Analysis of the dynamic sulfotyrosine proteome.

The survival of an organism depends upon the ability of different cell types to communicate with each other by assembling the correct complexes of proteins at the correct time in the correct place. One way this is achieved is to use the tricks of chemistry and biological catalysts (enzymes) called tyrosyl protein sulfotransferases, or TPSTs, to change the biological properties of polymers, such as proteins. TPSTs drive this process by adding small charged chemicals as a means of rapid (usually) reversible regulation. These events, more accurately called 'post-translational modifications', or PTMs, act as switches to change information flow and dictate the types of different biological outcomes elicited, such as cell movement, growth, survival or death.

Our proposal aims to exploit new tools and proteomics technology (using mass spectrometry, which looks at all proteins in cells in an unbiased way) to evaluate the addition of a specific chemical group, called sulfate, to proteins. Currently, the analysis of sulfation is unfocused, it attracts little strategic funding, and it is difficult to manipulate or study in a holistic manner, making efforts to study its global significance challenging. Since it underpins so much basic biology, yet detailed information as to the mechanism of this regulatory mechanism is still lacking, sulfation research requires concerted strategies to develop ways of analysing, compiling and distributing, the large amounts of biological data pertaining to how protein sulphation regulates cellular and acellular biology. Indeed, technology-based approaches for the analysis of a different chemical group on proteins, phosphate, led to a revolution in our understanding of how cells communicate, and has been critically important for biologists working in the areas of structural biology, cell signalling and communication and drug design for over 40 years, with knock-on effects on biotechnology, pharmaceutical industries and clinical intervention across the world.

To rapidly advance our understanding of cellular protein tyrosine sulfation (sTyr), our aims will be achieved through related, but distinct, work packages. These are:

WP1: Biochemical analysis of sTyr (and sThr/sSer) site-specificity in a variety of peptides and proteins

WP2: sTyr isolation and mass spectrometry-based quantitative sTyr analysis from complex cell-derived mixtures

WP3: Optimise cell-based approaches to manipulate TPST1/2 and perturb sTyr content in mammalian cells

WP4: Computational analysis of mass spectrometry data and dissection of tyrosine sulfation networks for public distribution and biological inference

WP5: A workflow for analysis of new cellular roles associated with Tyr sulphation

Tyrosine-O-sulfation (sTyr) catalyzed by tyrosine protein sulfotransferases (TPSTs) is an important, but poorly understood, post-translational modification (PTM) regulating enzyme activity and protein-protein interactions in eukaryotes. Vertebrates encode TPST1 and 2, which are Golgi-resident, membrane-bound (luminally-orientated) enzymes, which sulfate Tyr (and maybe Ser/Thr) residues in a variety of proteins, many of which are destined for membrane insertion or secretion into the extracellular space. sTyr was identified over 50 years ago in sulfated fibrinogen/gastrin, and involves transfer of the negative sulphate group from the universal donor PAPS (3'-phosphoadenosine-5'-phosphosulfate) to appropriate Tyr residue(s) in proteins. sTyr is governed by consensus motifs in substrates, and leads to biologically-relevant changes in, for example, host-pathogen interactions, chemotaxis, proteolytic peptide processing and viral entry via co-receptors.
Alongside chemical biology advances, the ascendency of high mass-accuracy mass spectrometry coupled to expert computational annotation place our research, for the first time, in a unique position to evaluate Tyr sulfation in proteins isolated from complex (a)cellular mixtures, including subcellular fractions. Nominally, Tyr sulfation and phosphorylation are 'isobaric', differing in mass by only ~10 mDa. However, this difference is detected by high mass-accuracy instruments, such as the Orbitrap Fusion Lumos, in high throughput proteomics experiments, permitting discrimination between peptides containing sTyr and pTyr prior to fragmentation/site localization. We propose the following work packages:
WP1: Enzymatic analysis of TPST1/2 Tyr site-specificity
WP2: sTyr isolation and MS-based quantitative sTyr analysis in complex cell mixtures
WP3: The extent of the cellular and extracellular human sTyr proteome (+WP1/2)
WP4: Computational Dissection of tyrosine sulfation (+WP1-3)
WP5: Analysis of new cellular roles for sTyr

This impact Summary answers three specific questions; who might benefit from this research? and how? and when?

Introduction: Currently, the analysis of protein sulfation is unfocused and attracts little funding. Critically, the scale of protein sulfation and the number of sulfated proteins is unknown. Where it has been analysed in any depth, Tyr sulfation has been shown to underpin/regulate aspects of basic cell biology, and we think that now is the ideal time to consolidate knowledge and reveal the extent and biological impact of sTyr in cells. We believe that our work will have impact on basic cell biology, since Tyr sulfation occurs from flies to plants and humans. Indeed, technology-based approaches for the analysis of a slightly different chemical group (phosphate) led to a revolution in understanding how cells communicate, and has been important for biologists working in the areas of structural biology, cell signalling and drug design, with critical effects on biotechnology and pharmaceutical industries. Major types of impact likely to emerge are:

1. Academic and Industrial Impact, major milestones: (0-3 years)
1.1 Publicise sTyr work through seminar/conference presentations, especially new technical outputs
1.2 Obtain PhD funding to support project through BBSRC DTP network
1.3 Invite interested parties to attend PI/CoI labs, for 'apprenticeship' to facilitate sTyr (or sThr/sSer) analytical training approaches, impacting methodology uptake and unbiased knowledge exchange
1.4 Report proteomics data, releasing information via ProteomeXchange, benefiting biologists, computational and proteomics-based scientists; data then released into universal freely-available websites (e.g UniProt/PeptideAtlas)
1.5 P Eyers recently received BBSRC GCRF funding aimed at capacity building in developing countries including Argentina, India and Brazil. We recognise the interest in sulfation in emerging markets
1.6 Engage with Industry: sulfation as a biomarker and/or (anti)target for intervention. Share TPST1/2 enzymes to discover/repurpose sulfotransferase inhibitors (with SGC)
1.7 Present and publicise work at (inter)national meetings in form of talks and/or posters and publish findings in journals.

2. Training impact for staff, major milestones: (1-3 years)
2.1 Dr Byrne and PDRA2 training in the emerging area of subcellular proteomics, learning new skills in Tyr-sulfation enzymology and sulfoproteomics. Specifically, 10 days at University of Cambridge hosted by K. Lilley to train in sub-cellular fractionation and MS-based hyperLOPIT. These could be excellent for career paths needed to become independent scientists in multiple fields
2.2 PDRA2: Training events for MS-based quantitative analysis (e.g. ProteoMMX:Strictly Quantitative, run by C Eyers)
2.3 Media and grant writing and fellowship skills training for PDRAs; Dr Byrne recently received his first independent internal funding from UoL

3. The general public (impacts on children in education and over >10 years): IIB (Athena Swan Gold) scientists have an enviable track-record in supporting outreach and equality.
3.1 Use our new static display and animations to embark on new educational sulfoproteomics presentations, public (and scientific) dissemination, and a new online sulfotransferase animation
3.2 Organise visits of members of the public/ children to labs and CPR, including primary/secondary children
3.3 Hosting A-level students, teachers and policy makers, explaining 'how enzymes work'
3.4 Speaking at charity/community events explaining how research drives impact
3.5 Evidence for public engagment via Twitter (@pseudoenzyme/@ClaireEEyers/@DaveFernig)

4. Longer term societal/economic impact (5-15 years)
4.1 Annotated sulfoproteomes, and a sulfation chemical 'toolbox', will impact on many groups of basic/translational scientists; by analogy with the kinase field, impact may take decades to be realised but might be faster given the lessons learnt.