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Oct 2019

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Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays
植物去泛素酶的体外活性标记和泛素链分解分析   

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Abstract

Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here, we provide protocols for the rapid analysis of DUB activity in vitro by activity-based labelling with the suicide probe, HA-ubiquitin vinyl sulfone (HA-UbVS), and ubiquitin chain disassembly assays. We have previously used these methods to analyse the activity of the Arabidopsis thaliana DUB, UBP6, but in principle, these protocols are applicable to any DUB of interest.

Keywords: Ubiquitin (泛素), Deubiquitinase (去泛素化酶), Ubiquitin vinyl sulfone (泛素乙烯砜), Proteasome (蛋白酶体), Cell signaling (细胞信号传导), Proteostasis (蛋白质稳态)

Background

Regulation of cellular protein homeostasis (proteostasis), the intricate balance between protein synthesis, modification, and degradation, is essential for the survival of all organisms (Balch et al., 2008). Proteostasis is maintained by a diverse range of post-translational modifications (PTMs) that increase the size and functionality of proteomes (Skelly et al., 2016). Ubiquitination, the covalent attachment of the small regulatory protein ubiquitin to target proteins, is a prevalent PTM involved in numerous cellular processes in eukaryotes. Ubiquitin is conjugated predominantly to lysine residues of target substrates via its C-terminus, which requires the sequential actions of three types of enzyme; E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. Ubiquitin itself contains seven lysine residues that can also be modified, facilitating formation of polyubiquitin chains of various linkage types. Different ubiquitin chain topologies have specific signalling roles and are associated with different cellular processes. For example, K48-linked chains generally target proteins for degradation by the proteasome, a large multi-protein complex that is responsible for the majority of proteolytic activity in eukaryotic cells. However, ubiquitin modifications can also be reversed by the action of a wide range of deubiquitinase (DUB) enzymes with diverse specificities for different ubiquitin linkage types (Mevissen and Komander, 2017). Since deubiquitination determines the stability, activity, or function of proteins, entire proteomes can be shaped by the activities of specific DUBs. Many human diseases, including cancers, are associated with dysregulated DUB activity, demonstrating the physiological and pathological importance of these enzymes (Harrigan et al., 2018; Clague et al., 2019).


To understand DUB function, their activity can be analysed by various in vitro methods to determine substrate specificity and regulation of activity (Cho et al., 2020). These methods include activity-based protein profiling using synthetic ubiquitin suicide probes that irreversibly label DUB active sites (Borodovsky et al., 2002; Gong et al., 2018; Schauer et al., 2020), as well as assays in which purified ubiquitin chains are incubated with DUBs and chain disassembly is monitored by western blotting or gel staining. Recently, we used these methods to uncover the signalling roles of the Arabidopsis thaliana proteasome-associated deubiquitinases, UBP6 and UBP7, in plant immunity (Skelly et al., 2019). Here, we describe protocols for analysing recombinant UBP6 activity by HA-UbVS labelling and in vitro deubiquitination assays. Importantly, these methods can be applied to any DUB of interest and in the case of HA-UbVS, labelling can also be applied to protein extracts from any organism to uncover proteome-wide DUB activity. Characterising the activity and regulation of DUBs is vital to understand how these enzymes act in vivo and will advance the fields of ubiquitin signalling and proteostasis across eukaryotes.


Materials and Reagents

  1. HA-ubiquitin-vinyl sulfone (Boston Biochem, catalog number: U-212)

  2. 26S proteasome (Ub-VS treated) (Ubiquigent, catalog number: 65-1020-010)

  3. Recombinant purified His6-T7-UBP6 (Skelly et al., 2019) or other DUB of interest

  4. Ubiquitin subtrates for deubiquitination assays:

    Poly-ubiquitin (Ub3-7) K48-linked (Boston Biochem, catalog number: UC-220)

    Poly-ubiquitin (Ub3-7) K63-linked (Boston Biochem, catalog number: UC-320)

    Di-ubiquitin K48-linked (Boston Biochem, catalog number: UC-200B)

    Di-ubiquitin K63-linked (Boston Biochem, catalog number: UC-300B)

  5. UltraPure Tris (Invitrogen, catalog number: 15504-020)

  6. MgCl2 hexahydrate (Sigma-Aldrich, catalog number: M2670)

  7. Adenosine triphosphate (ATP) (Sigma-Aldrich, catalog number: A26209)

  8. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 1114740005)

  9. Glycerol (Sigma-Aldrich, catalog number: G5516)

  10. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771)

  11. Bromophenol blue (Sigma-Aldrich, catalog number: B8026)

  12. Anti-HA antibody (ThermoFisher, catalog number: 26183)

  13. Anti-ubiquitin antibody (Santa Cruz Biotechnology, catalog number: sc-8017)

  14. Antibody against the DUB being analysed, e.g., anti-T7 for His6-T7-UBP6 (Millipore, catalog number: 69522)

  15. Double-distilled H2O (ddH2O)

  16. 15% SDS-PAGE gel (e.g., compatible with Bio-Rad Mini-PROTEAN system)

  17. SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher, catalog number: 34577)

  18. 10× DUB labelling buffer (store at -20°C) (see Recipes)

  19. 10× deubiquitination buffer (store at -20°C) (see Recipes)

  20. 2× SDS-PAGE loading buffer (see Recipes)

  21. DUBs, proteasomes, HA-UbVS, and ubiquitin substrates (see Recipes)

Equipment

  1. 1.5 ml microcentrifuge tubes (e.g., Starlab, catalog number: S1615-5500)

  2. Pipettes and tips (e.g., Gilson Pipettman, various volumes)

  3. Heat block (e.g., ThermoFisher, catalog number: 88870004)

  4. Protein gel electrophoresis and western blotting apparatus (e.g., Bio-Rad Mini-PROTEAN system)

Procedure

  1. HA-UbVS labelling

    1. Set up 10-μl reactions including the following: 1 μl 10× DUB labelling buffer, 5 μl 700 nM DUB, 1.25 μl 80 nM 26S proteasome (if required), 1 μl 7 μM HA-UbVS, and 1.75 μl ddH2O. Control samples should be included in which components are omitted and replaced with ddH2O.

    2. Incubate for 30 min at room temperature or in a heat block at 22°C.

    3. Add 10 μl 2× SDS-PAGE loading buffer to each sample.

    4. Incubate for 10 min in a heat block at 70°C.

    5. Perform SDS-PAGE with a 15% gel and western blotting using standard methods with:

      1. Anti-HA antibodies (at 1:5000 dilution) to detect free HA-UbVS (~9.8 kDa) and HA-UbVS-labelled DUB (Figure 1).

      2. Antibodies (at antibody-specific dilutions) against the appropriate DUB or epitope tag to detect both unlabelled and labelled DUB. Any standard imaging system can be used depending on the secondary antibodies (e.g., film or CCD imaging system for chemiluminescence).



      Figure 1. HA-UbVS labelling of recombinant purified His6-T7-UBP6. A typical western blot showing free and HA-UbVS-labelled recombinant His6-T7-UBP6. Adapted from Figure 6B of Skelly et al. (2019).


  2. In vitro deubiquitination assays

    1. Set up 10 μl reactions including the following: 1 μl 10× deubiquitination buffer, 1 μl 200 nM DUB, 1 μl 12.5 nM 26S proteasome (if required), 1 μl 4 μM ubiquitin substrate, and 6 μl ddH2O. Control samples should be included in which components are omitted and replaced with ddH2O.

    2. Incubate in a heat block at 30°C for the desired time [2-16 h for His6-T7-UBP6 (Skelly et al., 2019), but dependent on the DUB and substrate].

    3. Add 10 μl 2× SDS-PAGE loading buffer to each sample.

    4. Incubate for 10 min in a heat block at 70°C.

    5. Perform SDS-PAGE with a 15% gel and western blotting using standard methods with anti-ubiquitin antibodies to detect the original ubiquitin chain substrate and observe any DUB-mediated chain disassembly as lower-order ubiquitin species. Any standard imaging system can be used depending on the secondary antibodies (e.g., film or CCD imaging system for chemiluminescence).

Recipes

  1. 10× DUB labelling buffer (store at -20°C)

    500 mM Tris pH 7.4

    50 mM MgCl2

    10 mM DTT

    10 mM ATP

  2. 10× deubiquitination buffer (store at -20°C)

    500 mM Tris pH 7.4

    50 mM MgCl2

    10 mM DTT

    50 mM ATP

  3. 2× SDS-PAGE loading buffer (store at room temperature, add DTT fresh before use)

    20% glycerol

    120 mM Tris pH 6.8

    4% SDS

    0.02% Bromophenol Blue

    100 mM DTT

  4. DUBs, proteasomes, HA-UbVS, and ubiquitin substrates

    Diluted in ddH2O from stocks to the required concentrations described above

Acknowledgments

This work was supported by a Royal Society University Research Fellowship (UF090321), a BBSRC grant (BB/L006219/1), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 678511). This protocol was derived from Skelly et al. (2019).

Competing interests

The authors declare that no competing interests exist.

References

  1. Balch, W. E., Morimoto, R. I., Dillin, A. and Kelly, J. W. (2008). Adapting proteostasis for disease intervention. Science 319(5865): 916-919.
  2. Borodovsky, A., Ovaa, H., Kolli, N., Gan-Erdene, T., Wilkinson, K. D., Ploegh, H. L. and Kessler, B. M. (2002). Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family.Chem Biol 9(10): 1149-1159.
  3. Cho, J., Park, J., Kim, E. E. and Song, E. J. (2020). Assay Systems for Profiling Deubiquitinating Activity. Int J Mol Sci 21(16): 5638.
  4. Clague, M. J., Urbe, S. and Komander, D. (2019). Breaking the chains: deubiquitylating enzyme specificity begets function. Nat Rev Mol Cell Biol 20(6): 338-352.
  5. Gong, P., Davidson, G. A., Gui, W., Yang, K., Bozza, W. P. and Zhuang, Z. (2018). Activity-based ubiquitin-protein probes reveal target protein specificity of deubiquitinating enzymes. Chem Sci 9(40): 7859-7865.
  6. Harrigan, J. A., Jacq, X., Martin, N. M. and Jackson, S. P. (2018). Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov 17(1): 57-78.
  7. Mevissen, T. E. T. and Komander, D. (2017). Mechanisms of Deubiquitinase Specificity and Regulation. Annu Rev Biochem 86: 159-192.
  8. Schauer, N. J., Magin, R. S., Liu, X., Doherty, L. M. and Buhrlage, S. J. (2020). Advances in Discovering Deubiquitinating Enzyme (DUB) Inhibitors. J Med Chem 63(6): 2731-2750.
  9. Skelly, M. J., Frungillo, L. and Spoel, S. H. (2016). Transcriptional regulation by complex interplay between post-translational modifications. Curr Opin Plant Biol 33: 126-132.
  10. Skelly, M. J., Furniss, J. J., Grey, H., Wong, K. W. and Spoel, S. H. (2019). Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1. Elife 8: e47005.

简介



[摘要]泛素对蛋白质的翻译后修饰是所有真核生物中必不可少的细胞信号转导机制。泛素可通过多种具有不同活性和底物特异性的去泛素酶(DUB)酶从靶蛋白中去除。了解DUB在体外的功能是发现其细胞作用的至关重要的第一步。这里,我们提供了协议的快速ANALY SIS的DUB活性在体外由基于活动的拉贝升玲与自杀探针,HA-泛素乙烯基砜(HA-UbVS) , 和泛素链拆卸分析。我们在以前使用这些方法来ANALY小号E中的活性拟南芥DUB,UBP6,但在原则上,这些协议适用于任何感兴趣的DUB。


[背景]细胞蛋白稳态(蛋白质稳态)的调控,蛋白质合成,修饰之间的复杂的平衡,和降解,是所有生物的生存是必不可少的(鲍尔奇等人,2008) 。通过增加蛋白质组的大小和功能的多种翻译后修饰(PTM)可以维持蛋白质稳定(Skelly et al 。,2016)。泛素化是小调节蛋白泛素与靶蛋白的共价结合,是一种普遍的PTM,参与真核生物的许多细胞过程。泛素主要通过其C末端与靶底物的赖氨酸残基偶联,这需要三种酶的顺序作用。E1泛素激活酶,E2泛素结合酶,和E3泛素连接酶。遍在蛋白本身包含七个赖氨酸残基,这些赖氨酸残基也可以被修饰,从而促进各种连接类型的聚遍在蛋白链的形成。不同的泛素链拓扑结构具有特定的信号传导作用,并与不同的细胞过程相关。例如,K48连接的链通常靶向蛋白质以通过蛋白酶体降解,蛋白酶体是一种大型的多蛋白复合物,负责真核细胞中的大多数蛋白水解活性。但是,泛素修饰也可以通过对各种泛素键合类型具有不同特异性的多种去泛素酶(DUB)酶的作用来逆转(Mevissen和Komander,2017)。由于去泛素化决定了稳定性,活性,或蛋白质的功能,整个蛋白质组可以由特定DUB的活动被成形。许多人类疾病,包括癌症,都与DUB活性失调有关,证明了这些酶的生理和病理学重要性(Harrigan等人,2018 ; Clague等人,2019)。

为了理解DUB功能,它们的活性可以ANALY š通过各种编体外方法来确定底物特异性和活性的调节(CHO等人,2020) 。这些方法包括使用不可逆地标记DUB活性位点的合成泛素自杀探针进行基于活性的蛋白谱分析(Borodovsky等,2002; Gong等,2018; Schauer等,2020),以及其中纯化的泛素的测定将链与DUB一起孵育,并通过Western印迹或凝胶染色监测链的拆卸。最近,我们使用这些方法来揭开的所述信令角色拟南芥蛋白酶体相关deubiquitinases ,UBP6和UBP7 ,在植物免疫力(斯凯利等人。,2019) 。在这里,我们描述了用于ANALY协议小号通过HA-UbVS标记和ING重组UBP6活性在体外去泛素化测定法。重要的是,这些方法可以应用于任何感兴趣的DUB,在HA-UbVS的情况下,标记还可以应用于任何生物体的蛋白质提取物,以揭示整个蛋白质组的DUB活性。Characteri唱DUB的活性和调节至关重要的是要理解这些酶如何行动在体内和将推进的领域横跨泛真核生物信令和蛋白内稳态。

关键字:泛素, 去泛素化酶, 泛素乙烯砜, 蛋白酶体, 细胞信号传导, 蛋白质稳态

材料和试剂

HA-泛素-乙烯基砜(波士顿生物化学,目录号:U-212)
26S p roteasome(UB-VS处理的)(Ubiquigent,目录号:65-1020-010)
重组纯化的His 6 -T7-UBP6 (Skelly et al 。,2019)或其他感兴趣的DUB
泛素替代品用于去泛素化测定:
聚泛素(Ub3-7)K48连接(Boston Biochem,目录号:UC-220)

                            聚泛素(Ub3-7)K63连接(Boston Biochem,目录号:UC-320)

双泛素K48连接的(Boston Biochem,目录号:UC-200B)

双泛素K63连接的(Boston Biochem,目录号:UC-300B)

UltraPure Tris(Invitrogen,目录号:15504-020)
MgCl 2六水合物(Sigma-Aldrich,目录号:M2670)
三磷酸腺苷(ATP)(Sigma-Aldrich,目录号:A26209)
二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:1114740005)
甘油(Sigma-Aldrich,目录号:G5516)
十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
溴酚蓝(Sigma-Aldrich,目录号:B8026)
抗HA抗体(ThermoFisher,目录号:26183)
抗泛素抗体(圣克鲁斯生物技术,目录号:sc-8017)
抗体针对的DUB是ANALY小号版,例如。,他的6 -T7 -UBP6的抗T7 (Millipore,目录号:69522)
二次蒸馏H 2 O(ddH 2 O)
15%SDS-PAGE凝胶(例如,与Bio-Rad Mini-PROTEAN系统兼容)
SuperSignal West Pico PLUS化学发光底物(ThermoFisher,目录号:34577)
10 × DUB标记缓冲液(储存在-20 °C)(请参见食谱)
10 ×去泛素缓冲液(存储在-20 °C下)(请参阅食谱)
2 × SDS-PAGE加载缓冲区(请参阅食谱)
的DUB,蛋白酶体,HA-UbVS ,和泛素底物(见配方)


设备



1.5 ml微量离心管(例如,Starlab,目录号:S1615-5500)
移液器和吸头(例如,Gilson Pipettman,各种卷)
加热块(例如,ThermoFisher,目录号:88870004)
蛋白质凝胶电泳和蛋白质印迹仪(例如,Bio-Rad Mini-PROTEAN系统)


程序



HA-UbVS拉贝升玲
设置10 -微升反应,包括以下情况:1微升10 × DUB拉贝升玲缓冲液,5μl 700 nM的DUB,1.25微升80nM的26S p roteasome(如果需要),1微升7μMHA-UbVS ,和1.75微升双蒸水2个O.对照样品应当包括在哪些组件被省略,并用双蒸水代替2 O.                                                                                                                                                          
在室温下或在22 °C的加热块中孵育30分钟。
向每个样品中添加10μl2 × SDS-PAGE上样缓冲液。
在70 °C的加热块中孵育10分钟。
使用15%凝胶进行SDS-PAGE,并使用标准方法进行Western印迹分析,包括:
甲NTI-HA抗体(以1:5000稀释度),以检测游离HA-UbVS(〜9.8 kDa)的和HA-UbVS标签升编DUB(图1)。
甲ntibodies(在特异性抗体稀释液)到适当的DUB或表位标签同时检测unlabel升ED和拉贝升导致DUB。取决于二抗,可以使用任何标准成像系统(例如,用于化学发光的胶片或CCD成像系统)。





图1.重组纯化的His 6 -T7-UBP6的HA-UbVS标记。典型的蛋白质印迹,显示了游离的HA-UbVS标记的重组His 6 -T7-UBP6。改编自Skelly等人的图6B 。(2019)。



体外去泛素化验
设置10个微升反应,包括以下:1微升10 ×去泛素化缓冲液,1μl的200nM的DUB,1微升12.5 nM的26S p (如果需要)roteasome,1微升4μM泛素基板,和6微升的DDH 2 O.对照样品应包括省略了组件并用ddH 2 O代替的组件。
孵育在一个加热块在30 ℃下的所希望的时间[对他的2-16ħ 6 -T7-UBP6 (斯凯利等人。,2019) ,但依赖的DUB和基板。
向每个样品中添加10μl2 × SDS-PAGE上样缓冲液。
在70 °C的加热块中孵育10分钟。
用15%凝胶进行SDS-PAGE,并使用抗泛素抗体的标准方法进行蛋白质印迹,以检测原始的泛素链底物,并观察任何DUB介导的链分解为低阶泛素种类。取决于二抗,可以使用任何标准成像系统(例如,用于化学发光的胶片或CCD成像系统)。


菜谱



10 × DUB标记缓冲液(储存在-20 °C)
500 mM Tris pH 7.4

50毫米MgCl 2

10毫米DTT

10毫米ATP

10 ×去泛素缓冲液(储存在-20 °C)
500 mM Tris pH 7.4

50毫米MgCl 2

10毫米DTT

50毫米ATP

2 × SDS-PAGE加载缓冲液(储存在室温下,使用前新鲜添加DTT)
20%甘油

120 mM Tris pH 6.8

4%SDS

0.02%溴酚蓝

100毫米DTT

的DUB,蛋白酶体,HA-UbVS ,和泛素底物
在DDH稀释2 ö从库存到所需的浓度描述上述



致谢



这项工作是由英国皇家学会大学研究奖学金(UF090321),一个BBSRC资助(BB / L006219 / 1),并根据欧盟的欧洲研究委员会(ERC)的支持展望2020研究和创新计划(赠款协议号。678511 )。该协议源自Skelly等人。(2019)。



利益争夺



作者宣称不存在任何竞争利益。



参考           



Balch,WE,Ri.Morimoto,RI.Dillin和JW Kelly(2008年)。适应蛋白变性以进行疾病干预。科学319(5865):916-919。
Borodovsky,A.,Ovaa,H.,Kolli,N.,Gan-Erdene,T.,Wilkinson,KD,Ploegh,HL和Kessler,BM(2002)。基于化学的功能蛋白质组学揭示了去泛素化酶家族的新成员。化学生物学报9(10):1149-1159。
Cho,J.,Park,J.,Kim,EE和Song,EJ(2020)。分析去泛素化活性的测定系统。国际分子科学杂志21(16):5638。
克莱格,MJ,乌尔贝,S.和科曼德,D.(2019)。打破链条:去泛素化酶特异性产生功能。Nat Rev Mol Cell Biol 20(6):338-352。
Gong,P.,Davidson,GA,Gui,W.,Yang,K.,Bozza,WP and Zhuang,Z.(2018年)。基于活性的泛素蛋白探针可揭示脱泛素酶的靶蛋白特异性。化学Sci 9(40):7859-7865。
哈里根(JA),雅克(Jacq),X。马丁(NM)和杰克逊(SP)(2018)。去泛素化酶和药物发现:新的机会。Nat Rev Drug Discov 17(1):57-78。
TET的Mevissen和D.的Komander(2017)。去泛素酶特异性和调节的机制。生物学生物化学年鉴86:159-192。
新泽西州的绍尔,马金,RS,刘X.,多尔蒂,LM和布希拉格(SJ)(2020年)。发现去泛素化酶(DUB)抑制剂的进展。J Med Chem 63(6):2731-2750。
Skelly,MJ,Frungillo,L.和Spoel,SH(2016)。通过翻译后修饰之间复杂的相互作用进行转录调控。Curr Opin Plant Biol 33:126-132。
Skelly,MJ,Furniss,JJ,Grey,H.,Wong,KW和Spoel,SH(2019)。动态泛素化决定了植物免疫共激活因子NPR1的转录活性。Elife 8:e47005。
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Copyright Skelly and Spoel. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Skelly, M. J. and Spoel, S. H. (2021). Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays. Bio-protocol 11(9): e4015. DOI: 10.21769/BioProtoc.4015.
  2. Skelly, M. J., Furniss, J. J., Grey, H., Wong, K. W. and Spoel, S. H. (2019). Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1. Elife 8: e47005.
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