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The Catalytically Inactive Mutation of the Ubiquitin-Conjugating Enzyme CDC34 Affects its Stability and Cell Proliferation

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Abstract

The ubiquitin proteasome system (UPS) plays important roles in the regulation of protein stability, localization, and activity. A myriad of studies have focused on the functions of ubiquitin ligases E3s and deubiquitinating enzymes DUBs due to their specificity in the recognition of downstream substrates. However, the roles of the most ubiquitin-conjugating enzymes E2s are not completely understood except that they transport the activated ubiquitin and form E2–E3 protein complexes. Ubiquitin-conjugating enzyme CDC34 can promote the degradation of downstream targets through the UPS whereas its non-catalytic functions are still elusive. Here, we find that mutation of the catalytically active cysteine to serine (C93S) results in the reduced ubiquitination, increased stability, and attenuated degradation rate of CDC34. Through semi-quantitative proteomics, we identify the CDC34-interacting proteins and discover that the wild-type and mutant proteins have many differentially interacted proteins. Detailed examination finds that some of them are involved in the regulation of gene expression, cell growth, and cell proliferation. Cell proliferation assay reveals that both the wild-type and C93S proteins affect the proliferation of a cancer cell line. Database analyses show that CDC34 mRNA is highly expressed in multiple cancers, which is correlated with the reduced patient survival rate. This work may help to elucidate the enzymatic and non-enzymatic functions of this protein and might provide additional insights for drug discovery targeting E2s.

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Abbreviations

CCK-8:

Cell counting kit-8

CDC34:

Ubiquitin-conjugating enzyme E2 R1 or cell division cycle 34

CHX:

Cycloheximide

CSNK1G2:

Casein kinase I isoform γ2

GNL3:

Guanine nucleotide-binding protein-like 3

HEK:

Human embryonic kidney

MCM7:

DNA replication licensing factor MCM7

MS:

Mass spectrometry

NC:

Negative control

PCR:

Polymerase chain reaction

PEI:

Polyethyleneimine

PSM:

Peptide spectrum match

SCF:

Skp-cullin-1-F-box

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SEM:

Standard error of measurements

Ub:

Ubiquitin

UPS:

Ubiquitin proteasome system

C93S:

C93S mutant

WT:

CDC34 wild-type

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Acknowledgements

We thank Yarong Wang at the Mass Spectrometry core facility of the Medical School of Soochow University for the assistance during the MS analysis. We also appreciate Dr. Xiaoyan Qiu at Soochow University for her critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (31670833 & 31700722), Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX17_2040), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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  1. Xun Liu and Yang Zhang have contributed equally to this work.

Authors and Affiliations

  1. Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, 199 Ren’ai Road, Suzhou, 215123, Jiangsu, China

    Xun Liu, Yang Zhang, Zhanhong Hu, Qian Li, Lu Yang & Guoqiang Xu

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  1. Xun Liu
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Correspondence to Guoqiang Xu.

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Liu, X., Zhang, Y., Hu, Z. et al. The Catalytically Inactive Mutation of the Ubiquitin-Conjugating Enzyme CDC34 Affects its Stability and Cell Proliferation. Protein J 37, 132–143 (2018). https://doi.org/10.1007/s10930-018-9766-x

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