415 research outputs found
Activity- and reactivity-based proteomics: Recent technological advances and applications in drug discovery.
Activity-based protein profiling (ABPP) is recognized as a powerful and versatile chemoproteomic technology in drug discovery. Central to ABPP is the use of activity-based probes to report the activity of specific enzymes or reactivity of amino acid types in complex biological systems. Over the last two decades, ABPP has facilitated the identification of new drug targets and discovery of lead compounds in human and infectious disease. Furthermore, as part of a sustained global effort to illuminate the druggable proteome, the repertoire of target classes addressable with activity-based probes has vastly expanded in recent years. Here, we provide an overview of ABPP and summarise the major technological advances with an emphasis on probe development
The Rab-binding profiles of bacterial virulence factors during infection
Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection
Dynamic protein acylation: new substrates, mechanisms and drug targets
Post-translational attachment of lipids to proteins is found in all organisms, and is important for many biological processes. Acylation with myristic and palmitic acids are among the most common lipid modifications, and understanding reversible protein palmitoylation dynamics has become a particularly important goal. Linking acyltransferase enzymes to disease states can be challenging due to a paucity of robust models, compounded by functional redundancy between many palmitoyl transferases; however, in cases such as Wnt or Hedgehog signalling, small molecule inhibitors have been identified, with some progressing to clinical trials. In this review, we present recent developments in our understanding of protein acylation in human health and disease through use of chemical tools, global profiling of acylated proteomes, and functional studies of specific protein targets
Deconvoluting the biology and druggability of protein lipidation using chemical proteomics
Lipids are indispensable cellular building blocks, and their post-translational attachment to proteins makes them important regulators of many biological processes. Dysfunction of protein lipidation is also implicated in many pathological states, yet its systematic analysis presents significant challenges. Thanks to innovations in chemical proteomics, lipidation can now be readily studied by metabolic tagging using functionalized lipid analogs, enabling global profiling of lipidated substrates using mass spectrometry. This has spearheaded the first deconvolution of their full scope in a range of contexts, from cells to pathogens and multicellular organisms. Protein N-myristoylation, S-acylation, and S-prenylation are the most well-studied lipid post-translational modifications because of their extensive contribution to the regulation of diverse cellular processes. In this review, we focus on recent advances in the study of these post-translational modifications, with an emphasis on how novel mass spectrometry methods have elucidated their roles in fundamental biological processes
High-yielding 18F radiosynthesis of a novel oxytocin receptor tracer, a probe for nose-to-brain oxytocin uptake in vivo
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
Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation
This is the final version of the article. Available from Elsevier via the DOI in this record.Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.T.C. was funded by an EPSRC grant to the Institute of Chemical Biology, Imperial College London. A.K.-S. was funded by a European Union Seventh Framework Program (EU MCN Grant agreement 237942)
Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites
N-Myristoyltransferase (NMT) has been shown to be essential in Leishmania and subsequently validated as a drug target in Plasmodium. Herein, we discuss the use of antifungal NMT inhibitors as a basis for inhibitor development resulting in the first sub-micromolar peptidomimetic inhibitors of Plasmodium and Leishmania NMTs. High-resolution structures of these inhibitors with Plasmodium and Leishmania NMTs permit a comparative analysis of binding modes, and provide the first crystal structure evidence for a ternary NMT-Coenzyme A/myristoylated peptide product complex
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