TY - JOUR
T1 - The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis
AU - Vaughan, Natalie
AU - Scholz, Nico
AU - Lindon, Catherine
AU - Licchesi, Julien D.F.
N1 - Funding Information:
We are grateful to the Bio-Imaging and Cell Analysis Facility at the University of Bath for support with flow cytometry analysis and confocal microscopy, the MRC-LMB Mass Spectrometry Facility and in particular Sarah Maslen for sample processing and data analysis. We thank Dr Joshua E Flack (MRC-LMB) for contributing HECTD1 KO cell lines, Professor Irene Zohn (Children’s National Research Institute, USA) for the full-length mouse Hectd1 mammalian expression vector, and Professor David J Stephens (University of Bristol) for contributing hTERT-RPE cells. We thank Dr Julia Sero (University of Bath) for discussion and access to the IN Cell Analyzer 2000 High-Content Microscope.
Funding Information:
This work was supported by the University of Bath through a University Research Studentship to NV, the University of Bath Alumni Fund for seed funding our work on glioblastoma, and the Royal Society Grant ZR-Y0113. NS is funded by a GW4 BioMed MRC Doctoral Training Partnership. We acknowledge a SMRS award from Newnham College which enable visits of NV to Cambridge to carry out live cell imaging experiments. We also acknowledge funding MRC MR/M01102X/1 to CL.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/)82. The analysed mass spectrometry proteomics data is included in Supplementary Table 1. Uncropped western blot images are included in the Supplementary data, with cropped areas highlighted with a red box.
PY - 2022/12/31
Y1 - 2022/12/31
N2 - The cell cycle is tightly regulated by protein phosphorylation and ubiquitylation events. During mitosis, the multi-subunit cullin-RING E3 ubiquitin ligase APC/c functions as a molecular switch which signals for one cell to divide into two daughter cells, through the ubiquitylation and proteasomal degradation of mitotic cyclins. The contributions of other E3 ligase families during cell cycle progression remain less well understood. Similarly, the roles of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains during mitosis, also remain to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate HECTD1 function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell number and we established that this is mediated through loss of ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase) and we confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. In line with this data, HECTD1 depletion reduced the activity of the Spindle Assembly Checkpoint, and BUB3, a component of the Mitosis Checkpoint Complex, was identified as novel HECTD1 interactor. BUB3, BUBR1 or MAD2 protein levels remained unchanged in HECTD1-depleted cells. Overall, this study reveals a novel putative role for HECTD1 during mitosis and warrants further work to elucidate the mechanisms involved.
AB - The cell cycle is tightly regulated by protein phosphorylation and ubiquitylation events. During mitosis, the multi-subunit cullin-RING E3 ubiquitin ligase APC/c functions as a molecular switch which signals for one cell to divide into two daughter cells, through the ubiquitylation and proteasomal degradation of mitotic cyclins. The contributions of other E3 ligase families during cell cycle progression remain less well understood. Similarly, the roles of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains during mitosis, also remain to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate HECTD1 function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell number and we established that this is mediated through loss of ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase) and we confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. In line with this data, HECTD1 depletion reduced the activity of the Spindle Assembly Checkpoint, and BUB3, a component of the Mitosis Checkpoint Complex, was identified as novel HECTD1 interactor. BUB3, BUBR1 or MAD2 protein levels remained unchanged in HECTD1-depleted cells. Overall, this study reveals a novel putative role for HECTD1 during mitosis and warrants further work to elucidate the mechanisms involved.
UR - http://www.scopus.com/inward/record.url?scp=85135261430&partnerID=8YFLogxK
U2 - 10.1038/s41598-022-16965-y
DO - 10.1038/s41598-022-16965-y
M3 - Article
C2 - 35915203
AN - SCOPUS:85135261430
SN - 2045-2322
VL - 12
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 13160
ER -