A new, robust, and nonradioactive approach for exploring N-myristoylation

Rampoldi, Francesca; Sandhoff, Roger; Owen, Robert W.; Gröne, Hermann Josef; Porubský, Štefan

doi:10.1194/jlr.d026997

Cited by 12 publications

(18 citation statements)

References 55 publications

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“…The first detects fluorometrically the released CoA-SH and is thus generally sensitive to hydrolyzing compounds in the screening context [39], [53]. In the second assay a click-chemistry amenable myristate-analogue is utilized and detected by an ELISA-assay like procedure in both cellular and tissue samples [54].…”

Section: Resultsmentioning

confidence: 99%

Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins

et al. 2013

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Hundreds of eukaryotic signaling proteins require myristoylation to functionally associate with intracellular membranes. N-myristoyl transferases (NMT) responsible for this modification are established drug targets in cancer and infectious diseases. Here we describe NANOMS (NANOclustering and Myristoylation Sensors), biosensors that exploit the FRET resulting from plasma membrane nanoclustering of myristoylated membrane targeting sequences of Gαi2, Yes- or Src-kinases fused to fluorescent proteins. When expressed in mammalian cells, NANOMS report on loss of membrane anchorage due to chemical or genetic inhibition of myristoylation e.g. by blocking NMT and methionine-aminopeptidase (Met-AP). We used Yes-NANOMS to assess inhibitors of NMT and a cherry-picked compound library of putative Met-AP inhibitors. Thus we successfully confirmed the activity of DDD85646 and fumagillin in our cellular assay. The developed assay is unique in its ability to identify modulators of signaling protein nanoclustering, and is amenable to high throughput screening for chemical or genetic inhibitors of functional membrane anchorage of myristoylated proteins in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins

et al. 2013

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“…Thymus is the hub for T cell development and has been shown to have high NMT activities (41). The thymic microenvironment consists of two anatomical sites: the cortex and the medulla.…”

Section: The Role Of N-myristoylation In Hematopoiesismentioning

confidence: 99%

Myristoylation: An Important Protein Modification in the Immune Response

et al. 2017

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Protein N-myristoylation is a cotranslational lipidic modification specific to the alpha-amino group of an N-terminal glycine residue of many eukaryotic and viral proteins. The ubiquitous eukaryotic enzyme, N-myristoyltransferase, catalyzes the myristoylation process. Precisely, attachment of a myristoyl group increases specific protein–protein interactions leading to subcellular localization of myristoylated proteins with its signaling partners. The birth of the field of myristoylation, a little over three decades ago, has led to the understanding of the significance of protein myristoylation in regulating cellular signaling pathways in several biological processes especially in carcinogenesis and more recently immune function. This review discusses myristoylation as a prerequisite step in initiating many immune cell signaling cascades. In particular, we discuss the hitherto unappreciated implication of myristoylation during myelopoiesis, innate immune response, lymphopoiesis for T cells, and the formation of the immunological synapse. Furthermore, we discuss the role of myristoylation in inducing the virological synapse during human immunodeficiency virus infection as well as its clinical implication. This review aims to summarize existing knowledge in the field and to highlight gaps in our understanding of the role of myristoylation in immune function so as to further investigate into the dynamics of myristoylation-dependent immune regulation.

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“…The absence of myristoylation in T cells positive for Lck-Cre and carrying NMT1 and/or NMT2 floxed alleles was demonstrated by measuring the Nmt activity using a previously established ELISAbased method (15). T cells extracted from the Lck-Cre + Nmt1 f/f / Nmt2 f/f mice did not show Nmt activity, whereas a partial reduction of the activity could be observed in the single mutant mice.…”

Section: Generation Of Mice With T Cell-specific Depletion Of Nmt Actmentioning

confidence: 99%

“…We have previously demonstrated that thymus, in comparison with other organs, displays a strikingly high Nmt activity (15). To elucidate the role of protein myristoylation for the development and function of T cells, we have now generated mice with floxed Nmt1 and Nmt2 alleles and crossed them with mice expressing Cre under the control of the proximal Lck promoter, thus initiating recombinase activity during early thymocyte development (16).…”

Section: At This Stage Immature T Cells Are Called Doublenegative mentioning

confidence: 99%

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation

Rampoldi

Bonrouhi

Boehm

et al. 2015

The Journal of Immunology

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N-myristoylation refers to the attachment of myristic acid to the N-terminal glycine of proteins and substantially affects their intracellular targeting and functions. The thymus represents an organ with a prominent N-myristoylation activity. To elucidate the role of protein N-myristoylation for thymocyte development, we generated mice with a T cell lineage–specific deficiency in N-myristoyl transferase (Nmt)1 and 2. Depletion of Nmt activity in T cells led to a defective transmission of TCR signals, a developmental blockage of thymocytes at the transition from double-negative 3 to 4 stages, and a reduction of all the following stages. We could demonstrate that Lck and myristoylated alanine-rich C kinase substrate, two main myristoylated kinases in T cells, were mislocalized in the absence of Nmt activity. N-myristoylation was also indispensable for early and distal TCR signaling events such as CD3ζ, Zap70, and Erk activation and for release of cytokines such as IFN-γ and IL-2. As a consequence, the initiation and propagation of the TCR signaling cascade was severely impaired. Furthermore, we showed that the absence of myristoylation had an immunosuppressive effect on T cells in vivo after treatment with CpG and stimulation of the TCR with the staphylococcal enterotoxin B superantigen. Therefore, protein myristoylation is indispensable in T cell development and activation and its inhibition might offer a novel strategy to achieve immunosuppression.

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A new, robust, and nonradioactive approach for exploring N-myristoylation

Cited by 12 publications

References 55 publications

Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins

Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins

Myristoylation: An Important Protein Modification in the Immune Response

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation

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