Identifying the Ubiquitination Targets of E6AP by Orthogonal Ubiquitin Transfer

Overview

Journal Nat Commun

Specialty Biology

Date 2017 Dec 22

PMID 29263404

Citations 22

Authors

Yiyang Wang

Xianpeng Liu

Li Zhou

Duc Duong

Karan Bhuripanyo

Bo Zhao

Han Zhou

Ruochuan Liu

Yingtao Bi

Hiroaki Kiyokawa

Jun Yin

Affiliations

Soon will be listed here.

Abstract

E3 ubiquitin (UB) ligases are the ending modules of the E1-E2-E3 cascades that transfer UB to cellular proteins and regulate their biological functions. Identifying the substrates of an E3 holds the key to elucidate its role in cell regulation. Here, we construct an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in cancer and neurodevelopmental disorders. We use yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells affords 130 potential E6AP targets. Among them, we verify that MAPK1, CDK1, CDK4, PRMT5, β-catenin, and UbxD8 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E3 substrates and reveal the cellular circuits mediated by the E3 enzymes.

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References

Maspero E, Valentini E, Mari S, Cecatiello V, Soffientini P, Pasqualato S . Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming. Nat Struct Mol Biol. 2013; 20(6):696-701. DOI: 10.1038/nsmb.2566. View

Zhao B, Bhuripanyo K, Zhang K, Kiyokawa H, Schindelin H, Yin J . Orthogonal ubiquitin transfer through engineered E1-E2 cascades for protein ubiquitination. Chem Biol. 2012; 19(10):1265-77. PMC: 4104569. DOI: 10.1016/j.chembiol.2012.07.023. View

Glessner J, Wang K, Cai G, Korvatska O, Kim C, Wood S . Autism genome-wide copy number variation reveals ubiquitin and neuronal genes. Nature. 2009; 459(7246):569-73. PMC: 2925224. DOI: 10.1038/nature07953. View

Merbl Y, Kirschner M . Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays. Proc Natl Acad Sci U S A. 2009; 106(8):2543-8. PMC: 2633216. DOI: 10.1073/pnas.0812892106. View

Tan M, Lim H, Bennett E, Shi Y, Harper J . Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover. Mol Cell. 2013; 52(1):9-24. PMC: 3981468. DOI: 10.1016/j.molcel.2013.08.018. View

Huang L, Kinnucan E, Wang G, Beaudenon S, Howley P, Huibregtse J . Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science. 1999; 286(5443):1321-6. DOI: 10.1126/science.286.5443.1321. View

Hershko A, Ciechanover A . The ubiquitin system. Annu Rev Biochem. 1998; 67:425-79. DOI: 10.1146/annurev.biochem.67.1.425. View

Cutler A, Dammer E, Doung D, Seyfried N, Corbett A, Pavlath G . Biochemical isolation of myonuclei employed to define changes to the myonuclear proteome that occur with aging. Aging Cell. 2017; 16(4):738-749. PMC: 5506426. DOI: 10.1111/acel.12604. View

Olsen S, Lima C . Structure of a ubiquitin E1-E2 complex: insights to E1-E2 thioester transfer. Mol Cell. 2013; 49(5):884-96. PMC: 3625138. DOI: 10.1016/j.molcel.2013.01.013. View

10.

Mark K, Simonetta M, Maiolica A, Seller C, Toczyski D . Ubiquitin ligase trapping identifies an SCF(Saf1) pathway targeting unprocessed vacuolar/lysosomal proteins. Mol Cell. 2014; 53(1):148-61. PMC: 4032118. DOI: 10.1016/j.molcel.2013.12.003. View

11.

Zuin A, Bichmann A, Isasa M, Puig-Sarries P, Diaz L, Crosas B . Rpn10 monoubiquitination orchestrates the association of the ubiquilin-type DSK2 receptor with the proteasome. Biochem J. 2015; 472(3):353-65. DOI: 10.1042/BJ20150609. View

12.

Altun M, Besche H, Overkleeft H, Piccirillo R, Edelmann M, Kessler B . Muscle wasting in aged, sarcopenic rats is associated with enhanced activity of the ubiquitin proteasome pathway. J Biol Chem. 2010; 285(51):39597-608. PMC: 3000941. DOI: 10.1074/jbc.M110.129718. View

13.

Jin J, Li X, Gygi S, Harper J . Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature. 2007; 447(7148):1135-8. DOI: 10.1038/nature05902. View

14.

Jacobson A, Macfadden A, Wu Z, Peng J, Liu C . Autoregulation of the 26S proteasome by in situ ubiquitination. Mol Biol Cell. 2014; 25(12):1824-35. PMC: 4055262. DOI: 10.1091/mbc.E13-10-0585. View

15.

Lee I, Schindelin H . Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell. 2008; 134(2):268-78. DOI: 10.1016/j.cell.2008.05.046. View

16.

Rangasamy S, DMello S, Narayanan V . Epigenetics, autism spectrum, and neurodevelopmental disorders. Neurotherapeutics. 2013; 10(4):742-56. PMC: 3805864. DOI: 10.1007/s13311-013-0227-0. View

17.

Jang K, Lee K, Kim S, Jin T, Choi E, Jeon H . Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF-κB inactivation to regulate breast cancer cell invasion. J Cell Biochem. 2011; 112(12):3612-20. DOI: 10.1002/jcb.23292. View

18.

Kuslansky Y, Sominsky S, Jackman A, Gamell C, Monahan B, Haupt Y . Ubiquitin ligase E6AP mediates nonproteolytic polyubiquitylation of β-catenin independent of the E6 oncoprotein. J Gen Virol. 2016; 97(12):3313-3330. DOI: 10.1099/jgv.0.000624. View

19.

Kumar S, Talis A, Howley P . Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination. J Biol Chem. 1999; 274(26):18785-92. DOI: 10.1074/jbc.274.26.18785. View

20.

Xu G, Jaffrey S . The new landscape of protein ubiquitination. Nat Biotechnol. 2011; 29(12):1098-100. PMC: 3242807. DOI: 10.1038/nbt.2061. View