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      Global Analysis of Protein N-Myristoylation and Exploration of N-Myristoyltransferase as a Drug Target in the Neglected Human Pathogen Leishmania donovani

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          Summary

          N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen.

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          Highlights

          • •

            Alkyne-tagged probes for protein lipidation are applied in Leishmania donovani

          • •

            A global profile of protein lipidation in two life stages is presented

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            N-Myristoylated proteome defined by chemical knockdown and quantitative proteomics

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            Evidence for NMT as a drug target in the leishmaniases

          Abstract

          Wright et al. use metabolic tagging with an alkyne-myristate analog, click chemistry, and proteomics to identify lipidated proteins in two life stages of the parasite Leishmania donovani. Quantitative profiling of N-myristoyltransferase inhibition and identification of lipidation sites define the N-myristoylated proteome in this human pathogen.

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          Nonenzymatic protein acylation as a carbon stress regulated by sirtuin deacylases.

          Matthew Hirschey, Bradley Wagner (2014)
          Cellular proteins are decorated with a wide range of acetyl and other acyl modifications. Many studies have demonstrated regulation of site-specific acetylation by acetyltransferases and deacetylases. Acylation is emerging as a new type of lysine modification, but less is known about its overall regulatory role. Furthermore, the mechanisms of lysine acylation, its overlap with protein acetylation, and how it influences cellular function are major unanswered questions in the field. In this review, we discuss the known roles of acetyltransferases and deacetylases and the sirtuins as a conserved family of a nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacylases that are important for response to cellular stress and homeostasis. We also consider the evidence for an emerging idea of nonenzymatic protein acylation. Finally, we put forward the hypothesis that protein acylation is a form of protein "carbon stress" that the deacylases evolved to remove as a part of a global protein quality-control network. Copyright © 2014 Elsevier Inc. All rights reserved.
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            N-Myristoyltransferase inhibitors as new leads to treat sleeping sickness

            Julie A. Frearson, Stephen Brand, Stuart McElroy … (2010)
            African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease, when the parasite has infected the central nervous system. Here, we report the validation of a molecular target and discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT.
            Article 0 comments Cited 107 times – based on 0 reviews     Review now
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              Retooling Leishmania metabolism: from sand fly gut to human macrophage.

              Fred R. Opperdoes, Robert Olafson, Derek Smith … (2008)
              To survive extremely different environments, intracellular parasites require highly adaptable physiological and metabolic systems. Leishmania donovani extracellular promastigotes reside in a glucose-rich, slightly alkaline environment in the sand fly vector alimentary tract. On entry into human macrophage phagolysosomes, promastigotes differentiate into intracellular amastigotes. These cope with an acidic milieu, where glucose is scarce while amino acids are abundant. Here, we use an axenic differentiation model and a novel high-coverage, comparative proteomic methodology to analyze in detail protein expression changes throughout the differentiation process. The analysis identified and quantified 21% of the parasite proteome across 7 time points during differentiation. The data reveal a delayed increase in gluconeogenesis enzymes, coinciding with a decrease in glycolytic capacity. At the same time, beta-oxidation, amino acid catabolism, tricarboxylic acid cycle, mitochondrial respiration chain, and oxidative phosphorylation capacities are all up-regulated. The results indicate that the differentiating parasite shifts from glucose to fatty acids and amino acids as its main energy source. Furthermore, glycerol and amino acids are used as precursors for sugar synthesis, compensating for lack of exogenous sugars. These changes occur while promastigotes undergo morphological transformation. Our findings provide new insight into changes occurring in single-cell organisms during a developmental process.
              Article 0 comments Cited 97 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Megan H. Wright
                Edward W. Tate
                Journal
                Journal ID (nlm-ta): Chem Biol
                Journal ID (iso-abbrev): Chem. Biol
                Title: Chemistry & Biology
                Publisher: Elsevier
                ISSN (Print): 1074-5521
                ISSN (Electronic): 1879-1301
                Publication date PMC-release: 19 March 2015
                Publication date (Print): 19 March 2015
                Volume: 22
                Issue: 3
                Pages: 342-354
                Affiliations
                [1 ]Department of Chemistry, Imperial College London, London SW7 2AZ, UK
                [2 ]Centre for Immunology and Infection, Department of Biology, University of York, York YO10 5DD, UK
                Author notes
                [∗ ]Corresponding author megan.wright@ 123456tum.de
                [∗∗ ]Corresponding author e.tate@ 123456imperial.ac.uk
                [3]

                Present address: Department of Chemistry, Technische Universität München, Garching 85478, Germany

                [4]

                Present address: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK

                Article
                Publisher ID: S1074-5521(15)00034-4
                DOI: 10.1016/j.chembiol.2015.01.003
                PMC ID: 4372256
                PubMed ID: 25728269
                SO-VID: 73ed20a0-8542-4b03-8119-4e44b3a235ea
                Copyright © © 2015 The Authors
                License:

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 16 October 2014
                Date revision received : 16 December 2014
                Date accepted : 13 January 2015
                Categories
                Subject: Article

                ScienceOpen disciplines: Biochemistry
                Data availability:
                ScienceOpen disciplines: Biochemistry

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