之间的连接臂柔性不够,提示进一步研究的重点应该是对连接臂的优化。     

左旋多巴联合丹参注射液对鱼藤酮致分化PC12细胞凋亡的影响

目的:研究左旋多巴联合丹参注射液对鱼藤酮致分化PC12细胞凋亡的影响。方法:实验分为正常对照(正常细胞,等容培养液)组、联合用药对照(正常细胞,左旋多巴2μmol/L+丹参注射液3.125μg/ml)组、模型(模型细胞,等容培养液)组、神经生长因子(模型细胞,NGF,300μg/L)组、左旋多巴(模型细胞,2μmol/L)组、丹参注射液(模型细胞,3.125μg/ml)组、联合用药(模型细胞,左旋多巴2μmol/L+丹参注射液3.125μg/ml)组。以鱼藤酮诱导分化PC12细胞凋亡作为帕金森病体外模型,通过水溶性四氮唑(WST-1)法测细胞相对增殖率,Hoechst 33342标记染色法观察细胞荧光强度改变,酶联免疫吸附(ELISA)法检测细胞3-磷酸甘油醛脱氢酶(GAPDH)蛋白含量与20S蛋白酶体(PSM)活性。结果:与模型组比较,联合用药组细胞增殖率升高,细胞凋亡减小,GAPDH蛋白含量减少,20S PSM活性增强,且效果均优于左旋多巴组。结论:左旋多巴联合丹参注射液可拮抗鱼藤酮致分化PC12细胞凋亡,其机制与降低细胞内GAPDH含量及增强20S PSM活性有关,其效果优于左旋多巴单用。     

20S蛋白酶体表达与类风湿关节炎的关系探讨

目的 探讨类风湿关节炎（RA）患者20S蛋白酶体表达水平的变化及其与RA疾病活动性的关系.方法 测定RA患者及正常对照外周血单个核细胞20S蛋白酶体蛋白表达水平.结果 与正常对照相比,RA患者20S蛋白酶体表达升高[（2.92±1.41VS（0.65±0.42）,P＜0.01].20S蛋白酶本表达水平与DAS28、CRP、ERS之间无显著相关性.结论 RA患者PBMC中20S蛋白酶体表达升高,其水平与疾病活动无显著相关性.     

热毒宁注射液及其组分对流感病毒神经氨酸酶的抑制作用研究

目的研究热毒宁注射液及其组分对流感病毒神经氨酸酶(NA)抑制活性。方法通过测定流感病毒NA的荧光强度值来确定热毒宁注射液及其组分对流感病毒NA的抑制活性。结果热毒宁注射液对H1N1、H3N2、B流感病毒NA抑制活性较高,半数抑制浓度(IC50)分别为(46.49±2.25)、(49.77±1.77)、(45.33±5.32)μg/mL。金银花聚酰胺柱洗脱浓缩液、醋酸乙酯萃取液及残液、柱色谱提取物、隐绿原酸、异绿原酸A和异绿原酸B对H1N1、H3N2、B流感病毒NA酶活性均具有抑制作用。结论热毒宁注射液及其有机酸类成分对流感病毒神经氨酸酶活性均具有一定抑制作用。     

A new fluorogenic peptide determines proteasome activity in single cells

The ubiquitin-proteasome system plays a critical role in many diseases, making it an attractive biomarker and therapeutic target. However, the impact of results obtained in vitro using purified proteasome particles or whole cell extracts is limited by the lack of efficient methods to assess proteasome activity in living cells. We have engineered an internally quenched fluorogenic peptide with a proteasome-specific cleavage motif fused to TAT and linked to the fluorophores DABCYL and EDANS. This peptide penetrates cell membranes and is rapidly cleaved by the proteasomal chymotrypsin-like activity, generating a quantitative fluorescent reporter of in vivo proteasome activity as assessed by time-lapse or flow cytometry fluorescence analysis. This reporter is an innovative tool for monitoring proteasomal proteolytic activities in physiological and pathological conditions.     

肽醛类前药衍生物作为蛋白酶体抑制剂的研究(英文)

为了提高经典的肽醛类蛋白酶体抑制剂的稳定性和生物利用度,我们设计合成了4个肽缩醛和2个肽杂缩醛衍生物,并对其进行了酶水平和体外抗肿瘤实验。结果显示,4个肽缩醛化合物几乎没有显示任何活性,表明此类化合物有用作前药的潜质。而2个肽杂缩醛化合物在酶水平和细胞水平均表现出明显的活性,则表明此类化合物可能不是通过前药的形式而发挥作用,进一步优化杂环的结构,有可能发现更好的蛋白酶体抑制剂。     

Apoptosis induction of 2'-hydroxycinnamaldehyde as a proteasome inhibitor is associated with ER stress and mitochondrial perturbation in cancer cells

2'-Hydroxycinnamaldehyde (HCA), isolated from the stem bark of Cinnamomum cassia, and 2'-benzoyloxycinnamaldehyde (BCA), one of HCA derivatives, have antiproliferative activities on several human cancer cell lines. Our previous study suggested that reactive oxygen species (ROS) and caspase-3 are the major regulators of HCA-induced apoptosis. In the present study, we demonstrated a novel molecular target using in vitro pull-down assay by biotin-labeled HCA (biotin-HCA) in SW620 cells. We analyzed 11 differential spots of 2-dimensional gel prepared with pull-downed proteins by biotin-HCA. Among them, five spots were identified as proteasome subunits. An in vitro 26S proteasome function assay using specific fluorogenic substrates showed that HCA potently inhibits L3-like activity of the proteasome. In addition, HCA showed inhibitory action against chymotrypsin-like, trypsin-like, and PGPH-like activities. DNA microarray showed that HCA induced heat shock family and ER stress-responsive genes, which reflects the accumulation of misfolded proteins by proteasome inhibition. On western blot analysis, it was confirmed that HCA induces glucose-regulated protein, 78 kDa (GRP78) and some representative endoplasmic reticulum (ER) stress-responsive proteins. Furthermore, HCA treatment decreased mitochondrial membrane potential. The effect of HCA on cytochrome c and Bax translocation between cytosol and mitochondrial membrane was clarified using western blot analysis. These results suggest that HCA-induced apoptosis is associated with the inhibition of the proteasome activity that leads in turn to the increase of ER stress and mitochondrial perturbation.     

Inhibition of the proteasome activity by graphene oxide contributes to its cytotoxicity

Due to its hydrophobicity and other unique physicochemical properties, graphene oxide (GO) has been extensively utilized in various biological applications. However, introducing nanomaterials into the biological environment may raise serious risk in terms of nanotoxicity, leading to some unintended changes to the structure and the function of other biomolecules. This study investigates the interaction of GO with the ubiquitin–proteasome system, one of the essential machineries in the cellular metabolism, using a combination of experimental and computational approaches. The experimental results show that GO could adsorb the 20S proteasome, causing a dose-dependent suppression of the proteolytic activity of proteasome. This adverse effect eventually disturbed other important cellular activities relevant to cell cycle and survival. Meanwhile, the molecular dynamics simulations revealed that when 20S proteasome was adsorbed onto the graphene surface, the central gate in the outer ring (α-subunit) for the entry and the exit of the peptide ligand to the protease active site was effectively blocked. These findings of GO induced functional disturbance of 20S proteasome provides a novel perspective to understand the molecular mechanism of GO’s cytotoxicity, which might further promote applications of GO in potential therapies for various cancers due to the abnormal elevation of the relevant proteasome activities.     

Trypanocidal effect of alpha',beta'-epoxyketones indicates that trypanosomes are particularly sensitive to inhibitors of proteasome trypsin-like activity

Previous studies have shown that the proteasome of Trypanosoma brucei is a candidate for novel chemotherapy of African sleeping sickness. In this study, two potent and highly selective alpha',beta'-epoxyketones peptide proteasome inhibitors, epoxomicin and YU101, have been tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of T. brucei. Both inhibitors displayed promising anti-trypanosomal activities with ED(50) and ED(90) values in the low to mid nanomolar range. Based on MIC values, epoxomicin exhibited a selectivity index approaching those of commercially available drugs. Enzymatic analyses of proteasomal peptidase activities revealed that, compared with mammalian cells, trypanosomes are particular sensitive to inhibition of the trypsin-like activity of the proteasome. In conclusion, the data suggests that proteasome inhibitors targeting the trypsin-like activity are the rational choice for future anti-trypanosomal drug development.     

Down-regulation of estrogen receptor-alpha in MCF-7 human breast cancer cells after proteasome inhibition

The eukaryotic proteasome is a 26S ATP-dependent proteolytic complex, which possesses chymotrypsin-like, trypsin-like and peptidyl glutamyl peptide hydrolase (PGPH) activities, which enable the proteasome to degrade all short-lived and many long-lived proteins, and consequently regulate a myriad of activities in cells. In this study, we observed that inhibition of the proteasome, and more specifically, inhibition of the chymotrypsin-like activity of the proteasome, in MCF-7 human breast cancer cells resulted in selective down-regulation of the nuclear estrogen receptor-alpha (ERalpha). Our data indicated that estrogen had no effect, whereas the ERalpha antagonist, tamoxifen, reduced the amount of ERalpha that could be subjected to down-regulation after proteasome inhibition. Furthermore, our data demonstrated that protein synthesis was required for the down-regulation of ERalpha to occur. Collectively, these data indicate the existence of a proteasome-dependent mechanism that is utilized by MCF-7 cells to maintain a steady-state level of ERalpha.     

Dietary flavonoids as proteasome inhibitors and apoptosis inducers in human leukemia cells

It has been shown that proteasome activity is required for cancer cell survival and consumption of fruits and vegetables is associated with decreased cancer risk. Previously, we reported that grape extract could inhibit proteasome activity and induce apoptosis in tumor cells. In this study, we examined the flavonoids apigenin, quercetin, kaempferol and myricetin for their proteasome-inhibitory and apoptosis-inducing abilities in human tumor cells. We report that apigenin and quercetin are much more potent than kaempferol and myricetin at: (i) inhibiting chymotrypsin-like activity of purified 20S proteasome and of 26S proteasome in intact leukemia Jurkat T cells; (ii) accumulating putative ubiquitinated forms of two proteasome target proteins, Bax and Inhibitor of nuclear factor kappabeta-alpha in Jurkat T cells and (iii) inducing activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Jurkat T cells. The proteasome-inhibitory abilities of these compounds correlated with their apoptosis-inducing potencies. Results from computational modeling of the potential interactions of these flavonoids to the chymotrypsin site (beta5 subunit) of the proteasome were consistent with the obtained proteasome-inhibitory activities. We found that the C(4) carbon may be a site of nucleophilic attack by the OH group of N-terminal threonine of proteasomal beta5 subunit and that the C(3) hydroxyl may alter the ability of these flavonoids to inhibit the proteasome. Finally, apigenin neither effectively inhibited the proteasome activity nor induced apoptosis in non-transformed human natural killer cells. Our results suggested that the proteasome may be a target of these dietary flavonoids in human tumor cells and that inhibition of the proteasome by flavonoids may be one of the mechanisms responsible for their cancer-preventive effects.     

三肽四氮唑类20S蛋白酶体抑制剂的设计、合成与活性研究

泛素-蛋白酶体途径是细胞内降解蛋白质的一种主要方式,由20S蛋白酶体来完成蛋白质的降解。本文在已经报道的肽类抑制剂的基础上,设计合成了一类三肽四氮唑化合物,通过1H NMR、MS以及元素分析对化合物结构进行了表征。活性评价结果表明,有3个目标化合物(6b、6d和6h)具有较好的抑制20S蛋白酶体类胰凝乳蛋白酶的活性。分子对接研究显示,这类新型C端基肽类化合物能通过与活性位点非共价相互作用而与蛋白酶体结合。     

Targeting the proteasome pathway

Background: The ubiquitin–proteasome pathway functions as a main pathway in intracellular protein degradation and plays a vital role in almost all cellular events. Various inhibitors of this pathway have been developed for research purposes. The recent approval of bortezomib (PS-341, Velcade^®), a proteasome inhibitor, for the treatment of multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and other components of the ubiquitin–proteasome pathway. Objectives: We review the current understanding of the ubiquitin–proteasome pathway and inhibitors targeting this pathway, including proteasome inhibitors, as candidate drugs for chemical therapy. Methods: Preclinical and clinical data for inhibitors of the proteasome and the ubiquitin–proteasome pathway are discussed. Conclusions: The proteasome and other members in the ubiquitin–proteasome pathway have emerged as novel therapeutic targets.     

Recent advances in proteasome inhibitor discovery

Introduction: Proteasome inhibition is a quickly advancing subject of research and has a significant potential to become a potent therapeutic modality for many diseases and disorders. The aim of this review is to present the reader with the variety of approaches to the proteasome inhibitor discovery as well as highlight the diversity of scaffolds being considered for this task.

Areas covered: This review focuses on current developments in proteasome inhibitor discovery, including an account of research efforts covered in the literature from the years 2009 – 2012, although some of the earlier work is also mentioned. Specifically, presented are the type of experiments performed, the compounds and compound families investigated along with their activities and assessment for potential therapeutic value. In particular, authors highlight different paths to discovery of the proteasome inhibitors such as screening of large libraries, repurposing of existing therapeutics, development of compounds with known proteasome inhibitory activities as well as utilizing novel scaffolds.

Expert opinion: Discovery of therapeutically successful proteasome inhibitors depends on a number of factors and demands a multipronged approach. Screening protocols, choice of assays, desired mode of action, selection of a binding pocket, targeting and delivery strategy, all require careful consideration when attempting to target the proteasome.