The Experts below are selected from a list of 6165 Experts worldwide ranked by ideXlab platform
Toshihiko Utsumi - One of the best experts on this subject based on the ideXlab platform.
-
a strategy to identify protein n Myristoylation dependent phosphorylation reactions of cellular proteins by using phos tag sds page
PLOS ONE, 2019Co-Authors: Emiko Kinoshitakikuta, Koko Moriya, Aya Kiwado, Ayane Tanikawa, Takuro Hosokawa, Eiji Kinoshita, Tohru Koike, Toshihiko UtsumiAbstract:To establish a strategy for identifying protein-N-Myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-Myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-Myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-Myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin β1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.
-
identification and characterization of protein n Myristoylation occurring on four human mitochondrial proteins samm50 tomm40 mic19 and mic25
PLOS ONE, 2018Co-Authors: Toshihiko Utsumi, Kanako Matsuzaki, Aya Kiwado, Ayane Tanikawa, Yuki Kikkawa, Takuro Hosokawa, Aoi Otsuka, Yoshihito Iuchi, Hirotsugu Kobuchi, Koko MoriyaAbstract:Previously, we showed that SAMM50, a mitochondrial outer membrane protein, is N-myristoylated, and this lipid modification is required for the proper targeting of SAMM50 to mitochondria. In this study, we characterized protein N-Myristoylation occurring on four human mitochondrial proteins, SAMM50, TOMM40, MIC19, and MIC25, three of which are components of the mitochondrial intermembrane space bridging (MIB) complex, which plays a critical role in the structure and function of mitochondria. In vitro and in vivo metabolic labeling experiments revealed that all four of these proteins were N-myristoylated. Analysis of intracellular localization of wild-type and non-myristoylated G2A mutants of these proteins by immunofluorescence microscopic analysis and subcellular fractionation analysis indicated that protein N-Myristoylation plays a critical role in mitochondrial targeting and membrane binding of two MIB components, SAMM50 and MIC19, but not those of TOMM40 and MIC25. Immunoprecipitation experiments using specific antibodies revealed that MIC19, but not MIC25, was a major N-myristoylated binding partner of SAMM50. Immunoprecipitation experiments using a stable transformant of MIC19 confirmed that protein N-Myristoylation of MIC19 is required for the interaction between MIC19 and SAMM50, as reported previously. Thus, protein N-Myristoylation occurring on two mitochondrial MIB components, SAMM50 and MIC19, plays a critical role in the mitochondrial targeting and protein-protein interaction between these two MIB components.
-
Schematic representation of the intracellular localization and protein-protein interactions of SAMM50, TOMM40, and MIC19.
2018Co-Authors: Toshihiko Utsumi, Kanako Matsuzaki, Aya Kiwado, Ayane Tanikawa, Yuki Kikkawa, Takuro Hosokawa, Aoi Otsuka, Yoshihito Iuchi, Hirotsugu Kobuchi, Koko MoriyaAbstract:SAMM50, TOMM40, MIC19, and MIC25 were cotranslationally N-myristoylated in the cytoplasm. These proteins are transported into the intermembrane space of mitochondria through the TOM complex. For SAMM50 and MIC19, protein N-Myristoylation is required for mitochondrial targeting. An oligomeric form of N-myristoylated MIC19 seems to associate with N-myristoylated SAMM50. Protein N-Myristoylation of MIC19 is required for the binding of MIC19 to SAMM50.
-
Protein N-Myristoylation is required for cellular morphological changes induced by two formin family proteins, FMNL2 and FMNL3
Bioscience biotechnology and biochemistry, 2012Co-Authors: Koko Moriya, Takashi Suzuki, Takuo Yamamoto, Emi Takamitsu, Yukari Matsunaga, Mayumi Kimoto, Daichi Fukushige, Chihiro Kimoto, Toshihiko UtsumiAbstract:The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-Myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs coding for the proteins revealed that most of them were associated with the plasma membrane or the membranes of intracellular compartments, and did not affect cellular morphology. However, two proteins, formin-like2 (FMNL2) and formin-like3 (FMNL3), both of them are members of the formin family of proteins, were associated mainly with the plasma membrane and induced significant cellular morphological changes. Inhibition of protein N-Myristoylation by replacement of Gly2 with Ala or by the use of N-Myristoylation inhibitor significantly inhibited membrane localization and the induction of cellular morphological changes, indicating that protein N-Myristoylation plays critical roles in the cellular morphological changes induced by FMNL2 and FMNL3.
-
The consensus motif for N-Myristoylation of plant proteins in a wheat germ cell-free translation system
The FEBS journal, 2010Co-Authors: Seiji Yamauchi, Toshihiko Utsumi, Naoki Fusada, Hidenori Hayashi, Nobuyuki Uozumi, Yaeta Endo, Yuzuru TozawaAbstract:Protein N-Myristoylation plays key roles in various cellular functions in eukaryotic organisms. To clarify the relationship between the efficiency of protein N-Myristoylation and the amino acid sequence of the substrate in plants, we have applied a wheat germ cell-free translation system with high protein productivity to examine the N-Myristoylation of various wild-type and mutant forms of Arabidopsis thaliana proteins. Evaluation of the relationship between removal of the initiating Met and subsequent N-Myristoylation revealed that constructs containing Pro at position 3 do not undergo N-Myristoylation, primarily because of an inhibitory effect of this amino acid on elimination of the initiating Met by methionyl aminopeptidase. Our analysis of the consensus sequence for N-Myristoylation in plants focused on the variability of amino acids at positions 3, 6 and 7 of the motif. We found that not only Ser at position 6 but also Lys at position 7 affects the selectivity for the amino acid at position 3. The results of our analyses allowed us to identify several A. thaliana proteins as substrates for N-Myristoylation that had previously been predicted not to be candidates for such modification with a prediction program. We have thus shown that a wheat germ cell-free system is a useful tool for plant N-myristoylome analysis. This in vitro approach will facilitate comprehensive determination of N-myristoylated proteins in plants.
Anuraag Shrivastav - One of the best experts on this subject based on the ideXlab platform.
-
Schematic representation of protein N-Myristoylation.
2018Co-Authors: Ranjit Chauhan, Shailly Varma Shrivastav, David Datzkiw, Anuraag ShrivastavAbstract:Schematic representation of protein N-Myristoylation.
-
In silico identification of microRNAs predicted to regulate N-myristoyltransferase and Methionine Aminopeptidase 2 functions in cancer and infectious diseases
2018Co-Authors: Ranjit Chauhan, Shailly Varma Shrivastav, David Datzkiw, Anuraag ShrivastavAbstract:Protein Myristoylation is a key protein modification carried out by N-Myristoyltransferase (NMT) after Methionine aminopeptidase 2 (MetAP2) removes methionine from the amino-terminus of the target protein. Protein Myristoylation by NMT augments several signaling pathways involved in a myriad of cellular processes, including developmental pathways and pathways that when dysregulated lead to cancer or immune dysfunction. The emerging evidence pointing to NMT-mediated Myristoylation as a major cellular regulator underscores the importance of understanding the framework of this type of signaling event. Various studies have investigated the role that Myristoylation plays in signaling dysfunction by examining differential gene or protein expression between normal and diseased states, such as cancers or following HIV-1 infection, however no study exists that addresses the role of microRNAs (miRNAs) in the regulation of Myristoylation. By performing a large scale bioinformatics and functional analysis of the miRNAs that target key genes involved in Myristoylation (NMT1, NMT2, MetAP2), we have narrowed down a list of promising candidates for further analysis. Our condensed panel of miRNAs identifies 35 miRNAs linked to cancer, 21 miRNAs linked to developmental and immune signaling pathways, and 14 miRNAs linked to infectious disease (primarily HIV). The miRNAs panel that was analyzed revealed several NMT-targeting mRNAs (messenger RNA) that are implicated in diseases associated with NMT signaling alteration, providing a link between the realms of miRNA and Myristoylation signaling. These findings verify miRNA as an additional facet of Myristoylation signaling that must be considered to gain a full perspective. This study provides the groundwork for future studies concerning NMT-transcript-binding miRNAs, and will potentially lead to the development of new diagnostic/prognostic biomarkers and therapeutic targets for several important diseases.
-
Myristoylation: An Important Protein Modification in the Immune Response.
Frontiers in immunology, 2017Co-Authors: Daniel Ikenna Udenwobele, Sara V. Good, Terry B. Ball, Shailly Varma Shrivastav, Anuraag ShrivastavAbstract:Increasing biochemical and genetic evidence confirms that Myristoylation is an evolutionary conserved process of lipid modification in many eukaryotic and viral proteins. The discovery that the N-terminal blocking group of cyclic AMP-dependent protein kinase is a myristic acid, some 33 years ago, led to the birth of the field of ‘Myristoylation’, dedicated to understanding the significance of protein Myristoylation. Although the field is in its infancy, an increasing number of studies have appeared addressing the mechanisms and significance of N-Myristoylation in various aspects of cellular signaling. N-Myristoylation is known to occur in mammalian, plant, viral and fungal proteins. Many proteins involved in a variety of signal cascades and cellular differentiation are myristoylated. These include the catalytic subunit of cAMP-dependent protein kinase, the β-subunit of calcineurin, the α-subunit of several G-proteins, the cellular and transforming forms of pp60src. Given the indispensible role of Myristoylation in cellular functions, in this review, we examine the myriad functional implications of Myristoylation with respect to immune function and regulation.
Thierry Meinnel - One of the best experts on this subject based on the ideXlab platform.
-
Targeted profiling of A. thaliana sub-proteomes illuminates new co- and post-translationally N-terminal Myristoylated proteins
The Plant cell, 2018Co-Authors: Wojciech Majeran, Jean-pierre Le Caer, Lalit Ponnala, Thierry Meinnel, Carmela GiglioneAbstract:N-terminal Myristoylation, a major eukaryotic protein lipid modification, is difficult to detect in vivo and challenging to predict in silico. We developed a proteomics strategy involving subfractionation of cellular membranes, combined with separation of hydrophobic peptides by mass spectrometry-coupled liquid chromatography to identify the Arabidopsis thaliana myristoylated proteome. This approach identified a starting pool of 8837 proteins in all analyzed cellular fractions, comprising 32% of the Arabidopsis proteome. Of these, 906 proteins contain an N-terminal Gly at position 2, a prerequisite for Myristoylation, and 214 belong to the predicted myristoylome (comprising 51% of the predicted myristoylome of 421 proteins). We further show direct evidence of Myristoylation in 72 proteins; 18 of these myristoylated proteins were not previously predicted. We found one Myristoylation site downstream of a predicted initiation codon, indicating that posttranslational Myristoylation occurs in plants. Over half of the identified proteins could be quantified and assigned to a subcellular compartment. Hierarchical clustering of protein accumulation combined with Myristoylation and S-acylation data revealed that N-terminal double acylation influences redirection to the plasma membrane. In a few cases, MYR function extended beyond simple membrane association. This study identified hundreds of N-acylated proteins for which lipid modifications could control protein localization and expand protein function.
-
High-throughput profiling of N-Myristoylation substrate specificity across species including pathogens.
Proteomics, 2013Co-Authors: José A. Traverso, Carmela Giglione, Thierry MeinnelAbstract:One of the most critical modifications affecting the N-terminus of proteins is N-Myristoylation. This irreversible modification affects the membrane-binding properties of crucial proteins involved in signal transduction cascades. This cotranslational modification, catalyzed by N-myristoyl transferase, occurs both in lower and higher eukaryotes and is a validated therapeutic target for several pathologies. However, this lipidation proves very difficult to be evidenced in vivo even with state-of-the-art proteomics approaches or bioinformatics tools. A large part of N-myristoylated proteins remains to be discovered and the rules of substrate specificity need to be established in each organism. Because the peptide substrate recognition occurs around the first eight residues, short peptides are used for modeling the reaction in vitro. Here, we provide a novel approach including a dedicated peptide array for high-throughput profiling protein N-Myristoylation specificity. We show that Myristoylation predictive tools need to be fine-tuned to organisms and that their poor accuracy should be significantly enhanced. This should lead to strongly improved knowledge of the number and function of myristoylated proteins occurring in any proteome.
-
A continuous assay of myristoyl-CoA:protein N-myristoyltransferase for proteomic analysis.
Analytical Biochemistry, 2003Co-Authors: Bertrand Boisson, Thierry MeinnelAbstract:Protein N-Myristoylation is an important lipid modification that affects the activity and membrane-binding properties of crucial proteins belonging to signal transduction cascades. The aim of this work was to develop a rapid and easy diagnostic method to check for (i) effective N-Myristoylation of any given protein and (ii) easy proteome annotation. The N-Myristoylation reaction was coupled to that of pyruvate dehydrogenase, and NADH was continuously detected spectrophotometrically. This method was optimized for and applied to full-length Saccharomyces cerevisiae and Arabidopsis thaliana N-myristoyltransferases and two A. thaliana enzyme derivatives. The data were validated by comparison with a previously described discontinuous assay, modification of the chemical nature of the substrates, and use of specific inhibitors. The kinetics of N-Myristoylation were determined in vitro with various compounds including a full-length polypeptide substrate, a small G protein of the RAB family already known to be N-myristoylated in vivo. This automated assay can be used for proteomic studies to determine the N-Myristoylation state of any protein.
-
Unexpected protein families including cell defense components feature in the N-myristoylome of a higher eukaryote.
Journal of Biological Chemistry, 2003Co-Authors: Bertrand Boisson, Carmela Giglione, Thierry MeinnelAbstract:N-Myristoylation is an irreversible modification that affects the membrane binding properties of crucial cytoplasmic proteins from signal transduction cascades. We characterized the two putative N-myristoyltransferases of Arabidopsis thaliana as a means of investigating the entire N-Myristoylation proteome (N-myristoylome) in a higher eukaryote. AtNMT1 compensated for the nmt1 defect in yeast, whereas AtNMT2 and chimeras of the two genes did not. Only AtNMT1 modified known N-myristoylated proteins in vitro. AtNMT1 is therefore responsible for the A. thaliana N-myristoylome, whereas AtNMT2 does not seem to have usual Myristoylation activity. We began with the whole set of N-myristoylated G proteins in the A. thaliana proteome. We then used a reiterative approach, based on the in vitro N-Myristoylation of more than 60 different polypeptides, to determine the substrate specificity of AtNMT1. We found that the positive charge on residue 7 of the substrate was particularly important in substrate recognition. The A. thaliana N-myristoylome consists of 437 proteins, accounting for 1.7% of the complete proteome. We demonstrated the N-Myristoylation of several unexpected protein families, including innate immunity proteins, thioredoxins, components of the protein degradation pathway, transcription factors, and a crucial regulatory enzyme of glycolysis. The role of N-Myristoylation is discussed in each case; in particular, this process may underlie the "guard" hypothesis of innate immunity.
Nicholas K Tonks - One of the best experts on this subject based on the ideXlab platform.
-
Myristoylation of the dual specificity phosphatase c jun n terminal kinase jnk stimulatory phosphatase 1 is necessary for its activation of jnk signaling and apoptosis
FEBS Journal, 2010Co-Authors: Ulla Schwertassek, Deirdre A. Buckley, Andrew J. Lindsay, Mary W. Mccaffrey, Thomas A. Neubert, Nicholas K TonksAbstract:Activation of the c-JUN N-terminal kinase (JNK) pathway is implicated in a number of important physiological processes, from embryonic morphogenesis to cell survival and apoptosis. JNK stimulatory phosphatase 1 (JSP1) is a member of the dual-specificity phosphatase subfamily of protein tyrosine phosphatases. In contrast to other dual-specificity phosphatases that catalyze the inactivation of mitogen-activated protein kinases, expression of JSP1 activates JNK-mediated signaling. JSP1 and its relative DUSP15 are unique among members of the protein tyrosine phosphatase family in that they contain a potential Myristoylation site at the N-terminus (MGNGMXK). In this study, we investigated whether JSP1 was myristoylated and examined the functional consequences of Myristoylation. Using mass spectrometry, we showed that wild-type JSP1, but not a JSP1 mutant in which Gly2 was mutated to Ala (JSP1-G2A), was myristoylated in cells. Although JSP1 maintained intrinsic phosphatase activity in the absence of Myristoylation, the subcellular localization of the enzyme was altered. Compared with the wild type, the ability of nonmyristoylated JSP1 to induce JNK activation and phosphorylation of the transcription factor c-JUN was attenuated. Upon expression of wild-type JSP1, a subpopulation of cells, with the highest levels of the phosphatase, was induced to float off the dish and undergo apoptosis. In contrast, cells expressing similar levels of JSP1-G2A remained attached, further highlighting that the Myristoylation mutant was functionally compromised.
-
Myristoylation of the Dual Specificity Phosphatase JSP1 is necessary for its Activation of JNK Signaling and Apoptosis
FEBS Journal, 2010Co-Authors: Ulla Schwertassek, Deirdre A. Buckley, Chong-feng Xu, Andrew J. Lindsay, Mary W. Mccaffrey, Thomas A. Neubert, Nicholas K TonksAbstract:Activation of the c-JUN N-terminal kinase (JNK) pathway is implicated in a number of important physiological processes, from embryonic morphogenesis to cell survival and apoptosis. JNK stimulatory phosphatase 1 (JSP1) is a member of the dual specificity phosphatase subfamily of protein tyrosine phosphatases (PTPs). In contrast to other dual specificity phosphatases, which catalyze inactivation of mitogen-activated protein kinases, expression of JSP1 activates JNK-mediated signaling. JSP1 (and its relative DUSP15) are unique among members of the PTP family in that they contain a potential Myristoylation site at the N-terminus (MGNGMXK). In this study, we investigated whether JSP1 was myristoylated and examined the functional consequences of Myristoylation. Using mass spectrometry, we showed that wild type JSP1, but not a JSP1 mutant in which glycine 2 was mutated to alanine (JSP1-G2A), was myristoylated in cells. Abrogation of Myristoylation did not impair the intrinsic phosphatase activity of JSP1, but changed the subcellular localization of the enzyme. Compared to wild type, the ability of non-myristoylated JSP1 to induce JNK activation and phosphorylation of the transcription factor c-JUN was attenuated. Upon expression of wild type JSP1, a subpopulation of cells, with highest levels of the phosphatase, was induced to float off the dish and undergo apoptosis. In contrast, cells expressing similar levels of JSP1-G2A remained attached, further highlighting that the Myristoylation mutant was functionally compromised.
Carmela Giglione - One of the best experts on this subject based on the ideXlab platform.
-
Targeted profiling of A. thaliana sub-proteomes illuminates new co- and post-translationally N-terminal Myristoylated proteins
The Plant cell, 2018Co-Authors: Wojciech Majeran, Jean-pierre Le Caer, Lalit Ponnala, Thierry Meinnel, Carmela GiglioneAbstract:N-terminal Myristoylation, a major eukaryotic protein lipid modification, is difficult to detect in vivo and challenging to predict in silico. We developed a proteomics strategy involving subfractionation of cellular membranes, combined with separation of hydrophobic peptides by mass spectrometry-coupled liquid chromatography to identify the Arabidopsis thaliana myristoylated proteome. This approach identified a starting pool of 8837 proteins in all analyzed cellular fractions, comprising 32% of the Arabidopsis proteome. Of these, 906 proteins contain an N-terminal Gly at position 2, a prerequisite for Myristoylation, and 214 belong to the predicted myristoylome (comprising 51% of the predicted myristoylome of 421 proteins). We further show direct evidence of Myristoylation in 72 proteins; 18 of these myristoylated proteins were not previously predicted. We found one Myristoylation site downstream of a predicted initiation codon, indicating that posttranslational Myristoylation occurs in plants. Over half of the identified proteins could be quantified and assigned to a subcellular compartment. Hierarchical clustering of protein accumulation combined with Myristoylation and S-acylation data revealed that N-terminal double acylation influences redirection to the plasma membrane. In a few cases, MYR function extended beyond simple membrane association. This study identified hundreds of N-acylated proteins for which lipid modifications could control protein localization and expand protein function.
-
High-throughput profiling of N-Myristoylation substrate specificity across species including pathogens.
Proteomics, 2013Co-Authors: José A. Traverso, Carmela Giglione, Thierry MeinnelAbstract:One of the most critical modifications affecting the N-terminus of proteins is N-Myristoylation. This irreversible modification affects the membrane-binding properties of crucial proteins involved in signal transduction cascades. This cotranslational modification, catalyzed by N-myristoyl transferase, occurs both in lower and higher eukaryotes and is a validated therapeutic target for several pathologies. However, this lipidation proves very difficult to be evidenced in vivo even with state-of-the-art proteomics approaches or bioinformatics tools. A large part of N-myristoylated proteins remains to be discovered and the rules of substrate specificity need to be established in each organism. Because the peptide substrate recognition occurs around the first eight residues, short peptides are used for modeling the reaction in vitro. Here, we provide a novel approach including a dedicated peptide array for high-throughput profiling protein N-Myristoylation specificity. We show that Myristoylation predictive tools need to be fine-tuned to organisms and that their poor accuracy should be significantly enhanced. This should lead to strongly improved knowledge of the number and function of myristoylated proteins occurring in any proteome.
-
Unexpected protein families including cell defense components feature in the N-myristoylome of a higher eukaryote.
Journal of Biological Chemistry, 2003Co-Authors: Bertrand Boisson, Carmela Giglione, Thierry MeinnelAbstract:N-Myristoylation is an irreversible modification that affects the membrane binding properties of crucial cytoplasmic proteins from signal transduction cascades. We characterized the two putative N-myristoyltransferases of Arabidopsis thaliana as a means of investigating the entire N-Myristoylation proteome (N-myristoylome) in a higher eukaryote. AtNMT1 compensated for the nmt1 defect in yeast, whereas AtNMT2 and chimeras of the two genes did not. Only AtNMT1 modified known N-myristoylated proteins in vitro. AtNMT1 is therefore responsible for the A. thaliana N-myristoylome, whereas AtNMT2 does not seem to have usual Myristoylation activity. We began with the whole set of N-myristoylated G proteins in the A. thaliana proteome. We then used a reiterative approach, based on the in vitro N-Myristoylation of more than 60 different polypeptides, to determine the substrate specificity of AtNMT1. We found that the positive charge on residue 7 of the substrate was particularly important in substrate recognition. The A. thaliana N-myristoylome consists of 437 proteins, accounting for 1.7% of the complete proteome. We demonstrated the N-Myristoylation of several unexpected protein families, including innate immunity proteins, thioredoxins, components of the protein degradation pathway, transcription factors, and a crucial regulatory enzyme of glycolysis. The role of N-Myristoylation is discussed in each case; in particular, this process may underlie the "guard" hypothesis of innate immunity.
