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Expression and copper binding properties of the N-terminal domain of copper P-type ATPases of African trypanosomes.
Molecular and Biochemical Parasitology ( IF 1.4 ) Pub Date : 2019-11-18 , DOI: 10.1016/j.molbiopara.2019.111245
Murtala Bindawa Isah 1 , J P Dean Goldring 1 , Theresa H T Coetzer 1
Affiliation  

Copper is an essential component of cuproproteins but can be toxic to cells, therefore copper metabolism is very carefully regulated within cells. To gain insight into trypanosome copper metabolism, Trypanosoma spp. genomic databases were screened for the presence of copper-containing and -transporting proteins. Among other genes encoding copper-binding proteins, a copper-transporting P-type ATPase (CuATPase) gene was identified. Sequence and phylogenetic analyses suggest that the gene codes for a Cu+ transporter belonging to the P1B-1 ATPase subfamily that has an N-terminal domain with copper binding motifs. The N-terminal cytosolic domains of the proteins from Trypanosoma congolense and Trypanosoma brucei brucei were recombinantly expressed in Escherichia coli as maltose binding protein (MBP) fusion proteins. These N-terminal domains bound copper in vitro and within E. coli cells, more than the control MBP fusion partner alone. The copper binding properties of the recombinant proteins were further confirmed when they inhibited copper catalysed ascorbate oxidation. Native CuATPases were detected in a western blot of lysates of T. congolense IL3000 and T. b. brucei ILTat1.1 bloodstream form parasites using affinity purified IgY antibodies against N-terminal domain peptides. The CuATPase was also detected by immunofluorescence in T. b. brucei bloodstream form parasites where it was associated with subcellular vesicles. In conclusion, Trypanosoma species express a copper-transporting P1B-1-type ATPase and together with other copper-binding proteins identified in the genomes of kinetoplastid parasites may constitute potential targets for anti-trypanosomal drug discovery.



中文翻译:

非洲锥虫的铜P型ATP酶N末端域的表达和铜结合特性。

铜是铜蛋白的必需成分,但对细胞有毒性,因此铜的代谢在细胞内非常仔细地调节。为了深入了解锥虫铜代谢,锥虫属。筛选基因组数据库中是否存在含铜和转运蛋白。在编码铜结合蛋白的其他基因中,已鉴定出铜转运P型ATPase(CuATPase)基因。序列和系统发育分析表明,该基因编码属于P 1B-1 ATPase亚家族的Cu +转运蛋白,该亚家族具有一个具有铜结合基序的N末端结构域。锥虫锥虫蛋白的N末端胞质结构域布鲁氏锥虫布鲁氏锥虫分别作为麦芽糖结合蛋白(MBP)融合蛋白在大肠杆菌中重组表达。这些N末端结构域在体外大肠杆菌细胞内结合铜,比单独的对照MBP融合伴侣更多。当重组蛋白抑制铜催化的抗坏血酸盐氧化时,它们的铜结合特性得到进一步证实。在congolense IL3000和T.b.的裂解物的蛋白质印迹中检测到天然CuATPase 使用针对N的亲和纯化IgY抗体,布鲁斯ILTat1.1血流形成寄生虫-末端结构域肽。Cu.ATP酶也通过免疫荧光检测。Brucei血流形成与亚细胞囊泡相关的寄生虫。总之,锥虫物种表达一种铜转运蛋白P 1B-1型ATPase,并且与在运动质体寄生虫基因组中鉴定的其他铜结合蛋白一起可能构成抗锥虫药物发现的潜在靶标。

更新日期:2019-11-18
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