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Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways

Hill, Spencer and Reichermeier, Kurt and Scott, Daniel C. and Samentar, Lorena and Coulombe-Huntington, Jasmin and Izzi, Luisa and Tang, Xiaojing and Ibarra, Rebeca and Bertomeu, Thierry and Moradian, Annie and Sweredoski, Michael J. and Caberoy, Nora and Schulman, Brenda A. and Sicheri, Frank and Tyers, Mike and Kleiger, Gary (2019) Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways. eLife, 8 . Art. No. e51163. ISSN 2050-084X. PMCID PMC6975927.

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The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1a. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

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Moradian, Annie0000-0002-0407-2031
Sweredoski, Michael J.0000-0003-0878-3831
Kleiger, Gary0000-0003-3924-1680
Additional Information:© 2019, Hill et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Received: 17 August 2019; Accepted: 22 December 2019; Published: 23 December 2019. We thank Dr. Raymond Deshaies for thoughtful comments during the preparation of the manuscript, Eric Goldstein for artistic advice on Figure 8, and Casey Hall of the UNLV Genomics Core Facility for DNA sequencing. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Competing interests: Kurt Reichermeier: is an employee of the Genetech Biotechnology Compnay. Frank Sicheri: is a founder and consultant for Repare Therapeutics. The other authors declare that no competing interests exist. Author contributions: Spencer Hill, Kurt Reichermeier, Conceptualization, Formal analysis, Investigation; Daniel C Scott, Conceptualization, Resources; Lorena Samentar, Rebeca Ibarra, Formal analysis, Investigation, Methodology; Jasmin Coulombe-Huntington, Luisa Izzi, Xiaojing Tang, Thierry Bertomeu, Annie Moradian, Michael J Sweredoski, Conceptualization, Investigation; Nora Caberoy, Conceptualization, Supervision; Brenda A Schulman, Frank Sicheri, Resources, Supervision; Mike Tyers, Conceptualization, Formal analysis, Supervision; Gary Kleiger, Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology. Data availability: High through-put sequence data can be found at the GEO repository:
Funding AgencyGrant Number
NIH2 R15 GM117555-02
St. Jude Children's Research HospitalUNSPECIFIED
Canadian Institutes of Health Research (CIHR)FDN 143277
Canadian Institutes of Health Research (CIHR)FDN-167277
Institute for Data Valorisation (IVADO)UNSPECIFIED
Canada Research Chairs ProgramUNSPECIFIED
PubMed Central ID:PMC6975927
Record Number:CaltechAUTHORS:20200103-100005242
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Official Citation:Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways. eLife 2019;8:e51163; doi: 10.7554/eLife.51163
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:100491
Deposited By: Tony Diaz
Deposited On:05 Jan 2020 03:42
Last Modified:28 Jan 2020 18:39

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