Dr Mark Collins

Mark Collins

Lecturer
Deputy Director: biOMICS Mass Spectrometry Facility

Department of Biomedical Science
The University of Sheffield
Western Bank
Sheffield S10 2TN
United Kingdom

Room: E04 Florey building
Telephone: +44 (0) 114 222 2303
Email: mark.collins@sheffield.ac.uk

CMIAD


Neuroscience

General

Brief career history

  • 2013-present: Lecturer in Biological Mass Spectrometry, University of Sheffield
  • 2011-2013: Senior Staff Scientist, Wellcome Trust Sanger Institute
  • 2005-2011: Research Associate/Staff Scientist, Wellcome Trust Sanger Institute
  • 2001-2005: PhD, Division of Neuroscience, University of Edinburgh
  • 2000-2001: Research Assistant, Centre for Liver Disease, Mater Misericordiae University Hospital, Dublin.
  • 1996-2000: BSc, Department of Biochemistry, University College Dublin

Research interests

Cell Signalling & Proteome Dynamics
We are interested in how proteins are regulated by post-translational modifications and how cell signalling pathways are perturbed in disease. PTMs such as phosphorylation, ubiquitination, acetylation and palmitoylation regulate overlapping and distinct aspects of protein function, but the interplay of these modifications is not well understood.

We exploit biochemical and quantitative mass spectrometry-based approaches to understand how proteins are dynamically regulated by protein synthesis and degradation and an array of post-translational modifications.

Professional activities

  • Fellow of the Higher Education Academy (2016)
  • Peer reviewer for scientific journals and grant-awarding bodies

Full publications

Research

Cell Signalling and Proteome Dynamics

Banner

Our lab is interested in the regulation of signalling pathways by post-translational modifications (PTMs). We use a combination of molecular, cell and mass spectrometry based approaches to study PTMs on a global scale and we are particularly interested in understanding the regulation and activity of enzymes that dynamically regulate PTMs such as protein kinases, palmitoyl-acyltransferases and lysine deacetylases.

The key enabling technology for our research is shotgun proteomics; in which protein samples (whole cell lysates, organelles, protein complexes etc.) are digested with into peptides, separated using nanoflow chromatography and analysed using high-resolution tandem mass spectrometry (biOMICS facility). This approach permits unbiased, quantitative analysis of protein levels and PTMs, with unrivalled accuracy and sensitivity.

Figure 1

Figure 1. Proteome analysis using high-resolution tandem mass spectrometry

Much of our knowledge of the biochemistry of signalling pathways is an aggregate of data from studies of individual PTM sites in single protein experiments. Whilst very useful, this fails to give an integrated and unbiased view of what happens to all relevant proteins for example, when a specific receptor is activated.

The crosstalk of PTMs is emerging as an important mechanism to confer higher order regulation of signalling pathways. We are developing approaches to assay many PTMs from the same sample on a proteome-scale in order to understand how for example, phosphorylation, acetylation and palmitoylation interact to regulate protein function.

We also exploit affinity purification and proximity labelling strategies to purify and characterise multiprotein complexes formed by protein kinases and palmitoyl-acyltransferases to identify their substrates as well as regulatory proteins that  determine substrate specificity or target these enzymes to different subcellular compartments or membrane microdomains.

Research themes

Figure 2Regulation of membrane protein function by S-acylation (Palmitoylation)

Palmitoylation, the only known reversible lipid modification of proteins, is a critical regulator of protein trafficking, stability and signalling and is important for all cell types, and organisms from yeast to humans.

Proteins can be palmitoylated by a family of 23 protein acyl transferases (PATs) in humans and many of these enzymes have been shown to regulate important aspects of cell biology and in particular in neuronal cells in the brain where palmitoylation of receptors and associated proteins are essential for communication between brain cells and therefore functions such as learning and memory.

Indeed, several PATs have been implicated in the pathophysiology of neurological disorders from Huntington’s disease to intellectual disability and schizophrenia and well as other diseases such as diabetes and cancer.

The identification of determinants of substrate specificity of palmitoyl acyltransferases is an important goal toward understanding how palmitoylation regulates cell function. Development of strategies to enrich, identify and quantify proteins and PTMs is also a major focus of research in our lab.

Figure 2. site-specific-Acyl-Biotin-Exchange (ssABE) for comprehensive identification and quantification of sites of S-acylation


Figure 3

Figure 3. Concurrent identification of palmitoylation and phosphorylation sites by mass spectrometry (click image for enlarged version)

Figure 4Perturbed signalling pathways in neurodegenerative diseases

Motor Neurone Disease or Amyotrophic lateral sclerosis (ALS) is a disorder that results in fatal paralysis within a few years of symptom onset. Defects in a growing list of genes are associated with the development of ALS/MND.

Many of these gene defects result in the accumulation of aggregates in cells of patients with ALS and these aggregates are believed to cause neurons to die, resulting in the symptoms of the disease.

Recently, a number of large exome sequencing studies of ALS patients have independently identified several loss of function mutations in protein kinases but it is not known how dysregulation of these kinases might contribute to development of disease.

We are using protein biochemistry, immunofluorescence microscopy and  phosphoproteomic approaches to understand how human mutations lead to molecular and cellular changes that contribute to to the pathogenesis of ALS.

Figure 4. Monitoring autophagy levels in NSC-34 cells using a tandem mRFP-EGFP-LC3 fluorescent reporter. Autophagosomes are visualised as yellow  labelled puncta whilst autolysosomes appear red due to loss of acid sensitive GFP signal.


Collaborations

Campylobacter jejuni is a food-borne pathogen of worldwide importance but little is known about how the pathogenicity of this bacteria is regulated by PTMs. We collaborate with Prof Dave Kelly at the Department of Molecular Biology and Biotechnology, University of Sheffield to develop and apply proteomic approaches to probe the function of acetylation and phosphorylation in C. jejuni though two BBSRC White Rose Mechanistic Biology DTP PhD studentships (2015-2019 & 2017-2021).

Histone deacetylase 1 and 2 (HDAC1/2) containing complexes have important roles in almost all cellular processes, including cell cycle, DNA synthesis, DNA repair and gene expression. In collaboration with Dr Shaun Cowley at the Department of Molecular and Cell Biology, University of Leicester we are developing and apply proteomic approaches  (acetylomics and BioID) to probe the function of acetylation in embryonic stem cells  (BBSRC-SFI project grant 2017-2020).

Constitutive secretion is required for many biologically important processes such as the secretion of antibodies and the extracellular matrix. In collaboration with Dr Andrew Peden at the Department of Biomedical Science, University of Sheffield, we are investigating the regulation of SNAREs by palmitoylation as well as using quantitative proteomics to characterise novel machinery required for post-Golgi trafficking and antibody secretion.

Peptidoglycan (PG) is an essential component of the bacterial cell envelope, made of glycan strands and peptide stems containing unusual amino acids. In collaboration with Dr Stéphane Mesnage at the Department of Molecular Biology and Biotechnology, University of Sheffield, we are developing an automated, high-resolution analysis pipeline for bacterial peptidoglycan structural analyses though a BBSRC White Rose Mechanistic Biology DTP iCASE PhD studentship (2017-2021).

Team members:

  • Keith Woodley (PhD student , Protein palmitoylation)
  • Maria Davies  (PhD student , ALS signalling pathways)
  • Tom Puttick  (PhD student , Campylobacter acetylation

Funding:

  • BBSRC
  • Royal Society
Teaching

Undergraduate and postgraduate taught modules

Level 1

  • BMS109/157 Principles of Molecular Biology

Level 3

  • BMS369 Laboratory Research Project
  • BMS349/BMS359 Extended Library Project

Postgraduate

  • Mass Spectrometry-based Proteomics & Metabolomics Course for postgraduate research students
Opportunities

PhD Studentship Opportunities

An integrated molecular, cell and proteomic analysis of palmitoylation in neurons

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Palmitoylation, the only known reversible lipid modification of proteins, is an important regulator of protein localisation and function. It affects a plethora of cellular processes including protein trafficking, stability and signalling and is therefore important for all cell types, and organisms from yeast to humans. The extent to which this reversible post-translational modification is employed and how the specificity of the palmitoylation machinery is encoded remains to be determined.

Palmitoylation is mediated by a family of 23 enzymes (protein acyl transferases (PATs)) in humans. Many of these enzymes have been shown to regulate important aspects of cell biology and in particular in neuronal cells where palmitoylation of receptors and associated proteins regulates synaptic plasticity and therefore functions such as learning and memory. Indeed, several of these enzymes have been implicated in the pathophysiology of neurological disorders from Huntington’s disease to intellectual disability and schizophrenia. It is not well understood which proteins are modified by individual PATs in cells, how they can selectively recognise substrate proteins and what effect palmitoylation has on the function of these proteins.

This project will use proteomic approaches that we have recently developed to identify substrates of protein acyl transferases and investigate how palmitoylation of selected substrates regulates their stability, trafficking and function. This interdisciplinary project will utilise a range of experimental approaches including cell culture, molecular and cell biology, biochemistry and state-of-the-art quantitative mass spectrometry.

The student will be given in-depth training in all of these methods and will benefit from collaborations with other groups within the department. This research will further our understanding of how proteins dynamically associate with membranes and how this affects their function in health and disease.

References

  • Mark O. Collins Keith Woodley and Jyoti S. Choudhary. Global, site-specific analysis of neuronal protein S-acylation. Scientific Reports. 2017. Jul 5;7(1):4683.
  • Fukata Y, Fukata M. (2010). Protein palmitoylation in neuronal development and synaptic plasticity. Nature Reviews Neuroscience. Mar;11(3):161-75.
  • Jones ML, Collins MO, Goulding D, Choudhary JS & Rayner JC (2012). Analysis of protein palmitoylation reveals a pervasive role in Plasmodium development and pathogenesis. Cell Host & Microbe, 12(2), 246-258.
  • Cox J, Mann M. (2011). Quantitative, high-resolution proteomics for data-driven systems biology. Annual Review of Biochemistry 2011;80:273-99.

Keywords: Biochemistry, Cell Biology / Development, Genetics, Molecular Biology, Neuroscience/Neurology


Regulation of synaptic protein function by lysine acetylation

Supervisor 2: Dr Vincent Cunliffe

Funding status: Competition funded project European/UK students only

This project is eligible for a department scholarship. These scholarships are awarded on a competitive basis – find out more on our funding webpage.

Project Description

Recent proteomic studies have identified several thousand proteins in biochemically purified synapses and have uncovered multi-protein complexes essential for synapse function. Many of these synaptic genes are associated with >100 neurological diseases, highlighting the need for a better a molecular understanding of synapses.

Synaptic activity is regulated by a number of post-translational modifications such as protein phosphorylation. Recent studies have shown that acetylation regulates the localisation of synaptic scaffolding proteins and the surface expression of a major neurotransmitter receptor. The majority of proteins present in mouse/zebrafish synapses are acetylated but almost nothing is known about its function or the mechanism of regulation.

This project will exploit state-of-the-art methods including CRISPR/Cas9 technology in zebrafish, protein biochemistry and quantitative Orbitrap-based mass spectrometry to determine the synaptic targets of enzymes that regulate acetylation levels and to discover how acetylation, in turn, regulates the function of key synaptic proteins. The student will be given in-depth training in these methods and will benefit from collaborations with other groups within the department.

This is a multidisciplinary project between the Collins and Cunliffe labs that will require technology development and cutting-edge methods to generate high-quality quantitative data to understand complex signalling pathways regulating synaptic activity.

References

  • Àlex Bayés, Mark O Collins, Rita Reig-Viader, Gemma Gou, David Goulding, Abril Izquierdo, Jyoti S Choudhary, Richard D Emes & Seth GN Grant. Zebrafish synapse proteome complexity, evolution and ultrastructure. Nature Communications. 2017. Mar 2;8:14613.
  • Wang G, Li S, Gilbert J, Gritton HJ, Wang Z, Li Z, Han X, Selkoe DJ, Man HY. Crucial Roles for SIRT2 and AMPA Receptor Acetylation in Synaptic Plasticity and Memory. Cell Reports. 2017 Aug 8;20(6):1335-1347.

Keywords: Biochemistry, Cell Biology / Development, Genetics, Molecular Biology, Neuroscience/Neurology


Elucidating the signalling pathways important for plasma cell biology and antibody secretion

Co-supervisor: Dr Andrew Peden

Funding status: This project is also open to self funded students

Project Description

Plasma cells are the antibody secreting cells of the immune system thus are vital for fighting infection. However, dysregulation of plasma cell function is linked to a broad range of disease from Lupus to multiple myeloma. At present, it is not known how plasma cell differentiation and homeostasis is regulated.

The aim of this PhD project is to identify the key signaling pathways critical for this process. To identify these pathways we will use a combination of cutting edge in vitro cell culture models and novel mass-spectrometry based approaches. Once key pathways have been identified, we will manipulate them using chemical and genetic based approaches and determine how this affects plasma cell biology. In the long term, this information may help in the development of novel drugs for regulating plasma cell function in vivo. This project represents an exciting training opportunity where the student will become an expert in advanced proteomics, mechanistic cell biology and immunology.

Keywords: Biochemistry, Bioinformatics, Biotechnology, Cell Biology / Development, Immunology, Molecular Biology, Bioinformatics


Novel genes required for checkpoint control and their role in colo arectal cancer

Co-Supervisor: Professor Carl Smythe

Funding status: Awaiting funding decision/Possible external funding – find out more on our funding webpage.

Project Description

Background

Healthy aging is frequently affected by the onset of cancers. One in three of us will be affected over our lifetime, and 50% will have significantly reduced lifespan as a consequence. Cell cycle checkpoint genes are often mutated in cancers, while paradoxically, in appropriate circumstances, they may be attractive targets for rational drug design.

Our laboratory was one of the first labs to identify the significance of the Chk1 gene product in checkpoint control and have a significant interest in novel therapeutics and their identification. Colorectal cancer remains a significant challenge, often due to late presentation of symptoms and Chk1 inhibitors are currently in clinical trials for a subset of colorectal cancers. Recently we have undertaken a genome-wide siRNA screen in colorectal cancer cells to identify previously unknown checkpoint pathway genes in order to identify novel potential therapeutic targets. As a result we have identified novel genes whose roles in checkpoint regulation are completely unknown.

Aims

The aims of this project is to gain an understanding of the cellular function of one novel gene, which we have called MiCatS, in cellular checkpoint control. MiCatS-deficient cells appear to be unable to respond to cellular signalling pathways that indicate the presence of replication stress. This project will focus on developing an understanding of how MiCatS interferes with regulatory gene networks controlling genome integrity. Techniques: These will include a range of molecular cell biology techniques, tissue culture, confocal fluorescence microscopy, protein expression and functional characterisation approaches including proteomics, quantitative PCR, siRNA-mediated knockdown of gene expression, and immunoblotting.

References

(1) Feijoo, C., Hall-Jackson, C., Wu, R., Jenkins, D., Gilbert, D., and Smythe, C (2001). Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. The Journal of Cell Biology, 154(5), 913–924.

(2) Bowen, E., (2015). PhD Thesis. “A genomic screen for the identification of novel components of the S phase checkpoint”, University of Sheffield.

Keywords: Cancer / Oncology, Cell Biology / Development, Molecular Biology



For further information about projects within the department and how to apply, see our PhD Opportunities page:

PhD Opportunities

Selected publications

Journal articles