The Role of Half-Transporters in Multidrug Resistance

ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance. Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance. These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1. This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan. At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement. Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.     

Role of mycobacterial efflux transporters in drug resistance: an unresolved question

Two mechanisms are thought to be involved in the natural drug resistance of mycobacteria: the mycobacterial cell wall permeability barrier and active multidrug efflux pumps. Genes encoding drug efflux transporters have been isolated from several mycobacterial species. These proteins transport tetracycline, fluoroquinolones, aminoglycosides and other compounds. Recent reports have suggested that efflux pumps may also be involved in transporting isoniazid, one of the main drugs used to treat tuberculosis. This review highlights recent advances in our understanding of efflux-mediated drug resistance in mycobacteria, including the distribution of efflux systems in these organisms, their substrate profiles and their contribution to drug resistance. The balance between the drug transport into the cell and drug efflux is not yet clearly understood, and further studies are required in mycobacteria.     

ABC细胞膜转运蛋白及其介导的细胞多药耐药研究进展

ABC细胞膜转运蛋白是一个能转运多种底物的蛋白质家族,其在宿主对异物的防御机制和肿瘤细胞对抗癌药物的耐药性中发挥重要作用。ABC转运蛋白能将已进人细胞的外源性物质从胞内泵出胞外,是造成肿瘤细胞多药耐药的主要原因,其基因表达水平与细胞内药物浓度和耐药程度密切相关。近年来,肿瘤细胞多药耐药性研究炙手可热。我们简要综述ABC细胞膜转运蛋白的特点、分布、表达及其介导的细胞多药耐药方面的研究进展。     

真菌的多向耐药性ABC转运蛋白

真菌的多向耐药性ABC转运蛋白(ATP-binding cassette transporters)是导致多药耐药性和抗真菌药物治疗效果明显下降的主要原因。文章对酿酒酵母(Saccharomyces cerevisiae)和主要致病真菌白色假丝酵母(Candida albicans)、新型隐球酵母(Cryptococcus neoformans)和烟曲霉(Aspergillus fumigatus)中的多向耐药性ABC转运蛋白的种类、药物外排机制以及基因表达调控网络的研究进展作一综述,为深入了解真菌的多向耐药性机制以及探讨克服多向耐药性的策略和提高药效提供参考。     

半分子转运蛋白ABCG2的肿瘤多药耐药性研究及其应用

肿瘤常对临床上传统使用的多种化学治疗显示其内源性或获得性的药物耐受性即多药耐药性(multidrug resistance,MDR)．这种多药耐药性主要是由一类称为ABC(ATP-binding cassette)转运体蛋白超家族的跨膜蛋白引起的,它们结合并利用水解ATP提供的能量来转运药物,导致肿瘤细胞呈现抗药性．半分子转运蛋白ABCG2是近年来才发现的可归于ABC转运体大家族中的一个新成员,在肠、肝、胎盘和血脑屏障等部位大量表达,与全分子转运蛋白如P-gp (P-glycoprotein)和多药耐药蛋白(multi-drug resistance protein,MRP)相似,可以主动地把具有不同化学结构和作用于细胞内不同靶位点的化疗药物泵出胞外,从而引起肿瘤对多种抗癌药物(包括最新开发的药物)产生抗性．最近的一些十分有趣的研究还表明,ABCG2可能与干细胞分化状态和保护干细胞发育过程中免受周围环境的影响有关,而且还发现,它在侧群骨髓和神经干细胞内大量存在,因此,ABCG2可能在基因治疗中作为选择性的蛋白质标记正受到研究者们的极大关注．同时,ABCG2的单核苷酸多态性影响其结合并转运不同的底物/药物．鉴于ABCG2在肿瘤抗药性研究中的重要性以及它的一些新功能的初步研究表明,对ABCG2的生物学功能和作用机理以及在医学实践中的应用研究必将受到更大的关注．主要阐述了半分子ABC转运蛋白ABCG2的发现、重要的生化特性和生理功能及其相关的新研究进展和问题．     

MRP Subfamily Transporters and Resistance to Anticancer Agents

The MRP subfamily of ABC transporters from mammals consists of at least seven members, six of which have been implicated in the transport of amphipathic anions. MRP1, MRP2, and MRP3 bear a close structural resemblance, confer resistance to a variety of natural products as well as methotrexate, and have the facility for transporting glutathione and glucuronate conjugates. MRP1 is a ubiquitously expressed efflux pump for the products of phase II of xenobiotic detoxification, while MRP2, whose hereditary deficiency results in Dubin–Johnson syndrome, functions to extrude organic anions into the bile. MRP3 is distinguished by its capacity to transport the monoanionic bile constituent glycocholate, and may function as a basolateral back-up system for the detoxification of hepatocytes when the usual canalicular route is impaired by cholestatic conditions. MRP4 and MRP5 resemble each other more closely than they resemble MRPs 1–3 and confer resistance to purine and nucleotide analogs which are either inherently anionic, as in the case of the anti-AIDS drug PMEA, or are phosphorylated and converted to anionic amphiphiles in the cell, as in the case of 6-MP. Given their capacity for transporting cyclic nucleotides, MRP4 and MRP5 have also been implicated in a broad range of cellular signaling processes. The drug resistance activity and physiological substrates of MRP6 are unknown. However, its hereditary deficiency results in pseudoxanthoma elasticum, a multisystem disorder affecting skin, eyes, and blood vessels. It is hoped that elucidation of the resistance profiles and physiological functions of the different members of the MRP subfamily will provide new insights into the molecular basis of clinical drug resistance and spawn new strategies for combating this phenomenon.     

ABC转运蛋白结构及在植物病原真菌中的功能研究进展

ABC (ATP-binding cassette)转运蛋白是最大的膜转运蛋白超家族之一,其主要功能是利用ATP水解产生的能量将底物进行逆浓度梯度运输.所有生物体都含有大量ABC蛋白. ABC蛋白位于细胞的不同空间,如细胞膜、液泡、线粒体和过氧化物酶体.通常,ABC转运蛋白由跨膜结构域(TMD)和核苷酸结合结构域(NBD)组成,分别与底物和ATP结合.NBD执行与ATP结合和水解,是ABC转运蛋白的动力引擎,TMD识别特异性配体.大多数ABC转运蛋白最初是通过研究生物体耐药性而被发现的,包括多效耐药(PDR)和多药耐药(MDR).本文对ABC转运蛋白的结构及作用机制,以及植物病原真菌中ABC转运蛋白功能的研究进展进行综述.     

猪源大肠杆菌耐药质粒图谱及耐药性分析

目的:探讨猪大肠杆菌的耐药质粒图谱、耐药性及耐药基因之间的关系。方法:从湖南省株洲、益阳的四个猪场分离出9株大肠杆菌,进行质粒电泳图谱分析、用PCR法检测耐喹诺酮类耐药基因Gyr A、Par C和耐四环素类耐药基因Tet A、Tet B,并采用Kirby-bauer法对这9株大肠杆菌进行药敏(18种抗生素)试验。结果:其中9株大肠杆菌含有三条或者三条以上的质粒条带,且其质粒谱型均不相同;9株大肠杆菌均检测出4种耐药基因Gyr A、Par C、Tet A和Tet B;9株大肠杆菌对所选用的抗生素存在不同程度的耐药性,其中7株大肠杆菌对10种或10种以上的抗生素耐药,最高对13种抗生素耐药,氨苄西林、青霉素、阿莫西林、红霉素的耐药率达100%,对四环素、多西环素的耐药率达到88.9%,而多粘菌素B、阿奇霉素、大观霉素耐药率较低。结论:耐药性与质粒条带数、耐药基因之间并无明显的相关性;猪大肠杆菌呈多重耐药之势,在治疗大肠杆菌病时最好根据药敏实验结果选用合适的抗生素。     

生物膜耐药机制研究进展

生物膜形成是微生物耐药的重要原因之一,也是许多慢性感染性疾病反复发作和难以治疗的主要原因,微生物生物膜可以通过渗透限制,改变生物膜内外微环境,形成特殊的基因型等多种方式导致耐药。生物膜耐药机制的研究已成为医学、药学、微生物学等众多领域关注的课题。就生物膜耐药机制的最新进展进行综述。     

非编码RNA与细菌耐药

细菌非编码RNA是一类新发现的基因表达调控因子,通过与靶mRNA配对,导致mRNA翻译和稳定性的变化,从而影响细胞的各种生理功能,如个体发育、翻译激活与抑制、细菌毒性等,而且一个单独的非编码RNA就能调控大量基因并对细胞生理产生深远影响。近年来诸多研究证实,非编码RNA与细菌耐药性也存在一定的关系。我们对此进行简要综述,为细菌耐药性的研究奠定基础。     

Analysis of the inhibition potential of zosuquidar derivatives on selected bacterial and fungal ABC transporters

Abstract

The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue. Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role. In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD. For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar. One of these is a compound with a new catechol acetal structure that might represent a new lead compound. Furthermore, the determination of IC₅₀ values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 0.3 and 0.9 μM. Thus the identified compounds are among the most potent inhibitors known for Pdr5. For the ABC-type efflux pumps LmrA and LmrCD from L. lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found. Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect. Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.     

400株肠球菌临床耐药性分析

了解2001~2003年肠球菌对常用抗菌药物的耐药性,为临床治疗肠球菌感染提供参考。采用Kirby-Bauer纸片扩散法进行药敏试验。结果显示:2001~2003年肠球菌属的耐药率呈上升趋势。万古霉素和替考拉宁依然是敏感性最高的抗生素。青霉素、氨苄西林和呋喃妥因对粪肠球菌具有较好的抗菌活性,敏感率分别为62.5%、67.1%和92.4%。屎肠球菌对除糖肽类抗生素以外的所有临床常用抗菌药物显示高水平耐药。可见,动态监测肠球菌的耐药状况对指导临床治疗具有重要意义。     

ABC Proteins of Leishmania

ABC proteins were first characterized in the protozoan parasite Leishmania while studying mechanisms of drug resistance. PGPA is involved in resistance to arsenite and antimonite and it most likely confers resistance by sequestering metal–thiol conjugates into an intracellular vesicle. PGPA is part of gene family with at least four more members which are in search of a function. Leishmania also contains a P-glycoprotein, homologous to the mammalian MDR1, that is involved in multidrug resistance. The ongoing genome project of Leishmania has pinpointed several novel ABC transporters and experiments are carried out to study the function of the ABC proteins in drug resistance and in host–pathogen interactions.     

The PDR-type ABC transporters AtrA and AtrG are involved in azole drug resistance in Aspergillus oryzae

For strain improvement of Aspergillus oryzae, development of the transformation system is essential, wherein dominant selectable markers, including drug-resistant genes, are available. However, A. oryzae generally has a relatively high resistance to many antifungal drugs effective against yeasts and other filamentous fungi. In the course of the study, while investigating azole drug resistance in A. oryzae, we isolated a spontaneous mutant that exhibited high resistance to azole fungicides and found that pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporter genes were upregulated in the mutant; their overexpression in the wild-type strain increased azole drug resistance. While deletion of the gene designated atrG resulted in increased azole susceptibility, double deletion of atrG and another gene (atrA) resulted in further azole hypersensitivity. Overall, these results indicate that the ABC transporters AtrA and AtrG are involved in azole drug resistance in A. oryzae.     

Protein Linkers Provide Limits on the Domain Interactions in the ABC Importer GlnPQ and Determine the Rate of Transport

GlnPQ is an ATP-binding cassette importer with a unique domain organization and intricate transport behavior. The protein has two extracytoplamic substrate-binding domains (SBDs) per membrane subunit, each with different specificity for amino acids and different spacing to the translocator domain. We determined the effect of the length and structure of the linkers, which connect the SBDs to each other and to the membrane-embedded translocator domain, on the transport by GlnPQ. We reveal that varying the linker length impacts transport in a dual manner that depends on the conformational dynamics of the SBD. Varying the linker length not only changes the time for the SBD to find the translocator (docking) but also changes the probability to release the substrate again, thus altering the transport efficiency. On the basis of the experimental data and mathematical modeling, we calculate the docking efficiency as function of linker length and lifetime of the closed conformation. Importantly, not only linker length but also features in the sequence are important for efficient delivery of substrate from SBD to the translocator. We show that the linkers provide a platform for SBD docking and are not merely flexible structures.     

Conformational Cycle of the Vitamin B12 ABC Importer in Liposomes Detected by Double Electron-Electron Resonance (DEER)

Double electron-electron resonance is used here to investigate intermediates of the transport cycle of the Escherichia coli vitamin B₁₂ ATP-binding cassette importer BtuCD-F. Previously, we showed the ATP-induced opening of the cytoplasmic gate I in TM5 helices, later confirmed by the AMP-PNP-bound BtuCD-F crystal structure. Here, other key residues are analyzed in TM10 helices (positions 307 and 322) and in the cytoplasmic gate II, i.e. the loop between TM2 and TM3 (positions 82 and 85). Without BtuF, binding of ATP induces detectable changes at positions 307 and 85 in BtuCD in liposomes. Together with BtuF, ATP triggers the closure of the cytoplasmic gate II in liposomes (reported by both positions 82 and 85). This forms a sealed cavity in the translocation channel in agreement with the AMP-PNP·BtuCD-F x-ray structure. When vitamin B₁₂ and AMP-PNP are simultaneously present, the extent of complex formation is reduced, but the short 82–82 interspin distance detected indicates that the substrate does not affect the closed conformation of this gate. The existence of the BtuCD-F complex under these conditions is verified with spectroscopically orthogonal nitroxide and Gd(III)-based labels. The cytoplasmic gate II remains closed also in the vanadate-trapped state, but it reopens in the ADP-bound state of the complex. Therefore, we suggest that the substrate likely trapped in ATP·BtuCD-F can be released after ATP hydrolysis but before the occluded ADP-bound conformation is reached.     

Multi-scale Predictions of Drug Resistance Epidemiology Identify Design Principles for Rational Drug Design

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血培养病原菌构成及耐药性分析

了解2008年至2012年哈尔滨医科大学附属第一医院血培养常见病原菌构成及耐药性。对血培养分离出的病原菌进行鉴定,用MIC法、K-B法测定药物敏感性,用WHONET 5.6统计软件进行细菌菌谱及耐药性分析。共分离出病原菌4 245株;其中凝固酶阴性葡萄球菌最多,947株占22.3%;其次为大肠埃希菌822株,肺炎克雷伯菌520株,鲍曼不动杆菌195株,金黄色葡萄球菌142株;耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)和耐甲氧西林金黄色葡萄球菌(MRSA)检出率分别为81.1%、38.8%,未发现耐万古霉素、利奈唑胺及替考拉宁的凝固酶阴性葡萄球菌和金黄色葡萄球菌;大肠埃希菌及肺炎克雷伯菌产ESBLs检出率分别为55.2%、53.8%,对头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、头孢替坦、头孢西丁、阿米卡星的耐药率较低,对碳青霉烯类抗菌药物存在耐药现象;鲍曼不动杆菌对抗菌药物的耐药率普遍较高。及时、准确地对血培养分离出的病原菌进行监测,以便指导临床合理用药,控制耐药株的产生。