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Role of APC/C subunit APC7 in regulating the function of the APC/C E3 ubiquitin ligase

Primary Supervisor: Dr Andy Turnell, Institute of Cancer and Genomic Sciences

Secondary supervisor: Steve Smerdon

PhD project title: Role of APC/C subunit APC7 in regulating the function of the APC/C E3 ubiquitin ligase

University of Registration: University of Birmingham

Project outline:

The APC/C is a large macromolecular E3 ubiquitin ligase, that, through targeting protein substrates for polyubiquitylation and 26S proteasome-mediated degradation coordinates the progression of cells through mitosis (1). APC/C E3 ligase activity is stimulated by the temporally coordinated recruitment of one of two related activator proteins, Cdc20 or Cdh1 to specific APC/C subunits; APC/C activators also serve in conjunction with APC/C subunits to bind substrates (1). The atomic structure of the APC/C holoenzyme, including all APC/C subunits has recently been determined by cryo-EM at 3.2 Å resolution (2). Recent evidence indicates that APC/C phosphorylation plays a critical role in its regulation; CDK1-dependent phosphorylation of APC3 and APC1 allows for structural changes in the APC/C complex that facilitates binding of Cdc20 to the APC/C (3).

Previous work from our laboratory has established that APC/C subunits, APC5 and APC7 associate with CBP/p300 acetyltransferases and stimulate their activity (4), whilst the transcriptional repressor, TIF1g, regulates progression through mitosis by associating with APC7 and modulating the activity of APC/C-Cdc20 (5). We have also shown that that the DNA damage response (DDR) protein, MDC1 regulates APC/C-Cdc20 activity during mitosis by promoting Cdc20 association with the APC/C (6), and the DDR protein 53BP1, is both a regulator and substrate of the APC/C, and serves to ensure genomic stability in response to mitotic stress (7).

Despite recent insights into APC/C structure and function we still know very little about how individual APC/C subunits function. The primary aim of this studentship therefore, is to characterize the role of the APC/C subunit APC7 in coordinating cell cycle progression. Specifically, this study aims to: (i) identify the APC7 interactome by mass spectrometry and characterise APC7-interactors as APC/C substrates or regulators; (ii) identify APC7 post-translational modifications (PTMs), e.g. phosphorylation, by mass spectrometry and identify the modifiers responsible for residue-specific PTMs; (iii) Adopt a structure-function approach to establish how specific modifications affect APC7 structure and function; (iv) Use live cell imaging to characterise role of specific APC7 modifications in cell cycle progression. Results of this study will be important in understanding the specific role of APC7 in the regulation of APC/C function.

References:

  1. Peters JM. The anaphase promoting complex/cyclosome: a machine designed to destroy (2006) Nat Rev Mol Cell Biol. 7:644-56;
  2. Chang L et al. (2015). Atomic structure of the APC/C and its mechanism of protein ubiquitination Nature 522:450-454.
  3. Fujimitsu K et al. (2016) Cyclin-dependent kinase 1-dependent activation of APC/C ubiquitin ligase. Science 352:1121-1124.
  4. Turnell AS et al. (2005) The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature 438:690-695.
  5. Sedgwick GG et al. (2013) Transcriptional Intermediary Factor 1g binds to the Anaphase-Promoting Complex/Cyclosome and promotes mitosis. Oncogene 32: 4622-4633.
  6. Townsend K et al. (2009) MDC1 regulates mitotic progression. J. Biol. Chem. 284:33939-48.
  7. Kucharski TJ et al (2017) Reciprocal regulation between 53BP1 and the Anaphase-Promoting Complex/Cyclosome is required for genomic stability in response to mitotic stress. Cell Rep. 18(8):1982-1995.

BBSRC Strategic Research Priority: Integrated Understanding of Health: Ageing

    Techniques that will be undertaken during the project:

    • Protein Biochemistry: PAGE, IP/GST pulldowns, Western Blots; bacterial protein expression and purification; biochemical assays – Ubiquitination assays, kinase assays; mass spectrometry-interactomics and post-translational modifications.
    • Structural Biology: chemical cross-linking, hydrodynamic analysis (MALS/SAXS). crystallization, cryo-EM, structural bioinformatics, dynamics simulation and comparative computational analysis
    • Molecular Biology: cloning; sequencing; transformation; DNA and RNA purification; qPCR; mutagenesis.
    • Cell Biology: Tissue culture; Transfection- DNA and siRNA; generation of Tet-inducible cell lines- FlpIn system, retroviral transduction; confocal microscopy; flow cytometry; Live-cell imaging; use of UV irradiation and ionizing radiation.
    • Bioinformatics: Handling large data-sets generated from mass spectrometry; sequence analysis.

    Contact: Dr Andy Turnell, University of Birmingham