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Journal articleGoya Grocin A, Serwa R, Morales Sanfrutos J, et al., 2019,
N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging.
Journal articleLovell S, Sutherell CL, Tate EW, 2019,
Chemical Probes for Proteins and Networks, MASS SPECTROMETRY-BASED CHEMICAL PROTEOMICS, Pages: 127-158
Journal articleFurniss RCD, Low WW, Mavridou DAI, et al., 2018,
Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces, Journal of Biological Chemistry, Vol: 293, Pages: 17188-17199, ISSN: 0021-9258
Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing (A/E) lesion on the surface of infected cells causes significant remodelling of the host cell surface, however limited information is available about changes at the protein level. Here we employed "plasma membrane profiling", a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor CD55 exhibited the greatest reduction in cell surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE, and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.
Journal articleWang Z, Grosskurth SE, Cheung T, et al., 2018,
Pharmacological inhibition of PARP6 triggers multipolar spindle formation and demonstrates therapeutic effects in breast cancer, Cancer Research, Vol: 78, Pages: 6691-6702, ISSN: 1538-7445
PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity.
Journal articleDe Vita E, Schuler P, Lovell S, et al., 2018,
Journal articleBenns HJ, Tate EW, Child MA, 2018,
Parasites exist within most ecological niches, often transitioning through biologically and chemically complex host environments over the course of their parasitic life cycles. While the development of technologies for genetic engineering has revolutionised the field of functional genomics, parasites have historically been less amenable to such modification. In light of this, parasitologists have often been at the forefront of adopting new small-molecule technologies, repurposing drugs into biological tools and probes. Over the last decade, activity-based protein profiling (ABPP) has evolved into a powerful and versatile chemical proteomic platform for characterising the function of enzymes. Central to ABPP is the use of activity-based probes (ABPs), which covalently modify the active sites of enzyme classes ranging from serine hydrolases to glycosidases. The application of ABPP to cellular systems has contributed vastly to our knowledge on the fundamental biology of a diverse range of organisms and has facilitated the identification of potential drug targets in many pathogens. In this chapter, we provide a comprehensive review on the different forms of ABPP that have been successfully applied to parasite systems, and highlight key biological insights that have been enabled through their application.
Journal articleBeard R, Singh N, Grundschober C, et al., 2018,
High-yielding 18F radiosynthesis of a novel oxytocin receptor tracer, a probe for nose-to-brain oxytocin uptake in vivo, Chemical Communications, Vol: 54, Pages: 8120-8123, ISSN: 1359-7345
A novel Al18F labelled peptide tracer for PET imaging of oxytocin receptor has been accessed through a high radiochemical yield approach. This tracer showed comparable affinity and higher selectivity and stability compared to oxytocin, and was used to demonstrate direct nose-to-brain uptake following intranasal administration, a common yet controversial delivery route for oxytocin-based therapeutics.
Journal articleBeard R, Stucki A, Schmitt M, et al., 2018,
Oxytocin (OT) is an exciting potential therapeutic agent, but it is highly sensitive to modification and suffers extensive degradation at elevated temperature and in vivo. Here we report studies towards OT analogs with favorable selectivity, affinity and potency towards the oxytocin receptor (OTR), in addition to improving stability of the peptide by bridging the disulfide region with substituted dibromo-xylene analogs. We found a sensitive structure-activity relationship in which meta-cyclized analogs (dOTmeta) gave highest affinity (50 nM Ki), selectivity (34-fold), and agonist potency (34 nM EC50, 87-fold selectivity) towards OTR. Surprisingly, ortho-cyclized analogs demonstrated OTR and vasopressin V1a receptor subtype affinity (220 nM and 69 nM, respectively) and pharmacological activity (294 nM and 35 nM, respectively). V1a binding and selectivity for ortho-cyclized peptides could be improved 6-fold by substituting a neutral residue at position 8 with a basic amino acid, providing potent antagonists (14 nM IC50) that displayed no activation of the OTR. Furthermore, xylene-bridged analogs demonstrated increased stability compared to OT at elevated temperature, demonstrating promising therapeutic potential for these analogs which warrants further study.
Conference paperRiviere F, Dian C, Perez-Dorado I, et al., 2018,
Mechanistic insight into HsNMT1-mediated acylation, Publisher: WILEY, Pages: 421-422, ISSN: 2211-5463
Conference paperTate EW, 2018,
Protein N terminal modifications: from chemical biology to drug discovery, Publisher: WILEY, Pages: 72-73, ISSN: 2211-5463
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