斑马鱼Abcc2介导微囊藻毒素解毒功能探究

研究克隆了斑马鱼（Danio rerio）abcc2基因序列,探讨了其在微囊藻毒素（MC-LR）解毒中的潜在功能。结果表明:斑马鱼abcc2具有同哺乳动物ABCC2相似的介导荧光底物MCB外排的转运活性,MC-LR处理可显著诱导其在斑马鱼幼体中的转录表达;过表达Abcc2蛋白能显著增强ZF4细胞和斑马鱼胚胎对MC-LR的耐受性;Abcc2作为MC-LR的主要耐受因子,在组织防御和有毒物质的排泄中起重要作用,但其解毒功能还不清楚。研究结果为进一步揭示鱼类抗MC-LR积累的分子机理及培育低MC-LR残留的养殖新品种提供理论基础。     

PURA基因的结构与功能研究进展

富含嘌呤元件结合蛋白α(purine-rich element binding protein alpha,PURα)基因编码富含嘌呤的DNA和RNA结合蛋白。PURα在进化过程中高度保守。PURα蛋白既能直接或间接与DNA结合发挥转录因子的作用,促进或抑制下游基因的转录,也能通过与m RNA结合并影响其转运和翻译。PURα的结构变异与神经系统发育和HIV感染等多种疾病相关。近年发现PURα的结构和功能变异也与肿瘤发生发展相关。PURα通过影响DNA损伤修复而影响肿瘤细胞对化疗药物的敏感性。作为转录因子,PURα影响肿瘤相关基因的表达。PURA基因突变和PURα蛋白结构变异与肿瘤发生及肿瘤耐药性相关。     

转运体ABCC4和ABCC5介导的肿瘤多药耐药研究进展

：ABCC4、ABCC5是ABCC（ATP-binding cassette transporter family class C, ABCC）蛋白转运体家族的成员,介导众多内源性代谢产物和外源性药物从细胞内向外转运。ABCC4和ABCC5在体内分布广泛,参与机体对药物和内、外源物质的吸收、分布和排泄等。ABCC4、ABCC5的一些突变会引起转运体表达、功能的改变和机体对药物反应的改变。近年研究发现ABCC4、ABCC5与某些肿瘤的多药耐药相关,转运体的过表达可以引起肿瘤细胞对多种肿瘤化疗药物的耐药性,导致临床化疗效果不佳。本文就转运体ABCC4和ABCC5介导的肿瘤多药耐药研究进展进行综述。     

转运体ABCC4和ABCC5 介导的肿瘤多药耐药研究进展

ABCC4、ABCC5 是ABCC（ATP-binding cassette transporter family class C, ABCC）蛋白转运体家族的成员,介导众多内源性 代谢产物和外源性药物从细胞内向外转运。ABCC4和ABCC5 在体内分布广泛,参与机体对药物和内、外源物质的吸收、分布和 排泄等。ABCC4、ABCC5 的一些突变会引起转运体表达、功能的改变和机体对药物反应的改变。近年研究发现ABCC4、ABCC5 与某些肿瘤的多药耐药相关,转运体的过表达可以引起肿瘤细胞对多种肿瘤化疗药物的耐药性,导致临床化疗效果不佳。本文就 转运体ABCC4和ABCC5 介导的肿瘤多药耐药研究进展进行综述。     

胃癌HER2蛋白表达及基因拷贝分析

目的：探讨胃癌组织HER2蛋白表达和基因拷贝数增加频率,以及蛋白表达与基因拷贝数之间的相关性。方法：分别采用免疫组织化学（1HC）和显色原位杂交（ClSH）方法,检测80例青岛人胃癌组织中HER2蛋白表达和基因扩增情况。结果：HER2免疫组织化学0者51例、1＋者12例、2＋者12例、3＋者5例。通过CISH分析：这组患者中HER2基因拷贝数扩增者共7例（8．8％）,其中包括基因临界扩增3例（3．8％）。HER2的蛋白表达与基因拷贝数增加相关（P〈0．05）,这两个指标与肿瘤分化程度相关。HER2免疫组织化学3＋和基因扩增结果一致,与乳腺癌相似。结论：这组患者中,具有较高的HER2蛋白表达和基因拷贝数增加的比率,基因扩增可能是其蛋白过表达的主要分子机制。     

氯虫苯甲酰胺胁迫下小菜蛾ABCC1～ABCC5 mRNA的表达特征

【目的】为探讨小菜蛾Plutella xylostella ABC转运蛋白(ATP-binding cassette transporter)与氯虫苯甲酰胺代谢的关系【方法】采用实时荧光定量RT-PCR技术,测定了氯虫苯甲酰胺敏感和抗性小菜蛾及用LC50剂量的氯虫苯甲酰胺处理敏感种群不同时间后ABCC1~ABCC5 mRNA的表达量;并检测了其中过表达的ABCC3~ABCC5在小菜蛾不同发育阶段和4龄幼虫不同组织中的表达量。【结果】所检测的5个ABCC基因在氯虫苯甲酰胺抗性品系中均上调表达,最高为敏感品系的2倍;LC50的氯虫苯甲酰胺处理后ABCC3、ABCC4和ABCC5 mRNA的表达量分别上调2.16倍、2.81倍和1.85倍,ABCC1和ABCC2则分别下调为对照组的65.8%和37.2%。ABCC3的mRNA在幼虫期和雄性成虫中表达量显著高于其它时期;ABCC4的mRNA在各发育阶段表达量差异不大,其中在预蛹期最高;ABCC5的mRNA在3龄幼虫中表达量显著高于其它龄期。在4龄幼虫各组织中,ABCC3、ABCC4和ABCC5在中肠中的表达量均显著高于其它组织。【结论】小菜蛾ABCC3、ABCC4和ABCC5可能在其对氯虫苯甲酰胺的代谢中起作用,该研究结果为进一步探究小菜蛾对氯虫苯甲酰胺的抗性与ABCC1~ABCC5的关系奠定了基础。     

类泛素蛋白ISG15及其共价酶修饰系统与肿瘤

干扰素刺激基因15(interferon-stimulated gene 15,ISG15)编码的ISG15蛋白是最早发现的类泛素修饰蛋白。不仅干扰素和病毒感染能诱导该基因的表达,一些抗肿瘤药物也能刺激ISG15的表达。近年来,ISG15及其修饰系统与肿瘤的关系备受关注。研究证实,ISG15的激活酶E1即UBE1L与某些肿瘤的抑制有关,而其他一些成分如ISG15解聚酶UBP43则与肿瘤发生发展有关。此外,ISG15高表达与肿瘤的转移有关,ISG15对化疗药物的敏感性也有影响。本文较为全面地阐述ISG15及其修饰系统在肿瘤抑制或发生发展中的生物学作用,这将增强对ISG15与肿瘤关系的基础性认识,并为发展新的肿瘤靶向性治疗提供理论依据。     

核糖体蛋白基因表达与肿瘤的关系

核糖体蛋白是组成核糖体的主要成分,在细胞内蛋白质生物合成中发挥重要作用。近来人们发现,核糖体具有参与DNA修复、细胞发育调控和细胞分化等核糖体外功能。并且在胃癌、结直肠癌、食管癌和肝癌等肿瘤组织中一些核糖体蛋白基因高表达,通过对肿瘤组织中核糖体蛋白基因高表达的深入研究,可以进一步阐明肿瘤发生、发展的机制,了解核糖体蛋白基因高表达在恶性肿瘤中的作用,为肿瘤的基因诊断和基因治疗开辟一个新的研究领域。     

S100A4蛋白与肿瘤血管生成的研究进展

肿瘤血管生成是指肿瘤细胞诱导的微血管生长以及肿瘤中血液循环建立的过程。重要脏器的转移是恶性肿瘤致死的主要原因,而肿瘤生长、转移和复发都依赖于肿瘤血管生成.S100A4基因是近几年发现的一种具有促肿瘤作用的基因,该基因编码一种钙离子结合调节蛋白,通过与钙离子结合在肿瘤发生和发展中起重要作用。目前研究认为该蛋白在肿瘤的侵袭和转移中有促血管生成作用.本文主要就S100A4与肿瘤血管生成的有关研究进展加以综述。     

MicroRNA在肿瘤发生发展和诊断中作用的研究进展

microRNA是一类由内源基因编码的长度约为18-25个核苷酸的非编码单链RNA分子,可以与靶基因mRNA的3'非编码区结合,通过降解靶m RNA或(和)抑制靶m RNA转录后翻译调节靶蛋白的生成,从而发挥其生物学作用。目前,在人体基因组内发现的microRNA已经超过2500多个,可能调节着人类1/3的基因,在维持正常干细胞功能、调控细胞增殖分化及恶性肿瘤发生过程中均起重要作用。既往的研究表明microRNA与基因之间相互调控的失衡导致肿瘤的发生。从分子水平上研究microRNA与肿瘤发生的关系,检测microRNA与肿瘤相关基因表达情况的改变,分析肿瘤组织和血清中microRNA表达量与肿瘤分型的关系,将有利于肿瘤的病因学研究,早期发现和肿瘤治疗及预后判断。本文主要就microRNA在肿瘤发生发展和诊断中作用的研究进展进行了综述。     

Molecular model of the outward facing state of the human multidrug resistance protein 4 (MRP4/ABCC4)

ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4, ABCC4) is involved in multidrug resistance (MDR), which is an increasing challenge to the treatment of cancer and infections. We have constructed a molecular model of ABCC4 based on the outward facing Sav1866 crystal structure using molecular modeling techniques. Amino acids reported by ICMPocketFinder to take part in substrate translocation were among others Glu103 (TMH1), Ser328 (TMH5), Gly359 (TMH6), Arg362 (TMH6), Val726 (TMH7), and Leu987 (TMH12), and their corresponding amino acids in ABCB1 (P-glycoprotein) have been reported to be involved in drug binding according to site-directed mutagenesis studies. The ABCC4 model may be used as a working tool for experimental studies on ABCC4 and design of more specific membrane transport modulating agents (MTMA).     

Molecular Docking Simulations Provide Insights in the Substrate Binding Sites and Possible Substrates of the ABCC6 Transporter

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein (ABCC6), primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS) and leukotriene C4 (LTC4) were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3) was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.     

Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression

Background  

The ABCC5 gene encodes an organic anion pump of the ATP-binding cassette (ABC) transporter family, subclass C. The exact physiological function of ABCC5 however is not known. Here, we have isolated three novel ABCC5 splice variants and characterized their role in the regulation of ABCC5 gene expression.     

Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells

Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E²⁹⁵EVEALI³⁰¹, which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L³⁰⁰ and I³⁰¹ residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E²⁹⁵, E²⁹⁶, and E²⁹⁸ residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.     

Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure

The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published cross-linking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5), Ile340 (TMH6), Phe343 (TMH6), Phe728 (TMH7), and Val982 (TMH12), form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the site-directed mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.     

The amino terminus of the human multidrug resistance transporter ABCC1 has a U-shaped folding with a gating function

Multidrug resistance is a serious problem in successful cancer chemotherapy. Studies using model cell lines have demonstrated that overexpression of some members of the ATP-binding cassette (ABC) transporter superfamily, such as ABCC1, causes enhanced efflux and, thus, decreased accumulation of multiple anticancer drugs, which leads to increased cell survival. Unlike most other ABC transporters, ABCC1 has an additional membrane-spanning domain (MSD0) with a putative extracellular amino terminus of 32 amino acids. However, the function of MSD0 and the role of the extracellular amino terminus are largely unknown. In this study, we examined the structural folding and the function of the amino terminus. We found that it has a U-shaped folding with the bottom of the U-structure facing cytoplasm and both ends in extracellular space. We also found that this U-shaped amino terminus probably functions as a gate to regulate the drug transport activity of human ABCC1.     

cGMP transport by vesicles from human and mouse erythrocytes

cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.     

Collagen/β1 integrin signaling up-regulates the ABCC1/MRP-1 transporter in an ERK/MAPK-dependent manner

The mechanisms by which β1 integrins regulate chemoresistance of cancer cells are still poorly understood. In this study, we report that collagen/β1 integrin signaling inhibits doxorubicin-induced apoptosis of Jurkat and HSB2 leukemic T-cells by up-regulating the expression and function of the ATP-binding cassette C 1 (ABCC1) transporter, also known as multidrug resistance-associated protein 1. We find that collagen but not fibronectin reduces intracellular doxorubicin content and up-regulates the expression levels of ABCC1. Inhibition and knockdown studies show that up-regulation of ABCC1 is necessary for collagen-mediated reduction of intracellular doxorubicin content and collagen-mediated inhibition of doxorubicin-induced apoptosis. We also demonstrate that activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway is involved in collagen-induced reduction of intracellular doxorubicin accumulation, collagen-induced up-regulation of ABCC1 expression levels, and collagen-mediated cell survival. Finally, collagen-mediated up-regulation of ABCC1 expression and function also requires actin polymerization. Taken together, our results indicate for the first time that collagen/β1 integrin/ERK signaling up-regulates the expression and function of ABCC1 and suggest that its activation could represent an important pathway in cancer chemoresistance. Thus simultaneous targeting of collagen/β1 integrin and ABCC1 may be more efficient in preventing drug resistance than targeting each pathway alone.