Project PXD035503: "The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis"

Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, have provided unique insights which have contributed to the emergence of the ‘Ubiquitin code’. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains 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 remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its 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. To explore the underlying mechanisms, we used proteomics and identified the interactome of endogenous HECTD1 in cells synchronized in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity during mitosis.

Ubiquitin, E3 ubiquitin ligase, Cell proliferation, Trabid, HECDT1, Cell cycle

Cite this dataset as:
Vaughan, N., Scholz, N., Lindon, C., Licchesi, J., 2022. Project PXD035503: "The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis". PRIDE Archive. Available from:


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Natalie Vaughan
University of Bath

Nico Scholz
University of Bath

Catherine Lindon
University of Cambridge


Holger Kramer
Data Curator
MRC London Institute of Medical Science

Mark Skehel
Data Curator
Francis Crick Institute

University of Bath
Rights Holder


Data collection method:

HEK293T cells were synchronised in late G2 and mitosis as described above. Four μg of IgG or of a HECTD1 antibody (Ab101992) were coated onto Dynabead magnetic beads and incubated with either lysates from G2 or M-phase synchronised cells for 1 hr at RT prior to washes, and denaturation in 2xLDS sample buffer/100 mM DTT at 95°C for 5 min. Samples were then loaded onto a 4-12% Bis-Tris SDS polyacrylamide gel which was then stained with Imperial Protein Stain (ThermoFisher Scientific). Gel lanes were sliced into of 1-2 mm2 pieces and processed by in-gel trypsin digestion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an LTQ Orbitrap Velos (ThermoFisher Scientific) mass spectrometer coupled to an Ultimate 3000 nano-ultra performance liquid chromatography (UPLC) system (Dionex, Sunnyvale, CA, USA). The mass spectrometer was operated in data-dependent acquisition mode using a Top10 method and a 32-minute liquid chromatography gradient. The MS1 survey scans were acquired in the Orbitrap at a resolution of 30,000 and an m/z range of 300-2,000. MS2 data were acquired in the Ion Trap and precursor ion isolation was performed with an isolation width of 2.0 Da and CID activation with a normalised collision energy of 35.

Data processing and preparation activities:

The LC-MS/MS data were searched with the MASCOT search algorithm [1]. Mascot distiller was used to produce Mascot compatible (.mgf) files from the raw LC-MS/MS data. Mascot (Matrix Science, algorithm version 2.2.04) was used to perform database searches of the Uniprot database without taxonomy restriction (14,423,061 entries). Variables were set to: 2+ and 3+ ions, peptide mass tolerance 10 ppm, fragment mass tolerance 0.8 Da, number of missed cleavages: one, instrument type: ESI-TRAP, variable modifications: Carbamidomethylation (Cys), Oxidation (Met). N-terminal pyroglutamate (Gln->pyroGly), phosphorylation (Ser, Thr, Tyr). MS/MS data were further validated using Scaffold ( [1] Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data., Electrophoresis. 20, 3551-3567.


Research Studentship

University of Bath Alumni Fund


Medical Research Council

GW4 BioMed MRC Doctoral Training Partnership

Medical Research Council

Ubiquitin-mediated events in cell fate decisions

Newnham College, University of Cambridge

SMRS Award

Publication details

Publication date: 3 August 2022
by: PRIDE Archive

Version: 1

Official landing page URL:

URL for this record:

Related papers and books

Vaughan, N., Scholz, N., Lindon, C., and Licchesi, J. D. F., 2022. The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis. Scientific Reports, 12(1). Available from:

Contact information

Please contact the Research Data Service in the first instance for all matters concerning this item.

Contact person: Julien Licchesi


Life Sciences
Biology & Biochemistry