Current research

Andrew is currently based at Cancer Research UK's Cambridge Institute where he leads a research team.

Andrew's research programme focuses on the role of the oestrogen receptor (ER) in breast cancer. In 70% of all breast cancer cases, this receptor drives the growth and proliferation of the tumour, and is a key target for the drug Tamoxifen. The ER is known to associate with pioneer factors and numerous co-factors, including many with enzymatic activity that can modify ER and other co-factors. Activity of ER and co-factors is regulated by growth factor signalling pathways. ER binding also depends on epigenetic factors like DNA methylation and histone modifications. This process is central to the physiological and pathological behaviour of the cell, but it is unknown how signalling and epigenetic determinants converge to regulate ER dynamic.

Andrew aims to investigate these dynamics and in the long term to produce a model could be used to predict the effects of perturbing components of the pathway with drugs and potentially lead to improved combinatorial treatment regimes.

Proteomics

Proteomics

Proteomics uses mass spectrometry to detect the levels of proteins within the cell. Building on the technologies Andrew developed at the MRC Laboratory of Molecular Biology, his research programme makes use of state-of-the-art instrumentation to investigate how the Oestrogen Receptor activates genes while it is bound to the DNA in the cell's nucleus. This allows for the identification of novel protein interactions. If these interactions are involved in the regulation of the gene activation they could be of therapeutic interest.


Genomics

Genomics

By combining high-throughput sequence and computational methods we are able to analyse the activation of specific genes by the Oestrogen receptor. This technology is used in tandem with proteomics to provide a complete understanding of both which genes are activated and how they are activated in response to a specific stimulus.

Team

Current Members

Amanda Donnelly
PhD student
MSc Mol. Medicine, Imperial College London, UK, 2013
Funding: Cancer Research UK

Amy Cullen
Research Assistant
BSc (Hons), Imperial College London, UK, 2016
Funding: Cancer Research UK

Anze Godicelj
Research Assistant
MSci BA (Hons), Univesrity of Cambridge, UK, 2017
Previously Part III Student in lab, 2016, University of Cambridge: Biochemistry awarded overall First Class.

Ryan Blake
MPhil Project Placement
University of Essex
Funding: University of Essex

Previous Members

Jannik Lauenstein
Work Experiance Research Assistant
Apprenticeship in biology laboraty technician, Lise-Meitner-Schule, Berlin, Germany
Funding: Lise-Meitner-Schule, Berlin

George Wood
Visiting Student
Natural Sciences Tripos Undergraduate, University of Cambridge
Funding: Downing College — Bill Willetts and Mays Wild funds

Marisa Di Monaco
MPhil Project Placement
University of Essex
Funding: University of Essex

Previous research

While based at the Medical Research Council (MRC) in the Laboratory of Molecular Biology, Andrew developed methods for the structural analysis of protein complexes. The research involved the study of protein-protein binding by way of using small isotopically-labelled linker molecules. These linker molecules bind between residues that are within range of each other and then resultant the cross-linked protein complex is digested and analysed by mass spectrometry. The software developed to interrogate the mass spectrometry data was named Hekate and is freely available.


Download Hekate


More details are available on the Hekate page of this site and the results of this work were published as:

  1. Holding et. al (2013) J. Proteome Res. [PubMed]

  2. Liu et. al (2013) Genes Dev. [PubMed]

  3. Rego et. al (2013) EMBO J. 32. 1334-1343. [Pubmed] [F1000Prime]


Figure showing XCMS workflow


Figure 1. Cartoon image outlining the method of protein cross-linking. A protein sample is cross-linked with a homobifunctional reagent that links residues within a certain distance of each other. The protein sample is then digested into small peptides and the cross-linked fragments are detected by mass spectrometry. These peptides are then fragmented to provide an amino acid sequence detailing where in the protein the cross-linkers occur, and thereby we can work out the structure of the protein and how multiple proteins interact.

Published Research

Journal Articles

  1. Andrew N. Holding
    XL–MS: Protein cross-linking coupled with mass spectrometry
    Methods. 2015. [Science Direct]

  2. Jennifer Lee, ShuJing Ding, Thomas B. Walpole, Andrew N. Holding, Martin G. Montgomery, Ian M. Fearnley and John E. Walker (2015)
    Organisation of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.
    J. Biol. Chem. 290. 13308-20. [PubMed]

  3. Andrew N Holding, Meindert H Lamers, Elaine Stephens, J Mark Skehel (2013)
    Hekate: software suite for the mass spectrometric analysis and three-dimensional visualization of cross-linked protein samples.
    J Proteome Res. 12. 5923-33. [PubMed]

  4. Yang Liu,1 Hannah K Salter, 1 Andrew N Holding, Christopher M Johnson, Elaine Stephens, Peter J Lukavsky, John Wlashaw, Simon L Bullock (2013)
    Bicaudal-D uses a parallel, homodimeric coiled coil with heterotypic registry to coordinate recruitment of cargos to dynein.
    Genes Dev. 27. 1233-46. [PubMed]

  5. Andrew N Holding,1 Ana Toste Rego,1 Helen Kent and Meindert H Lamers (2013)
    Architecture of the Pol III—clamp—exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair.
    EMBO J. 32. 1334-1343. [Pubmed] [F1000Prime] - recommended as being of special significance.

  6. Holding AN, Spencer JB. (2008)
    Investigation into the mechanism of phenolic couplings during the biosynthesis of glycopeptide antibiotics.
    ChemBioChem. 9. 2209-14. [Pubmed]

  7. O'Hare HM, Huang F, Holding A, Choroba OW, Spencer JB. (2006)
    Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution.
    FEBS Lett. 580. 3445-50. [Pubmed]

Oral Presentations

  1. Utilising mass spectrometry for the structural and functional analysis of complex systems in medical research
    Invited speaker, January 2016, University of Essex, Colchester, UK.
  2. Visualizing gene regulation: a combined proteomic and genomic approach for the structural analysis of steroid hormone receptor complexes
    Invited speaker, 4th International Congress on Analytical Proteomics, 2015, Lisbon, Portugal.
  3. Bringing Proteogenomics to the study of ER+ breast cancer
    Invited speaker, 2014, MRC Cancer Unit, Cambridge, UK.
  4. Interactions within the E. coli Replisome
    Mini-symposium on Biophysics, 2012, MRC Laboratory of Molecular Biology.
  5. Interactions within the E. coli Replisome
    Proteomics Methods Forum, 2012, Barts Cancer Institue, London, UK.
  6. Cross-linking in mass spectrometry
    Proteomics Methods Forum, 2010, Department of Biochemistry, University of Cambridge.
  7. Cross-linking in mass spectrometry
    Mini-symposium on Biophysics, 2010, MRC Laboratory of Molecular Biology.

Poster Presentations

  1. ChIP-MS/TMT: a quantative proteomic analysis of steroid hormone receptor activation.
    A. N. Holding, F. Markowetz
    64th ASMS Conference on Mass Spectrometry and Allied Topics, 2016, San Antonio, Texas, USA
  2. Visualising gene regulation: A combined proteomic and genomic approach for the analysis of steroid hormone receptor complexes.
    A. N. Holding, J. C. Carroll, F. Markowetz
    29th Annual Symposium of the Protein Society, Barcelona, Spain
  3. Visualising gene regulation: A combined proteomic and genomic approach for the analysis of steroid hormone receptor complexes.
    A. N. Holding, J. C. Carroll, F. Markowetz
    Cambridge Cancer Centre Symposium, 2015, Cambridge, UK.
  4. Hekate – Software suite for the mass spectrometric analysis & three-dimensional visualization of cross-linked protein samples.
    A. N. Holding, Y. Liu, H. K. Salter, M. L. Lamers, S. L. Bullock, M. J. Skehel
    Postdoctoral Career Development Day, Cancer Research UK, 2014,London, UK
  5. A shared binding site for different cargo adaptors in the asymmetric coiled-coil structure of the dynein co-factor Bicaudal-D
    H. K. Salter, Y. Liu, A. N. Holding, C. Johnson, E. Stephens, J. Walshaw, S. L. Bullock
    The American Society For Cell Biology Annual Meeting, 2012, San Franciso, CA, USA.
  6. Resolving interactions involved in binding of the dynein co-factor Bicaudal-D (BicD) to Rab6
    Andrew N Holding, Elaine Stephens, Mark Skehel.
    EuPA/BSPR Prteomic Meeting, 2012, Glasgow, Scotland.
  7. Resolving interactions involved in binding of the dynein co-factor Bicaudal-D (BicD) to Rab6
    Andrew N Holding, Elaine Stephens.
    60th ASMS Conference on Mass Spectrometry and Allied Topics, 2012, Vancouver Convention Centre, Vancouver, BC, Canada.
  8. XL-SAXS and DNA replication
    Ana Toste Rêgo, Andrew Holding, Michal Hammel, Elaine Stephens, John Kuriyan and Meindert H. Lamers.
    Current Opinion in Structural Biology & DNA Repair, 2011, Amsterdam, The Netherlands
  9. Development of arginine and guanine specific isotopically-labelled linkers for the characterisation of binding interactions by mass spectrometry
    Andrew N Holding, Elaine Stephens.
    59th ASMS Conference on Mass Spectrometry and Allied Topics, 2011, Colorado Convention Center, Denver, CO, USA.
  10. Sdo1/Efl1 interaction site mapped by photocrosslinking
    Andrew N Holding, Nicholas Basse, Alan Warren, Elaine Stephens.
    Memorial symposium for Dr. Jonathan Spencer, 2010, University of Cambridge.
  11. Investigation into the mechanism of phenolic couplings during the biosynthesis of glycopeptide antibiotics
    Andrew N Holding, Jonathan B Spencer.
    Zing Conference in Natural Products, 2009, Antigua.

Degrees

PhD in Chemical Biology
Studies on the biosynthesis of non-ribosomal peptides
University of Cambridge, UK

Masters in Chemistry
University of Oxford, UK

Vita

2015
Senior Research Associate
Cancer Research UK Cambridge Research Institute

2013 - 2014
Research Associate
Cancer Research UK Cambridge Research Institute

2009 - 2013
Career Development Fellow
MRC Laboratory of Molecular Biology

2005 - 2009
PhD student
Deparment of Chemistry, University of Cambridge, UK.
Advisors: Dr. J. Spencer and Dr. F. Leeper

Awards and fellowships

2015 CRUK Rising Star in Research Engagement Prize

2015 Fellow Downing College

2014 Unltd Try-It Award

2013 College Lecturer and Bye-Fellow Downing College

2012 Wellcome Trust People Award

2012 British Mass Spectrometry Society Travel Award

2012 MRC Award via the Special Awards Scheme for contributions to the MRC

2012 British Science Association Media Fellowship

2011 UnLtd Catalyst Award for Social Entrepreneurship

2009 MRC Career Development Fellowship

2005 BBSRC PhD Studentship

Professional Memberships

2015 Fellow of the Higher Education Academy

2014 Member Biochemical Society

2009 Member Royal Society of Chemistry


PhD thesis

Studies on the biosynthesis of non-ribosomal peptides

Abstract: The problem of bacterial resistance is of growing concern within the medical community. Eventually, even with responsible use of antibiotics, new compounds will be required to bypass the resistance that bacteria have acquired. Thus the expansion of knowledge and understanding of antibiotics is key in the development of new compounds in the fight against infection. One attractive starting point for the development of new compounds are those natural products generated by non-ribosomal peptide synthetases (NRPS), which include a range of clinically relevant glycopeptide antibiotics.

Several aspects of the biosynthesis of glycopeptide antibiotics were examined: first, the investigation to identify, by the use of directed evolution if 4-hydroxymandelic acid synthase (HmaS) from the gene cluster of the antibiotic chloroeremomycin may have evolved from its homologue 4-hydroxyphenylpyruvate dioxygenase (HppD). The summation of this work is published in FEBS Lett. 2006; 580:3445. Following on from this work was an investigation into the hypothesis that HmaS catalyses the turnover of the non-natural phenylpyruvic acid to produce a product with an inverted chiral centre compared to that of the natural substrate due to differences in substrate binding. Results showed that, while not the major product, the inverted product was detected via chiral GCMS. Secondly, it is shown that all three cytochrome P450 enzymes (OxyA-C) that catalyse the sequential formation of three essential oxidative cross-links within the chloroeremomycin molecule do so with the retention of the oxygen atom on the peptide backbone and without the incorporation of oxygen in the air. This portion of the work is published in ChemBioChem 2008; 9:2209. The final part of the study was the development of a high-throughput screening method for NRPS A-domains, with the aim of both rapid characterisation and directed evolution of novel substrate specificity. This led to the identification that the amino acid loaded by the first A-domain of the teicoplanin NRPS was shown to load the d-amino acid in preference to the l-amino acid. This is in contrast to the equivalent domain in the chloroeremomycin gene cluster that loads the l-amino acid.

Download: Studies on the biosynthesis of non-ribosomal peptides.