蛋白酶体抑制剂Syrbactins家族的生物合成

微生物来源的syrbactins属于十二元内酰胺短肽类化合物,包括丁香霉素(syringolins)、滑杆菌素(glidobactins)、cepafungins和luminmycins等。其中,syringolins、glidobactins、luminmycins等几个化合物生物合成基因簇已被克隆、测序和异源表达。研究发现它们的十二元内酰胺环骨架都是由非核糖体肽合成酶(NRPS)-聚酮合酶(PKS)复合体采用模块化组装的方式,将系列底物组装而成。这类化合物因具有优异的蛋白酶体抑制活性而受到广泛关注。本文从syrbactins的分子结构、生物合成、作用机理等几个方面进行综述,介绍了近年来syrbactins的研究进展。     

蛋白酶体抑制剂MG132对青扦花粉萌发和花粉管生长的影响

泛素／蛋白酶体系统（UPP）是真核细胞内蛋白质选择性降解的主要途径,而蛋白酶体是UPP中蛋白质降解的场所。本文应用细胞学、统计学方法以及FTIR技术研究了蛋白酶体抑制剂MG132对青扦（Pecea wilsonii）花粉萌发、花粉管生长的影响。结果表明：MG132显著抑制青扦花粉萌发和花粉管生长,并导致花粉管形态异常,主要表现为花粉管亚顶端出现液泡化,并且液泡随着培养时间的延长而扩大到整个花粉管,花粉管濒临死亡;而DMSO以及非蛋白酶体抑制剂E-64不产生类似结果;半薄切片结果表明,MG132处理后不仅花粉管细胞质发生液泡化,生殖细胞也发生液泡化;FTIR分析进一步表明,MG132处理后,花粉管顶端的细胞壁蛋白和果胶质含量大幅度下降。上述结果表明：MG132通过抑制蛋白酶体活性显著影响青扦花粉萌发及花粉管生长;UPP在青扦花粉萌发、花粉管极性生长模式的建立和维持过程中起重要作用;抑制蛋白酶体活性将导致青扦花粉管的程序性死亡。     

用于多发性骨髓瘤的首个口服蛋白酶体抑制剂 Ixazomib

自武田（Takeda）公司产品硼替佐米（bortezomib,Velcade）于 2003 年获准上市以来,蛋白酶体抑制剂已成为多发性骨髓瘤 治疗的主要用药。武田公司日前又在美国和欧洲递交了同类新药 ixazomib 的上市申请,使其上市进程更近一步,而该药物可能成为此类药 物中的首个口服治疗药物。虽然武田公司非常希望能在 2017 年 5 月 Velcade 专利到期前大力推动 ixazomib 替代 Velcade 的进程,以维 护其在多发性骨髓瘤治疗市场的地位,但要让医生接受这一新型口服药物,还需要更多临床数据,尤其是一线治疗数据的支持。主要介绍 ixazomib 用于治疗复发性多发性骨髓瘤以及作为多发性骨髓瘤一线治疗药物的若干临床研究,解析并展望多发性骨髓瘤治疗市场。     

一种基于绿色荧光蛋白的蛋白酶体抑制剂细胞筛选模型

为建立基于绿色荧光蛋白(GFP)的药物筛选模型,并用此模型从包括中药提取物在内的化合物中筛选新型蛋白酶体抑制剂,本研究构建了pGC-E1-ZU1-GFP融合蛋白慢病毒表达载体并感染A549细胞,筛选稳定表达细胞株,用已知蛋白酶体抑制剂PS-341处理细胞,荧光显微镜检测处理前后细胞GFP水平变化。结果获得了稳定表达pGC-E1-ZU1-GFP的A549细胞,这些细胞用PS-341处理24h后用荧光显微镜检测,发现细胞绿色荧光强度相对于对照组明显增强。利用这一模型对一些化合物进行筛查,发现了一些新的蛋白酶体抑制剂。     

蛋白酶体结构和功能研究进展

蛋白酶体是真核细胞内依赖ATP的蛋白质水解途径的重要成分,负责大多数细胞内蛋白质的降解. 20 S蛋白酶体有多种肽酶活性,其活性位点为Thr. 19 S复合物与20 S蛋白酶体结合成为26 S复合物,能降解泛素化蛋白.近几年来,蛋白酶体的分子组成、亚基、生化机理、胞内功能等方面的研究取得了明显进展.     

蓝光和蛋白酶体抑制剂MG132对蛹虫草形态的影响

蓝光是影响生物生长发育过程的重要因素,同时生物个体的生长发育过程中不断有蛋白质的泛素化降解。采用蓝光和蛋白酶体抑制剂MG132处理蛹虫草菌,观察蛹虫草菌落、菌丝体和子实体形态的变化。研究结果表明,黑暗条件下正常生长的菌落边缘圆滑一致,菌落之间融合无边界;MG132处理后,菌落之间出现明显的界限,边缘菌丝稀疏。蓝光条件下无MG132处理时,菌落较为单薄,转色明显;MG132处理时,菌落中间橙色,边缘颜色变淡。扫描电镜观察,黑暗条件下无MG132处理的菌丝自然弯曲,菌丝表面较为光滑,菌丝粗细差别不大且分枝较少,分生孢子表面较光滑。黑暗条件下MG132处理菌丝体,菌丝较直且分枝较多,菌丝容易断裂,分生孢子表面仍较光滑。蓝光条件下菌丝体弯曲减少,菌丝表面较为粗糙,单条菌丝常出现部分区段膨大呈不规则状,菌丝粗细差异较大,菌丝断裂较多;分生孢子呈扁平的椭圆状,表面有纹理,且粗糙。蓝光条件下MG132处理的菌丝体,菌丝较直,表面粗糙,菌丝整体变得更细,菌丝断裂较多;分生孢子呈不规则形状,表面纹理更深,粗糙有褶皱。此外,MG132可导致蛹虫草子实体畸形生长。结果表明,蓝光和MG132均可以影响蛹虫草的形态变化。     

蛋白酶体抑制剂MG132的浓度对人白血病细胞K562凋亡的影响

探讨蛋白酶体抑制剂MG132 在诱导人白血病K562细胞凋亡过程中作用.分别以不同浓度的蛋白酶体抑制剂MG132 处理人白血病细胞K562,通过MTT法检测K562细胞活力,应用Annexin Ⅴ和PI 双染的细胞流式法检测K562细胞凋亡率和细胞内活性氧(ROS) 水平,应用酶标仪法检测K562细胞内Caspase- 3活性变化的情况.结果表明,随着MG132浓度的增加,各个指标与对照组比较差异均有显著性(P<0.05):K562细胞增殖明显受到抑制;细胞凋亡率明显增加,且当MG132浓度为900 nmol/L时,细胞凋亡率达36.5 %;同时,ROS 水平和caspase- 3活性明显升高.因次,蛋白酶体抑制剂MG132可显著抑制人白血病细胞K562增殖并促进其凋亡.     

蛋白酶体调节颗粒的结构生物学特征及其功能

蛋白酶体调节颗粒(regulatory particle,RP)参与调控许多重要信号通路的蛋白质降解,在维持细胞稳态中发挥重要作用.近年来,真核细胞蛋白酶体在癌症治疗中的作用机制及药物研发已引起了广泛关注,并有3种蛋白酶体抑制剂已用于临床治疗.随着蛋白酶体功能研究的不断深入,以及晶体学和冷冻电镜技术在其结构生物学研究中...     

植物泛素/26S蛋白酶体通路的生理功能和分子生物学

介绍泛素/26S蛋白酶体通路的分子生物学研究最新进展,并对其同源异形性及在植物激素信号、抗病和衰老、光形态建成、植物逆境信号转导中的功能进行了讨论.     

细菌分裂蛋白及FtsZ抑制剂的研究进展

近年来,由于抗生素的滥用,耐药性细菌广泛出现,对人体健康的威胁日益严峻.随着临床用药的选择不断减少,迫切需要开发新的抗菌药物,特别是新作用机制的抗菌分子来对抗出现的耐药菌.细胞分裂温度敏感.突变体Z (filamenting temperature-setnsitive mutant Z,FtsZ)作为细菌分裂的必需蛋白质,是目前研究最热门的作用靶点之一.FtsZ是一高度保守的蛋白质,在大多数原核细胞的细胞分裂中发挥着关键作用,本文回顾了细菌分裂蛋白的结构特点及其生物学功能,并综述了以FtsZ为靶点的抗菌药物研究的进展.     

Cell-specific inhibition of paramyxovirus maturation by proteasome inhibitors

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)-infected LLC-MK2 cells with 50 microM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose-dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV-like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC-MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell-specific and partly virus-specific.     

Differential effects of proteasome inhibitors on cell cycle and apoptotic pathways in human YT and Jurkat cells

Herein, we report differential effects of various proteasome inhibitors including clasto-lactacystin-beta-lactone, (-)-epigallocatechin gallate (EGCG) and N-Acetyl-Leu-Leu-Norleu-al (LLnL) on proteasomal activities of YT and Jurkat cells, human natural killer (NK) and T cell lines, respectively. The inhibitory rates of these inhibitors on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact cells did not show significant differences between the two cell lines. The viability of both cell lines was reduced in the presence of LLnL. Subsequent studies revealed a reduction of the mitochondrial membrane potential and caspase-3 activation in these two cell lines upon treatment with proteasome inhibitors; however, caspase-3 activation occurred much earlier in Jurkat cells. Cell cycle analysis indicated a sub-G(1) apoptotic cell population in Jurkat cells and G(2)/M arrest in YT cells after they were treated by proteasome inhibitors. Moreover, pretreatment of YT cells by a caspase inhibitor followed by a proteasome inhibitor did not increase the percentage of G(2)/M phase cells. In addition, accumulation of p27 and IkappaB-alpha was detected only in Jurkat cells, but not YT cells. In summary, proteasome inhibitors may act differentially in cell cycle arrest and apoptosis of tumors of NK and T cell origin, and may have similar effects on normal NK and T cells.     

Capillary electrophoresis for screening of 20S proteasome inhibitors

A method for studying 20S proteasome inhibitors by capillary electrophoresis (CE) has been developed. Proteasome plays a fundamental role in degrading key regulatory proteins. The 20S proteasome can degrade intrinsically disordered proteins in an ATP-independent manner without additional “helper” molecules. The discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy. In this study, the inhibitory effects of MG132 and MG115 on the 20S proteasome were evaluated by CE for the first time. The optimized CE conditions were as follows: fused-silica capillary of 30 cm effective length and 75 μm internal diameter, pressure injection of 0.5 psi for 5 s, 50 mM Hepes buffer (pH 7.6) with 2% dimethyl sulfoxide, constant voltage of 20 kV, and detection wavelength at 340 nm. Also, the new method was used to study the inhibitory effects of 30 novel peptidyl vinyl ester derivatives of MG132. The 50% inhibition concentrations (IC₅₀ values) of MG132 and MG115 were 40.0 and 84.7 nM, respectively. Two new compounds, XP32 and XP35, showed considerable inhibitory effects on the 20S proteasome. When the concentrations of them were fixed at 172 nM, their inhibition rates were 36.2% and 29.1%, respectively. The results showed that the CE method was powerful, sensitive, and fast and required little sample. It could be employed as one of the reliable drug screening methods for 20S proteasome inhibitors.     

Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.     

The power of proteasome inhibition in multiple myeloma

ABSTRACT

Introduction: Proteasome inhibitors (PIs) are therapeutic backbones of multiple myeloma treatment, with PI-based therapies being standards of care throughout the treatment algorithm. Proteasome inhibition affects multiple critical signaling pathways in myeloma cells and interacts synergistically with mechanisms of action of other conventional and novel agents, resulting in substantial anti-myeloma activity and at least additive effects.

Areas covered: This review summarizes the biologic effects of proteasome inhibition in myeloma and provides an overview of the importance of proteasome inhibition to the current treatment algorithm. It reviews key clinical data on three PIs, specifically bortezomib, carfilzomib, and ixazomib; assesses ongoing phase 3 trials with these agents; and looks ahead to the increasingly broad role of both approved PIs and PIs under investigation in the frontline and relapsed settings.

Expert commentary: Progress to date with PIs in multiple myeloma has been impressive, but there remain unmet needs and challenges, as well as increasing opportunities to optimize the use of these agents. Understanding discrepancies between PIs in terms of efficacy and safety profile is a key goal of ongoing research, along with proteomics-based efforts to identify potential biomarkers of sensitivity and resistance, thereby enabling increasingly personalized treatment approaches in the future.     

Screening of marine actinobacteria for amylase enzymes inhibitors

Amylase inhibitor producing actinobacteria were isolated and characterized from terrestrial environment and there is no much report found from marine environment, hence in the present study, 17 strains isolated from the rhizosphere sediments of mangroves were tested for their amylase inhibition ability. Seawater requirement test for the growth of actinobacteria found that the strains SSR-3, SSR-12 and SSR-16 requires at least 50% and SSR-6 requires at least 25% seawater for their growth. The inhibition activity of both prokaryotic and eukaryotic amylase was tested by using Bacillus subtilis and Aspergillus niger. The maximum amylase activity (40mm) produced by the A. niger was taken as positive control, when the test actinobacteria strains grown in the medium they inhibited amylase activity and was evidenced by the reduction in inhibition zone (14–37 mm) similarly the amylase produced by the Bacillus subtilis was also recorded maximum (35 mm) amylase activity and was taken as positive control, and the test atinobacterial strains reduced enzyme action(12–33 mm) it varied levals. This indicates that the actinobacteria strains were controlled amylase enzyme activity in both the cases. The strain SSR-10 was highly effective and SSR-8 was less effective in inhibiting eukaryotic amylase produced by A. niger. The strain SSR-2 was effective and SSR-6 showed very less effect in inhibiting the prokaryotic amylase produced by the B subtilis.     

Synthesis and activity of isoxazoline vinyl ester pseudopeptides as proteasome inhibitors

The ubiquitin–proteasome pathway (UPP) influences essential cellular functions including cell growth, differentiation, apoptosis, signal transduction, antigen processing and inflammatory responses. The main proteolytic component of the UPP is the 26S proteasome, which is responsible for the turnover of many cellular proteins and represents an attractive target for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Natural and synthetic proteasome inhibitors having different chemical structures and potency have been discovered. We report herein the synthesis, proteasome inhibition and modelling studies of novel C‐terminal isoxazoline vinyl ester pseudopeptides. Some new compounds that contain a C‐terminal extended conjugation inhibit β1 and especially β5 proteasomal catalytic subunits with IC₅₀ values ranging from 10 to 100 µm . These results will permit further optimization based on these structural moieties to develop more active and selective molecules. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.