Structure and activity of the essential UCH family deubiquitinase DUB16 from Leishmania donovani.

IF 4.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
James A Brannigan, Mohd Kamran, Nathaniel G Jones, Elisa M Nightingale, Eleanor J Dodson, Sarfaraz A Ejazi, Jeremy C Mottram, Nahid Ali, Anthony J Wilkinson
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引用次数: 0

Abstract

In Leishmania parasites, as for their hosts, the ubiquitin (Ub) proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease-type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here, we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the Ub C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of Ub consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric Ub from small molecule Ub adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type DUB fold, and a relatively short and disordered cross-over loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits DUB activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal Ub and the C-terminus of the distal Ub. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 μM, comparable with the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. While it is highly likely that IMP-1710 has additional targets, these results suggest that DUB16 is a potential target for the development of new anti-leishmanial compounds.

多诺瓦利什曼原虫UCH家族去泛素酶DUB16的结构和活性。
在利什曼原虫中,对于它们的宿主来说,泛素蛋白酶体系统对细胞生存能力很重要。作为一项系统基因缺失研究的一部分,研究人员发现,包括DUB16在内的四种半胱氨酸蛋白酶型去泛素酶(DUBs)对寄生虫在promastigote阶段的生存至关重要。我们从多诺瓦利什曼原虫中纯化并鉴定了重组DUB16,它属于泛素c端水解酶(UCH)家族。DUB16有效地水解泛素的c端氨基香豆素和罗丹明缀合物,这与提出的uch型dub在由非定代谢过程产生的小分子泛素加合物再生游离单体泛素的细胞作用一致。DUB16的晶体结构显示出典型的uch型去泛素酶折叠,以及一个相对较短且无序的交叉环,似乎限制了催化半胱氨酸的进入。在接近化学计量酶与底物的比率时,DUB16对通过近端泛素的Lys11、Lys48或Lys63残基与远端泛素的c端之间的异肽键连接的双泛素表现出去泛素酶活性。DUB16以高100-1000倍的周转率切割泛素-核糖体L40融合蛋白,得到成熟产物。一种针对DUB16的半胱氨酸反应性氰吡咯烷化合物IMP-1710抑制DUB16活性。promastigote细胞活力测定显示IMP-1710具有杀灭寄生虫的活性,EC50值为1-2M,与抗利什曼病药物米替福辛相当。过量生产DUB16的L. mexicana寄生虫显示对IMP-1710的抗性适度增加,这证明IMP-1710在体内抑制DUB16。虽然IMP-1710极有可能具有其他靶点,但这些结果表明DUB16是开发新的抗利什曼化合物的潜在靶点。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Biochemical Journal
Biochemical Journal 生物-生化与分子生物学
CiteScore
8.00
自引率
0.00%
发文量
255
审稿时长
1 months
期刊介绍: Exploring the molecular mechanisms that underpin key biological processes, the Biochemical Journal is a leading bioscience journal publishing high-impact scientific research papers and reviews on the latest advances and new mechanistic concepts in the fields of biochemistry, cellular biosciences and molecular biology. The Journal and its Editorial Board are committed to publishing work that provides a significant advance to current understanding or mechanistic insights; studies that go beyond observational work using in vitro and/or in vivo approaches are welcomed. Painless publishing: All papers undergo a rigorous peer review process; however, the Editorial Board is committed to ensuring that, if revisions are recommended, extra experiments not necessary to the paper will not be asked for. Areas covered in the journal include: Cell biology Chemical biology Energy processes Gene expression and regulation Mechanisms of disease Metabolism Molecular structure and function Plant biology Signalling
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