Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.
Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.
复制标题
DOI：
10.1021/mp500230b
复制
发表时间：
2014-10-06
影响因子：
4.9
通讯作者：
Cannon RD
中科院分区：
文献类型：
--
作者：
Keniya MV;Holmes AR;Niimi M;Lamping E;Gillet JP;Gottesman MM;Cannon RD
ABCB 5是一种ATP结合盒（ABC）转运蛋白，在黑色素瘤细胞中高度表达，可能通过抗癌药物的外排导致黑色素瘤对化疗的极端耐药性。我们的目标是确定我们是否可以在模型酵母酿酒酵母中功能性表达人ABCB 5，以证明在缺乏来自其他人转运蛋白的背景泵活性的情况下ABCB 5的外排功能。异源表达也将促进针对这一重要靶点的药物发现。将编码ABCB 5序列的DNA克隆到S.酿酒酵母菌株，其中七个内源性ABC转运蛋白已被删除。通过使用特异性抗ABCB 5抗体和交叉反应性抗ABCB 1抗体的免疫检测来监测酵母细胞中的蛋白质表达。在酵母生长测定中，通过确定与宿主酵母菌株相比，重组酵母细胞对已知泵底物的抗性是否增加来测量ABCB 5在重组酵母细胞中的功能。在酵母中制备了三个ABCB 5构建体。一种来源于ABCB 5-β mRNA，其在人体组织中高度表达，但却是典型的全长ABC转运蛋白的截短物。两个构建体含有全长ABCB 5序列：来自cDNA的天然序列或与S.啤酒。通过免疫检测确认所有三种构建体在酵母中的表达。表达的密码子协调的全长ABCB 5 DNA赋予增加的电阻，相对于宿主酵母菌株，推定的底物罗丹明123，柔红霉素，四甲基罗丹明，FK 506，或氯吉林。我们的结论是全长ABCB 5可以在S.酿酒酵母并赋予耐药性。
ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.
登录
查看更多内容
影响因子：
4.9
作者：
Holmes, Ann R.;Keniya, Mikhail V.;Cannon, Richard D.
通讯作者：
Cannon, Richard D.
影响因子：
4.8
作者：
Frank, NY;Pendse, SS;Frank, MH
通讯作者：
Frank, MH
DOI：
10.1073/pnas.0600213103
发表时间：
2006-06-27
影响因子：
11.1
作者：
Chen, Kevin G.;Valencia, Julio C.;Gottesman, Michael M.
通讯作者：
Gottesman, Michael M.
影响因子：
11.2
作者：
Frank, NY;Margaryan, A;Frank, MH
通讯作者：
Frank, MH
DOI：
10.1126/science.1168750
发表时间：
2009-03-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Aller SG;Yu J;Ward A;Weng Y;Chittaboina S;Zhuo R;Harrell PM;Trinh YT;Zhang Q;Urbatsch IL;Chang G
通讯作者：
Chang G