Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Subcellular mapping of protein re-localisation in response to ionising radiation: A new approach to understanding DDR (#478)

Josie Christopher 1 , Mercedes Vazquez­-Chantada 2 , Derek Barratt 2 , Hilary Lewis 3 , Kathryn Lilley 1
  1. Cambridge Centre for Proteomics (CCP), University of Cambridge, Cambridge, UK
  2. Discovery Science , AstraZeneca, Cambridge , UK
  3. Oncology Biosciences , AstraZeneca, Cambridge , UK

Genomic instability is an enabling hallmark of cancer, often triggered by abnormal expression and behaviour of DNA damage repair (DDR) proteins [1] High levels of mutations and aberrant post­translational modifications (PTMs) can lead to changes in interacting partners and/or subcellular location, affecting the efficiency of DDR and causing subsequent mutagenesis [2,3]. These deficiencies in DDR can sensitise cancer cells to DNA damaging agents, such as chemotherapeutics and radiotherapy. Despite this, cancer cells use alternative DDR pathways to bypass cell death and become resistant to such therapeutics. Therefore, contributing genes and proteins to these reliant pathways have become prime candidates for oncology drug design and diagnostics. The prime examples are PARP1 and BRCA1/2, , targeted by the therapeutic inhibitor olaparib and diagnostic markers for breast and ovarian cancers, respectively [4].

Ionising radiation (IR) is detrimental for DNA and causes prolific cellular changes, such as cell morphology and activation of multiple signalling cascades. Whether response to IR is pro­ or anti­apoptotic, trafficking between organelles is required for a proportion of proteins to function, as it is well documented that some proteins have localisation­dependent roles and ‘moonlight’ between subcellular compartments [3,5]. Previous work studying re­localisation have been performed using low­throughput techniques, which rely on effective fluorescent antibodies or GFP­tagging [6]. To investigate this trafficking, we applied a holistic and novel spatial proteomics workflow, LOPIT­DC. This technique uses the combination of differential ultracentrifugation for organelle separation, multiplexed quantitative mass spectrometry and machine learning to produce subcellular proteome maps [7]. Currently, we have successfully produced extremely reproducible, high-resolution spatial proteome maps of dynamic protein re-localisation in a lung carcinoma cell line (A549) in response to ionising radiation. We hope this will give us a deeper understanding of these proteins’ roles and behaviour to this stimulus and to aid DDR-targeting drug design.

  1. Hanahan D, Weinberg RA (2011) "Hallmarks of cancer: the next generation". Cell 144: 646–674.
  2. Alshareeda AT, Negm OH, Green AR, Nolan CC, Tighe P, Albarakati N, Sultana R, Madhusudan S, Ellis IO, Rakha EA (2015) "KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer". Br J Cancer 112: 1929–1937.
  3. Li R, Hannon GJ, Beach D, Stillman B (1996) "Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage". Curr Biol 6: 189–199.
  4. O’Connor MJ (2015) "Targeting the DNA Damage Response in Cancer". Mol Cell 60: 547–560.
  5. Boisvert F-O-M, Lamond AI (2010) "p53-Dependent subcellular proteome localization following DNA damage". Proteomics 10: 4087–4097.
  6. Thul PJ, Åkesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, Alm T, Asplund A, Björk L, Breckels LM, et al. (2017) "A subcellular map of the human proteome". Science (80- ) 356: eaal3321.
  7. Geladaki A, Kočevar Britovšek N, Breckels LM, Smith TS, Vennard OL, Mulvey CM, Crook OM, Gatto L, Lilley KS (2019) "Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics". Nat Commun 10: 331.