Characterisation of the Trichinella spiralis Deubiquitinating Enzyme, TsUCH37, an Evolutionarily Conserved Proteasome Interaction Partner
Characterisation of the Trichinella spiralis Deubiquitinating Enzyme, TsUCH37, an Evolutionarily Conserved Proteasome Interaction Partner
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旋毛虫去泛素化酶 TsUCH37（一种进化上保守的蛋白酶体相互作用伴侣）的表征
DOI：
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发表时间：
2011
影响因子：
3.8
通讯作者：
Katerina Artavanis
中科院分区：
文献类型：
--
作者：
Rhiannon R. White;S. Miyata;Eliseo Papa;E. Spooner;K. Gounaris;M. Selkirk;Katerina Artavanis
旋毛虫是一种引起旋毛虫病的人畜共患寄生线虫，除南极洲外，其他各大洲均已发现旋毛虫病。在慢性感染期间，T.旋毛虫幼虫感染骨骼肌纤维，严重破坏它们的分化状态。方法与结果用HA Ub VME探针检测T.旋毛虫L1幼虫通过免疫印迹和免疫沉淀分析结果，鉴定出一些潜在的DUB。对免疫沉淀的蛋白质进行LC/MS/MS，产生与5种保守的人DUB：UCH-L5、UCH-L3、HAUSP、OTU 6B和共济失调蛋白-3具有序列同源性的肽。克隆了预测的编码推定的UCH-L5同源物TsUCH 37的基因，并表达和纯化重组蛋白。通过Ub-AMC实验验证了该酶的去泛素化活性。重组TsUCH 37蛋白的共沉淀表明，该蛋白与推定的T. spiralis蛋白酶体组分，包括酵母Rpn 13同源物ADRM 1。此外，UCH抑制剂LDN-57444表现出对重组TsUCH 37的特异性抑制，并降低了培养的L1幼虫的活力。结论本研究首次鉴定出T. spiralis DUB，一种与人蛋白hUCH-L5直接相邻的半胱氨酸蛋白酶。结果表明，这种蛋白质与蛋白酶体的相互作用在整个进化过程中是保守的。我们显示了使用抑制剂化合物来阐明UCH酶在T。旋毛虫感染及其作为旋毛虫病治疗靶点研究
Background Trichinella spiralis is a zoonotic parasitic nematode that causes trichinellosis, a disease that has been identified on all continents except Antarctica. During chronic infection, T. spiralis larvae infect skeletal myofibres, severely disrupting their differentiation state. Methodology and Results An activity-based probe, HA-Ub-VME, was used to identify deubiquitinating enzyme (DUB) activity in lysate of T. spiralis L1 larvae. Results were analysed by immuno-blot and immuno-precipitation, identifying a number of potential DUBs. Immuno-precipitated proteins were subjected to LC/MS/MS, yielding peptides with sequence homology to 5 conserved human DUBs: UCH-L5, UCH-L3, HAUSP, OTU 6B and Ataxin-3. The predicted gene encoding the putative UCH-L5 homologue, TsUCH37, was cloned and recombinant protein was expressed and purified. The deubiquitinating activity of this enzyme was verified by Ub-AMC assay. Co-precipitation of recombinant TsUCH37 showed that the protein associates with putative T. spiralis proteasome components, including the yeast Rpn13 homologue ADRM1. In addition, the UCH inhibitor LDN-57444 exhibited specific inhibition of recombinant TsUCH37 and reduced the viability of cultured L1 larvae. Conclusions This study reports the identification of the first T. spiralis DUB, a cysteine protease that is putatively orthologous to the human protein, hUCH-L5. Results suggest that the interaction of this protein with the proteasome has been conserved throughout evolution. We show potential for the use of inhibitor compounds to elucidate the role of UCH enzymes in T. spiralis infection and their investigation as therapeutic targets for trichinellosis.
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影响因子：
16
作者：
Yao, Tingting;Song, Ling;Jin, Jingji;Cai, Yong;Takahashi, Hidehisa;Swanson, Selene K.;Washburn, Michael P.;Florens, Laurence;Conaway, Ronald C.;Cohen, Robert E.;Conaway, Joan W.
通讯作者：
Conaway, Joan W.
影响因子：
--
作者：
Borodovsky, A;Ovaa, H;Kessler, BM
通讯作者：
Kessler, BM
影响因子：
2.6
作者：
Fabre, M. V.;Beiting, D. P.;Bliss, S. K.;Appleton, J. A.
通讯作者：
Appleton, J. A.
影响因子：
--
作者：
Liu, YC;Lashuel, HA;Lansbury, PT
通讯作者：
Lansbury, PT
影响因子：
14.8
作者：
Love, Kerry Routenberg;Catic, Andre;Ploegh, Hidde L.
通讯作者：
Ploegh, Hidde L.