20 research outputs found
Chemical tools to validate N-myristoyltransferase as a new target in cancer therapy
N-myristoylation is the irreversible attachment of myristate, a C14-lipid, to the N-terminal glycine of a protein. This modification is catalysed by myristoyl CoA: protein N-myristoyltransferase (NMT). N-myristoylation has been shown to be essential for the viability and survival of many organisms, including plants, parasites and humans. In humans, two isoforms of NMT, HsNMT1 and HsNMT2, were identified and it was suggested that they possess overlapping substrate specificities. NMT was identified as a potential chemotherapeutic target for cancer 18 years ago and subsequent studies showed that NMT was up-regulated in several cancers. However, no study demonstrated that NMT could be a therapeutic target in cancer therapy.
In this study, chemically-tagged analogues of myristic acid were synthesised and applied to cancer cell lines to study protein N-myristoylation. The analogues can be incorporated on the N-terminal glycine of NMT substrates. The tag in this system enables a second extremely selective and high-yielding chemical ligation to any reporter of choice, for instance a dye label. Visualisation of the tagged proteins was carried out by in-gel fluorescence. Tagged-proteins could also be enriched and analysed by mass spectrometry-based proteomics. The combination of chemical proteomics with a potent NMT inhibitor is described in this thesis as a new method to identify with high confidence a large number of NMT substrates.
In search for novel and potent human NMT inhibitors, various compounds were tested using an in vitro assay and a cell cytotoxicity assay. On-target inhibition of NMT in cells was assessed by in-gel fluorescence using analogues of myristic acid.
The best inhibitors were employed as tools to study NMT inhibition in cancer cells. Western blot analysis and flow cytometry were used to assess the phenotype of NMT inhibition.
Finally, the substrate specificity of the two isoforms was studied in cells using siRNAs against HsNMT1 or HsNMT2, and in vitro by screening for activity libraries of peptides against NMT1 or NMT2.Open Acces
Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry
Global profiling of protein lipidation using chemical proteomic technologies
Protein lipidation is unique amongst post-translational modifications (PTMs) in enabling direct interaction with cell membranes, and is found in every form of life. Lipidation is important in normal function and in disease, but its intricate interplay with disease context presents a challenging for drug development. Global whole-proteome profiling of protein lipidation lies beyond the range of standard methods, but is well-suited to metabolic tagging with small ‘clickable’ chemical reporters that do not disrupt metabolism and function; chemoselective reactions are then used to add multifunctional labels exclusively to tagged-lipidated proteins. This chemical proteomic technology has opened up the first quantitative whole-proteome studies of the known major classes of protein lipidation, and the first insights into their full scope in vivo
<i>N</i>‑Myristoyltransferase Inhibition Induces ER-Stress, Cell Cycle Arrest, and Apoptosis in Cancer Cells
<i>N</i>-Myristoyltransferase (NMT) covalently attaches
a C14 fatty acid to the N-terminal glycine of proteins and has been
proposed as a therapeutic target in cancer. We have recently shown
that selective NMT inhibition leads to dose-responsive loss of <i>N</i>-myristoylation on more than 100 protein targets in cells,
and cytotoxicity in cancer cells. <i>N</i>-myristoylation
lies upstream of multiple pro-proliferative and oncogenic pathways,
but to date the complex substrate specificity of NMT has limited determination
of which diseases are most likely to respond to a selective NMT inhibitor.
We describe here the phenotype of NMT inhibition in HeLa cells and
show that cells die through apoptosis following or concurrent with
accumulation in the G1 phase. We used quantitative proteomics to map
protein expression changes for more than 2700 proteins in response
to treatment with an NMT inhibitor in HeLa cells and observed down-regulation
of proteins involved in cell cycle regulation and up-regulation of
proteins involved in the endoplasmic reticulum stress and unfolded
protein response, with similar results in breast (MCF-7, MDA-MB-231)
and colon (HCT116) cancer cell lines. This study describes the cellular
response to NMT inhibition at the proteome level and provides a starting
point for selective targeting of specific diseases with NMT inhibitors,
potentially in combination with other targeted agents
Selective Enrichment and Direct Analysis of Protein S‑Palmitoylation Sites
S-Fatty-acylation is the covalent
attachment of long chain fatty
acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine
(Cys) residues via a thioester linkage on proteins. This post-translational
and reversible lipid modification regulates protein function and localization
in eukaryotes and is important in mammalian physiology and human diseases.
While chemical labeling methods have improved the detection and enrichment
of S-fatty-acylated proteins, mapping sites of modification and characterizing
the endogenously attached fatty acids are still challenging. Here,
we describe the integration and optimization of fatty acid chemical
reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated
proteins and direct tagging of modified Cys residues to selectively
map lipid modification sites. This afforded improved enrichment and
direct identification of many protein S-fatty-acylation sites compared
to previously described methods. Notably, we directly identified the
S-fatty-acylation sites of IFITM3, an important interferon-stimulated
inhibitor of virus entry, and we further demonstrated that the highly
conserved Cys residues are primarily modified by palmitic acid. The
methods described here should facilitate the direct analysis of protein
S-fatty-acylation sites and their endogenously attached fatty acids
in diverse cell types and activation states important for mammalian
physiology and diseases
Selective Enrichment and Direct Analysis of Protein S‑Palmitoylation Sites
S-Fatty-acylation is the covalent
attachment of long chain fatty
acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine
(Cys) residues via a thioester linkage on proteins. This post-translational
and reversible lipid modification regulates protein function and localization
in eukaryotes and is important in mammalian physiology and human diseases.
While chemical labeling methods have improved the detection and enrichment
of S-fatty-acylated proteins, mapping sites of modification and characterizing
the endogenously attached fatty acids are still challenging. Here,
we describe the integration and optimization of fatty acid chemical
reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated
proteins and direct tagging of modified Cys residues to selectively
map lipid modification sites. This afforded improved enrichment and
direct identification of many protein S-fatty-acylation sites compared
to previously described methods. Notably, we directly identified the
S-fatty-acylation sites of IFITM3, an important interferon-stimulated
inhibitor of virus entry, and we further demonstrated that the highly
conserved Cys residues are primarily modified by palmitic acid. The
methods described here should facilitate the direct analysis of protein
S-fatty-acylation sites and their endogenously attached fatty acids
in diverse cell types and activation states important for mammalian
physiology and diseases
Selective Enrichment and Direct Analysis of Protein S‑Palmitoylation Sites
S-Fatty-acylation is the covalent
attachment of long chain fatty
acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine
(Cys) residues via a thioester linkage on proteins. This post-translational
and reversible lipid modification regulates protein function and localization
in eukaryotes and is important in mammalian physiology and human diseases.
While chemical labeling methods have improved the detection and enrichment
of S-fatty-acylated proteins, mapping sites of modification and characterizing
the endogenously attached fatty acids are still challenging. Here,
we describe the integration and optimization of fatty acid chemical
reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated
proteins and direct tagging of modified Cys residues to selectively
map lipid modification sites. This afforded improved enrichment and
direct identification of many protein S-fatty-acylation sites compared
to previously described methods. Notably, we directly identified the
S-fatty-acylation sites of IFITM3, an important interferon-stimulated
inhibitor of virus entry, and we further demonstrated that the highly
conserved Cys residues are primarily modified by palmitic acid. The
methods described here should facilitate the direct analysis of protein
S-fatty-acylation sites and their endogenously attached fatty acids
in diverse cell types and activation states important for mammalian
physiology and diseases
Selective Enrichment and Direct Analysis of Protein S‑Palmitoylation Sites
S-Fatty-acylation is the covalent
attachment of long chain fatty
acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine
(Cys) residues via a thioester linkage on proteins. This post-translational
and reversible lipid modification regulates protein function and localization
in eukaryotes and is important in mammalian physiology and human diseases.
While chemical labeling methods have improved the detection and enrichment
of S-fatty-acylated proteins, mapping sites of modification and characterizing
the endogenously attached fatty acids are still challenging. Here,
we describe the integration and optimization of fatty acid chemical
reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated
proteins and direct tagging of modified Cys residues to selectively
map lipid modification sites. This afforded improved enrichment and
direct identification of many protein S-fatty-acylation sites compared
to previously described methods. Notably, we directly identified the
S-fatty-acylation sites of IFITM3, an important interferon-stimulated
inhibitor of virus entry, and we further demonstrated that the highly
conserved Cys residues are primarily modified by palmitic acid. The
methods described here should facilitate the direct analysis of protein
S-fatty-acylation sites and their endogenously attached fatty acids
in diverse cell types and activation states important for mammalian
physiology and diseases
Selective Enrichment and Direct Analysis of Protein S‑Palmitoylation Sites
S-Fatty-acylation is the covalent
attachment of long chain fatty
acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine
(Cys) residues via a thioester linkage on proteins. This post-translational
and reversible lipid modification regulates protein function and localization
in eukaryotes and is important in mammalian physiology and human diseases.
While chemical labeling methods have improved the detection and enrichment
of S-fatty-acylated proteins, mapping sites of modification and characterizing
the endogenously attached fatty acids are still challenging. Here,
we describe the integration and optimization of fatty acid chemical
reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated
proteins and direct tagging of modified Cys residues to selectively
map lipid modification sites. This afforded improved enrichment and
direct identification of many protein S-fatty-acylation sites compared
to previously described methods. Notably, we directly identified the
S-fatty-acylation sites of IFITM3, an important interferon-stimulated
inhibitor of virus entry, and we further demonstrated that the highly
conserved Cys residues are primarily modified by palmitic acid. The
methods described here should facilitate the direct analysis of protein
S-fatty-acylation sites and their endogenously attached fatty acids
in diverse cell types and activation states important for mammalian
physiology and diseases
Selective Enrichment and Direct Analysis of Protein S‑Palmitoylation Sites
S-Fatty-acylation is the covalent
attachment of long chain fatty
acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine
(Cys) residues via a thioester linkage on proteins. This post-translational
and reversible lipid modification regulates protein function and localization
in eukaryotes and is important in mammalian physiology and human diseases.
While chemical labeling methods have improved the detection and enrichment
of S-fatty-acylated proteins, mapping sites of modification and characterizing
the endogenously attached fatty acids are still challenging. Here,
we describe the integration and optimization of fatty acid chemical
reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated
proteins and direct tagging of modified Cys residues to selectively
map lipid modification sites. This afforded improved enrichment and
direct identification of many protein S-fatty-acylation sites compared
to previously described methods. Notably, we directly identified the
S-fatty-acylation sites of IFITM3, an important interferon-stimulated
inhibitor of virus entry, and we further demonstrated that the highly
conserved Cys residues are primarily modified by palmitic acid. The
methods described here should facilitate the direct analysis of protein
S-fatty-acylation sites and their endogenously attached fatty acids
in diverse cell types and activation states important for mammalian
physiology and diseases