RESUMO
Protein S-fatty-acylation, the covalent addition of a long-chain fatty acid, predominantly palmitate (S-palmitoylation), to cysteine, is a highly dynamic and regulated process that controls protein function and localization of membrane-associated proteins in eukaryotes. The analysis of S-fatty acylated peptides by mass spectrometry remains challenging due to the hydrophobic and potentially labile thioester linkage of the S-fatty acylated peptides.Here we describe an optimized protocol for the global analysis of S-palmitoylated proteins based on the combination of an alkyne-tagged chemical reporter of palmitoylation, alk-16 with hydroxylamine-selective hydrolysis of thioester bonds. This protocol decreased the number of false positive proteins and was applied to identify S-fatty acylation sites, providing modification sites for 44 proteins out of the 106 S-fatty acylated proteins identified.
Assuntos
Lipoproteínas , Lipoilação , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Acilação , Células HeLa , Humanos , Lipoproteínas/análise , Lipoproteínas/metabolismoRESUMO
BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations.
Assuntos
Aquaporinas/metabolismo , Água Corporal/metabolismo , Cálcio/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Processamento de Proteína Pós-Traducional , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Western Blotting , Caspases/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Cristalino/citologia , Células MCF-7/metabolismo , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Dados de Sequência Molecular , Miristatos/metabolismo , Oócitos , Domínios Proteicos , Transfecção , Xenopus laevis , Adulto JovemRESUMO
Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR-Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Vesículas Transportadoras/fisiologia , Células A549 , Animais , Antígenos de Diferenciação/metabolismo , Antivirais , Endossomos/fisiologia , Células HeLa , Humanos , Lisossomos/fisiologia , Imagem Óptica/métodos , Transporte Proteico , Vírion/patogenicidade , Internalização do VírusRESUMO
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.
Assuntos
Cisteína/metabolismo , Lipoilação , Ácido Palmítico/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Acilação , Animais , Sítios de Ligação , Ácidos Graxos/metabolismo , Humanos , Hidroxilamina , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Coloração e RotulagemRESUMO
The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S-fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods developed over the past decade have improved the detection and profiling of S-fatty acylation, these are hampered in their ability to characterize endogenous protein S-fatty acylation levels under physiological conditions. Furthermore, understanding the contribution of specific sites and levels of S-fatty acylation remains a major challenge. To evaluate S-fatty acylation of endogenous proteins as well as to determine the number of S-fatty acylation events, we developed the acyl-PEG exchange (APE) that utilizes cysteine-specific chemistry to exchange S-fatty acylation sites with mass-tags of defined size, which can be readily observed by western blotting. APE provides a readily accessible approach to investigate endogenous S-fatty acylation from any sample source, with high sensitivity and broad applicability that complements the current toolbox of methods for thioester-based post-translational modifications. © 2017 by John Wiley & Sons, Inc.
Assuntos
Acrilatos/química , Cisteína/química , Ácidos Graxos/química , Polietilenoglicóis/química , Proteínas/química , Acilação , Linhagem Celular , Células HEK293 , Humanos , Maleimidas/química , Ligação ProteicaRESUMO
We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Animais , Técnicas Biossensoriais , Células COS , Chlorocebus aethiops , Humanos , Imagem Óptica , Software , Produtos do Gene gag do Vírus da Imunodeficiência Humana/químicaRESUMO
N-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 N-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. N-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.
Assuntos
Aciltransferases/antagonistas & inibidores , Apoptose , Pontos de Checagem do Ciclo Celular , Estresse do Retículo Endoplasmático , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , ProteomaRESUMO
Dietary unsaturated fatty acids, such as oleic acid, have been shown to be covalently incorporated into a small subset of proteins, but the generality and diversity of this protein modification has not been studied. We synthesized unsaturated fatty-acid chemical reporters and determined their protein targets in mammalian cells. The reporters can induce the formation of lipid droplets and be incorporated site-specifically onto known fatty-acylated proteins and label many proteins in mammalian cells. Quantitative proteomics analysis revealed that unsaturated fatty acids modify similar protein targets to saturated fatty acids, including several immunity-associated proteins. This demonstrates that unsaturated fatty acids can directly modify many proteins to exert their unique and often beneficial physiological effects in vivo.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Proteínas/análise , Proteínas/química , Proteômica , Acilação , Animais , Linhagem Celular , Ácidos Graxos Insaturados/química , Células HeLa , Humanos , Camundongos , Proteínas/metabolismoRESUMO
Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.
Assuntos
Cisteína/metabolismo , Ácidos Graxos/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Coloração e Rotulagem/métodos , Acilação , Animais , CamundongosRESUMO
In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed "RU-SKI") class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article "Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase" (Lanyon-Hogg et al., 2015) [1]. (1)H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.
RESUMO
Protein fatty-acylation in eukaryotes has been associated with many fundamental biological processes. However, the diversity, abundance and regulatory mechanisms of protein fatty-acylation in vivo remain to be explored. Herein, we review the proteomic analysis of fatty-acylated proteins, with a focus on N-myristoylation and S-palmitoylation. We then highlight major challenges and emerging methods for direct site identification, quantitation, and lipid structure characterization to understand the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.
Assuntos
Ácidos Graxos/metabolismo , Proteínas/metabolismo , Proteômica/métodos , Acilação , Animais , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl ß-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.
Assuntos
Aciltransferases/metabolismo , Proteínas Hedgehog/metabolismo , Processamento de Proteína Pós-Traducional , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Aciltransferases/genética , Biotinilação , Química Click , Detergentes/química , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Ácidos Graxos Insaturados/farmacologia , Células HEK293 , Proteínas Hedgehog/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Lipoilação/efeitos dos fármacos , Maltose/análogos & derivados , Maltose/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Palmitoil Coenzima A/análogos & derivados , Palmitoil Coenzima A/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Estreptavidina/química , Estreptavidina/metabolismo , Especificidade por SubstratoRESUMO
Proteins are acylated by a variety of metabolites that regulates many important cellular pathways in all kingdoms of life. Acyl groups in cells can vary in structure from the smallest unit, acetate, to modified long-chain fatty acids, all of which can be activated and covalently attached to diverse amino acid side chains and consequently modulate protein function. For example, acetylation of Lys residues can alter the charge state of proteins and generate new recognition elements for protein-protein interactions. Alternatively, long-chain fatty-acylation targets proteins to membranes and enables spatial control of cell signalling. To facilitate the analysis of protein acylation in biology, acyl analogues bearing alkyne or azide tags have been developed that enable fluorescent imaging and proteomic profiling of modified proteins using bioorthogonal ligation methods. Herein, we summarize the currently available acylation chemical reporters and highlight their utility to discover and quantify the roles of protein acylation in biology.
Assuntos
Acilação/genética , Proteínas/metabolismo , Proteômica , Humanos , Redes e Vias Metabólicas/genética , Mapas de Interação de Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteínas/químicaRESUMO
Nearly isosteric oxo to thioxo substitution was employed to interrogate the structure of foldamers with a urea backbone and explore the relationship between helical folding and hydrogen-bonding interactions. A series of oligomers with urea bonds substituted by thiourea bonds at discrete or all positions in the sequence have been prepared and their folding propensity was studied by using a combination of spectroscopic methods and X-ray diffraction. The outcome of oxo to thioxo replacements on the helical folding was found to depend on whether central or terminal ureas were modified. The canonical helix geometry was not affected upon insertion of thioureas close to the negative end of the helix dipole, whereas thioureas close to the positive pole were found to increase the terminal flexibility and cause helix fraying. Perturbation was amplified when a selenourea was incorporated instead, leading to a structure that is only partly folded.
Assuntos
Compostos Organosselênicos/química , Tioureia/química , Ureia/análogos & derivados , Ureia/química , Dicroísmo Circular , Modelos Moleculares , Estrutura MolecularRESUMO
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.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas/química , Acilação , Animais , Colesterol/análise , Colesterol/metabolismo , Glicosilfosfatidilinositóis/análise , Glicosilfosfatidilinositóis/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipídeos/análise , Proteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteômica/métodosRESUMO
Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells.
Assuntos
Proteoma/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Cristalografia por Raios X , Células HeLa , Humanos , Ácido Mirístico/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/genéticaRESUMO
Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Multimerização Proteica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Automação , Células HeLa , Humanos , Estrutura Quaternária de ProteínaRESUMO
N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.
Assuntos
Aciltransferases/análise , Aciltransferases/antagonistas & inibidores , Aciltransferases/genética , Aciltransferases/metabolismo , Coenzima A , Cumarínicos , Fluorescência , Corantes Fluorescentes , Humanos , Cinética , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.