Cloning and Functional Analysis of Deubiquitinating Enzyme Gene UCHs from Hevea brasiliensis

doi:10.7525/j.issn.1673-5102.2023.04.015

Abstract

Abstract:

To investigate the potential function of ubiquitin carboxyl terminal hydrolases（UCHs） in the ubiquitination process of laticiferous of Hevea brasiliensis， the full-length sequences of two UCHs family members（HbUCH-L3 and HbUCH-L5） were isolated from Hevea brasiliensis， and both had typical UCHs domains. The open reading frame of HbUCH-L3 and HbUCH-L5 were 993 bp and 558 bp， and encoded 330 and 185 amino acids， respectively. The results of qRT-PCR showed that HbUCH-L3 and HbUCH-L5 were constitutively expressed in all tissues， but was low in latex. In vitro ubiquitination substrates cleavage of recombinant HbUCHs showed that both HbUCH-L3 and HbUCH-L5 had the function of hydrolyzing ubiquitin. HbUCHs significantly reduced the overall ubiquitination level of C-serum proteins； while， the deubiquitinating activity of HbUCH-L3 was higher than that of HbUCH-L5. Therefore， it was speculated that UCHs played a role in maintaining the dynamic balance of laticiferous ubiquitination and thus played a specific biological function， but the exact mechanism was still unclear.

Key words: Hevea brasiliensis, ubiquitin carboxy-terminal hydrolases, laticiferous ubiquitination, in vitro ubiquitination substrate cleavage

CLC Number:

S794.1

Mingyue YUAN, Tianzhong ZHOU, Ma YU, Bin HU, Xiangyu LONG, Hua CHEN. Cloning and Functional Analysis of Deubiquitinating Enzyme Gene UCHs from Hevea brasiliensis[J]. Bulletin of Botanical Research, 2023, 43(4): 622-630.

Figures/Tables 7

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References 33

1	金华斌，田维敏，史敏晶.我国天然橡胶产业发展概况及现状分析［J］.热带农业科学，2017，37（5）：98-104.
	JIN H B， TIAN W M， SHI M J.Current situation and industrial development of natural rubber in China［J］.Chinese Journal of Tropical Agriculture，2017，37（5）：98-104.
2	安锋，林位夫，谢贵水，等.国内外巴西橡胶树替代作物及技术研发现状［J］.热带作物学报，2012，33（6）：1134-1141.
	AN F， LIN W F， XIE G S，et al.Development of alternative natural rubber producing crops and techniques［J］.Chinese Journal of Tropical Crops，2012，33（6）：1134-1141.
3	ZHU J H， ZHANG Z L.Ethylene stimulation of latex production in Hevea brasiliensis ［J］.Plant Signaling & Behavior，2009，4（11）：1072-1074.
4	ZHANG Y.Transcriptional regulation by histone ubiquitination and deubiquitination［J］.Genes & Development，2003，17（22）：2733-2740.
5	崔超，赵阿慧，李芳，等.泛素/26S蛋白酶体途径调节作物种子大小的研究进展［J］.中国农业科技导报，2021，23（9）：19-29.
	CUI C， ZHAO A H， LI F，et al.Research progress of ubiquitin/26S proteasome pathway regulating crop seed size［J］.Journal of Agricultural Science and Technology，2021，23（9）：19-29.
6	曹孝强，于菲菲，谢旗.泛素化修饰调控植物非生物逆境响应［J］.中国基础科学，2020，22（1）：7-10.
	CAO X Q， YU F F， XIE Q.Protein ubiquitination-mediated abiotic stress response in plants［J］.China Basic Science，2020，22（1）：7-10.
7	田爱梅，于晖，曹家树.植物E3泛素连接酶的分类与功能［J］.中国细胞生物学学报，2020，42（5）：907-915.
	TIAN A M， YU H， CAO J S.Classification and function of E3 ubiquitin ligase in plants［J］.Chinese Journal of Cell Biology，2020，42（5）：907-915.
8	李招娣，兰秋艳，李衍常，等.定量蛋白质组学揭示酵母去泛素化酶Ubp14生物学功能［J］.生物工程学报，2022，38（10）：3901-3913.
	LI Z D， LAN Q Y， LI Y C，et al.Quantitative proteomics reveal the potential biological functions of the deubiquitinating enzyme Ubp14 in Saccharomyces cerevisiae ［J］.Chinese Journal of Biotechnology，2022，38（10）：3901-3913.
9	程岚，刘春燕，刘琼，等.植物UBP类去泛素化酶功能研究进展［J］.植物生理学报，2023，59（3）：433-441.
	CHEN L， LIU C Y， LIU Q，et al.Advances in functional studies of plant UBP deubiquitinating enzymes［J］.Plant Physiology Journal，2023，59（3）：433-441.
10	KULATHU Y， KOMANDER D.Atypical ubiquitylation：the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages［J］.Nature Reviews Molecular Cell Biology，2012，13（8）：508-523.
11	NIJMAN S M B， LUNA-VARGAS M P A， VELDS A，et al.A genomic and functional inventory of deubiquitinating enzymes［J］.Cell，2005，123（5）：773-786.
12	HUANG T T， NIJMAN S M B， MIRCHANDANI K D，et al.Regulation of monoubiquitinated PCNA by DUB autocleavage［J］.Nature Cell Biology，2006，8（4）：341-347.
13	DOELLING J H， YAN N， KUREPA J，et al.The ubiquitin‐specific protease UBP14 is essential for early embryo development in Arabidopsis thaliana ［J］.The Plant Journal，2001，27（5）：393-405.
14	YANG P Z， SMALLE J， LEE S，et al.Ubiquitin C‐terminal hydrolases 1 and 2 affect shoot architecture in Arabidopsis［J］.The Plant Journal，2007，51（3）：441-457.
15	LONG X Y， FANG Y J， QIN Y X，et al.Latex-specific transcriptome analysis reveals mechanisms for latex metabolism and natural rubber biosynthesis in laticifers of Hevea brasiliensis［J］.Industrial Crops and Products，2021，171：113835.
16	APPEL R D， BAIROCH A， HOCHSTRASSER D F.A new generation of information retrieval tools for biologists：the example of the ExPASy WWW server［J］.Trends in Biochemical Sciences，1994，19（6）：258-260.
17	TANZ S K， CASTLEDEN I， SMALL I D，et al.Fluorescent protein tagging as a tool to define the subcellular distribution of proteins in plants［J］.Frontiers in Plant Science，2013，4：214.
18	ALMAGRO ARMENTEROS J J， TSIRIGOS K D， SØNDERBY C K，et al.SignalP 5.0 improves signal peptide predictions using deep neural networks［J］.Nature Biotechnology，2019，37（4）：420-423.
19	KROGH A， LARSSON B， VON HEIJNE G，et al.Predicting transmembrane protein topology with a hidden Markov model：application to complete genomes［J］.Journal of Molecular Biology，2001，305（3）：567-580.
20	KUMAR S， TAMURA K，NEI M.MEGA：molecular evolutionary genetics analysis software for microcomputers［J］.Bioinformatics，1994，10（2）：189-191.
21	WANG D H， SONG W， WEI S W，et al.Characterization of the ubiquitin C-terminal hydrolase and ubiquitin-specific protease families in rice（Oryza sativa）［J］.Frontiers in Plant Science，2018，9：1636.
22	RONG C， ZHOU R， WAN S，et al.Ubiquitin carboxyl-terminal hydrolases and human malignancies：the novel prognostic and therapeutic implications for head and neck cancer［J］.Frontiers in Oncology，2021，10：592501.
23	黄浩.泛素羧基端水解酶UCH家族水解反应动力学研究［D］.苏州：苏州大学，2009.
	HUANG H.Study on the activity and dynamics of ubiquitin C-terminal hydrolase［D］.Suzhou：Soochow University，2009.
24	LEE Y T C， CHANG C Y， CHEN S Y，et al.Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome［J］.Scientific Reports，2017，7（1）：1-14.
25	任禹静，王丰.泛素羧基末端水解酶L3的研究进展［J］.生命科学仪器，2020，18（2）：14-22.
	REN Y J， WANG F.Advances in the study of ubiquitin C-terminal hydrolase L3［J］.Life Science Instruments，2020，18（2）：14-22.
26	牛彤旭，王张，宝鲁日，等.UCH-L3和HIF-1α在乳腺良恶性病变中的表达和临床意义［J］.海南医学院学报，2021，27（3）：174-179.
	NIU T X， WANG Z， BAO L R，et al.Expression and clinical significance of UCH-L3 and HIF-1a in breast benign and malignant lesions［J］.Journal of Hainan Medical University，2021，27（3）：174-179.
27	陈立杰，方远鹏，杜巧丽，等.水稻去泛素化酶OsUCH-L5基因的克隆及表达分析［J］.种子，2021，40（3）：45-51.
	CHEN L J， FANG Y P， DU Q L，et al.Cloning and expression analysis of OsUCH-L5 gene encoded rice deubiquitin enzyme［J］.Seed，2021，40（3）：45-51.
28	OLDAK L， CHLUDZINSKA-KASPERUK S， MILEWSKA P，et al.MMP-1，UCH-L1，and 20S proteasome as potential biomarkers supporting the diagnosis of brain glioma［J］.Biomolecules，2022，12（10）：1477.
29	尉娜，路坦，谭军.脑梗死患者静脉溶栓疗程中泛素羧基末端水解酶L1和缺血修饰白蛋白水平的动态变化及临床意义［J］.中国心血管杂志，2022，27（3）：263-268.
	WEI N， LU T， TAN J.Dynamic changes and clinical significance of ubiquitin carboxyl-terminal hydrolase L1 and ischemia-modified albumin levels during intravenous thrombolysis in patients with cerebral infarction［J］.Chinese Journal of Cardiovascular Medicine，2022，27（3）：263-268.
30	丁珊，任禹静，姜凌，等.磷酸化对UCHL3体外去泛素化酶活性的影响［J］.生物技术进展，2019，9（5）：527-535.
	DING S， REN Y J， JIANG L，et al.Effect of phosphorylation on deubiquitinase activity of UCHL3 in vitro［J］.Current Biotechnology，2019，9（5）：527-535.
31	WOLBERGER C.Mechanisms for regulating deubiquitinating enzymes［J］.Protein Science，2014，23（4）：344-353.
32	PICKART C M， ROSE I A.Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides［J］.Journal of Biological Chemistry，1985，260（13）：7903-7910.
33	魏芳，校现周，刘实忠.适合巴西橡胶树胶乳C-乳清蛋白双向电泳技术的建立［J］.热带作物学报，2009，30（11）：1618-1623.
	WEI F， XIAO X Z， LIU S Z.Establishment of a two-dimensional electrophoresis technology for latex C-serum of Hevea brasiliensis［J］.Chinese Journal of Tropical Crops，2009，30（11）：1618-1623.

引物名称 Primer name	引物序列（5′→3′） Primer sequence（5′→3′）	备注 Note
HbUCH-L3-F	ATTGTGTAGTTCCTTTGGGGCTTAG	HbUCH-L3基因克隆全长引物 Cloning of full-length primers of HbUCH-L3 gene
HbUCH-L3-R	CAGAAAATTATTCCCTGCATGTCT
HbUCH-L5-F	AATCCTGAAGATCCAAGCATTTTCC	HbUCH-L5基因克隆全长引物 Cloning of full-length primers of HbUCH-L5 gene
HbUCH-L5-R	TGCCAAAAATATGCTAAAAAAGTC
HbUCH-L3-qp-F	TGGAGCGTGCTGCATATCTT	HbUCH-L3实时定量PCR引物 HbUCH-L3 real-time quantitative PCR primer
HbUCH-L3-qp-R	AGTTCTCCTTCCACACACGC
HbUCH-L5-qp-F	TGGACTGGTTGCGTATGGTG	HbUCH-L5实时定量PCR引物 HbUCH-L5 real-time quantitative PCR primer
HbUCH-L5-qp-R	CCTCCTTTGCAGCTCCTTGA
HbYLS8-qp-F	CCTCGTCGTCATCCGATTC	实时定量PCR内参引物 Real-time quantitative PCR internal reference primers
HbYLS8-qp-R	CAGGCACCTCAGTGATGTC
HbUCH-L3-MBP-F	TTGCGGCCGCATGGTTCCGAAGCCTGTTCTT	HbUCH-L3原核表达引物 HbUCH-L3 prokaryotic expression primer
HbUCH-L3-MBP-R	CGGTCGACTTAAATTGCACCTTCAGCTTTC	HbUCH-L3原核表达引物 HbUCH-L3 prokaryotic expression primer
HbUCH-L5-MBP-F	TTGCGGCCGCATGTCTTGGTGCACTATTGAGT	HbUCH-L5原核表达引物 HbUCH-L5 prokaryotic expression primer
HbUCH-L5-MBP-R	CGGTCGACTCATGTTTTTTGCTTGGCCTTC	HbUCH-L5原核表达引物 HbUCH-L5 prokaryotic expression primer

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