scholarly journals SETD3 protein is the actin-specific histidine N-methyltransferase

2018 ◽  
Author(s):  
Sebastian Kwiatkowski ◽  
Agnieszka K. Seliga ◽  
Maria Veiga-da-Cunha ◽  
Didier Vertommen ◽  
Marianna Terreri ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modification ◽  
Mass Spectrometry Analysis ◽  
Set Domain ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Molecular Identity ◽  
Glycolytic Activity ◽  
Human Ortholog ◽  
First Time

AbstractProtein histidine methylation is rarely studied posttranslational modification of unknown biochemical importance. In vertebrates, only a few methylhistidne-containing proteins have been reported so far, including β-actin as an essential example. The evolutionary conserved methylation of β-actin H73 residue is catalyzed by a specific histidine N-methyltransferase that has never been identified molecularly. In the present investigation, we have purified actin-specific histidine N-methyltransferase from rat muscles about 1200-fold. Its activity was studied by the radiochemical assay employing either homogeneous recombinant human β-actin produced in E. coli or its mutated form exhibiting substitution of H73 by Ala residue (H73A) as substrates. Three polypeptides of ≈65, 75 and 90 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of SETD3 methyltransferase as the only plausible candidate. Rat SETD3 and its human ortholog were expressed in COS-7 cells, purified to homogeneity and shown to catalyze methylation of β-actin at H73 residue as confirmed by mass spectrometry analysis. The SETD3 enzyme was active towards a synthetic peptide corresponding to residues 69-77 of β-actin, but not to its mutated form exhibiting His-to-Ala substitution. Finally, Setd3-deficient HAP1 cells were devoid of methylated H73 in β-actin and exhibited phenotypic changes, including a decrease in F-actin content and an increased glycolytic activity. We conclude that SETD3 is the actin-specific histidine N-methyltransferase. The data show for the first time the molecular identity of protein histidine N-methyltransferase in vertebrates and throw new light on the substrate specificity of SET-domain-containing enzymes.

scholarly journals Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 629-635 ◽  
Author(s):  
J. M. Nieto ◽  
C. Madrid ◽  
E. Miquelay ◽  
J. L. Parra ◽  
S. Rodríguez ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein A ◽  
Nitrilotriacetic Acid ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Protein Protein Interaction ◽  
New Family ◽  
Missense Mutant ◽  
First Time

ABSTRACT Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin α-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni2+-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha′ proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

scholarly journals Modifications of proteins by 4-hydroxy-2-nonenal in the ventilatory muscles of rats

2006 ◽  
Vol 290 (5) ◽  
pp. L996-L1003 ◽  
Author(s):  
Sabah N. A. Hussain ◽  
Ghassan Matar ◽  
Esther Barreiro ◽  
Maria Florian ◽  
Maziar Divangahi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid Peroxidation ◽  
Triosephosphate Isomerase ◽  
Adduct Formation ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Protein Adduct ◽  
Dose Dependent Fashion

Although 4-hydroxy-2-nonenal (HNE, a product of lipid peroxidation) is a major cause of oxidative damage inside skeletal muscles, the exact proteins modified by HNE are unknown. We used two-dimensional electrophoresis, immunoblotting, and mass spectrometry to identify selective proteins targeted by HNE inside the diaphragm of rats under two conditions: severe sepsis [induced by E. coli lipopolysaccharides (LPS)] and during strenuous muscle contractions elicited by severe inspiratory resistive loading (IRL). Diaphragm HNE-protein adduct formation (detected with a polyclonal antibody) increased significantly after 1 and 3 h of LPS injection with a return to baseline values thereafter. Similarly, HNE-protein adduct formation inside the diaphragm rose significantly after 6 but not 3 h of IRL. Mass spectrometry analysis of HNE-modified proteins revealed enolase 3b, aldolase and triosephosphate isomerase 1, creatine kinase, carbonic anyhdrase III, aconitase 2, dihydrolipoamide dehydrogenase, and electron transfer flavoprotein-β. Measurements of in vitro enolase activity in the presence of pure HNE revealed that HNE significantly attenuated enolase activity in a dose-dependent fashion, suggesting that HNE-derived modifications have inhibitory effects on enzyme activity. We conclude that lipid peroxidation products may inhibit muscle contractile performance through selective targeting of enzymes involved in glycolysis, energy production as well as CO2 hydration.

scholarly journals A validated method for the analysis and profiling of ‘nyaope’ using gas chromatography – mass spectrometry

2021 ◽  
Vol 117 (11/12) ◽  
Author(s):  
Pabalala M. Mthembi ◽  
Ellen M. Mwenesongole ◽  
Michael D. Cole
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Principal Component ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Drug Cocktail ◽  
Spectrometry Analysis ◽  
Chromatography Mass Spectrometry ◽  
First Time ◽  
Problem Drug

Nyaope, a Tswana word for a mixture or ‘mish-mash’, describes a drug cocktail consisting of heroin, cannabis, and on occasion other controlled substances and warfarin. It is highly addictive with extremely unpleasant side effects caused by withdrawal from the drug. It is a problem drug especially in townships in South Africa. However, its prevalence in neighbouring southern African states and further afield is not yet known. There is currently no validated method for the analysis and comparison of nyaope. We describe a validated method for the gas chromatography – mass spectrometry analysis of nyaope so that within-batch and between-batch comparisons of nyaope can successfully be made for the first time. The validated method managed an accuracy within the range 80–120%, the precision was less than 20% for all analytes and managed linearity with R2≥0.99. The detection limits for diamorphine, efavirenz, nevirapine and Δ9-tetrahydrocannabinol were 14.2, 18.6, 18.7 and 9.94 pg on column, respectively, and the limits of quantitation were 43.1, 56.3, 56.6 and 30.1 pg on column, respectively. The simulated and casework samples were successfully discriminated into original batches using the identified nyaope components, the unsupervised chemometric methods principal component analysis and hierarchical clustering, as well as chromatographic profiles.

scholarly journals Flagellin from Listeria monocytogenes Is Glycosylated with β-O-Linked N-Acetylglucosamine

2004 ◽  
Vol 186 (20) ◽  
pp. 6721-6727 ◽  
Author(s):  
M. Schirm ◽  
M. Kalmokoff ◽  
A. Aubry ◽  
P. Thibault ◽  
M. Sandoz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Listeria Monocytogenes ◽  
Posttranslational Modification ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Central Surface ◽  
Cytoplasmic Proteins ◽  
Exposed Region

ABSTRACT Glycan staining of purified flagellin from Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b suggested that the flagellin protein from this organism is glycosylated. Mass spectrometry analysis demonstrated that the flagellin protein of L. monocytogenes is posttranslationally modified with O-linked N-acetylglucosamine (GlcNAc) at up to six sites/monomer. The sites of glycosylation are all located in the central, surface-exposed region of the protein monomer. Immunoblotting with a monoclonal antibody specific for β-O-linked GlcNAc confirmed that the linkage was in the β configuration, this residue being a posttranslational modification commonly observed in eukaryote nuclear and cytoplasmic proteins.

scholarly journals Effect of Cadmium on the Level of Isoprenoid-Derived Phytohormones in Duckweed Wolffia arrhiza

2020 ◽  
Vol 39 (4) ◽  
pp. 1518-1530
Author(s):  
Magdalena Chmur ◽  
Andrzej Bajguz ◽  
Alicja Piotrowska-Niczyporuk
Keyword(s):  
Mass Spectrometry ◽  
Aquatic Plant ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Endogenous Level ◽  
Wolffia Arrhiza ◽  
First Time ◽  
Dose Dependent

AbstractWolffia arrhiza (L.) Horkel ex Wimm. is an aquatic plant belonging to the Lemnaceae family. It does not have leaves, stems, and roots, flowers rarely occur, while body size can reach 1 mm of width and 1.3 mm of length. The present study demonstrates the endogenous level of isoprenoid-derived phytohormones and their changes under the influence of different cadmium (Cd) concentrations (0.1, 1, 10, and 100 µM). A liquid chromatography quadrupole-time-of-flight mass spectrometry analysis indicated the presence of abscisic acid, eight brassinosteroids (6-deoxocastasterone, 6-deoxotyphasterol, cathasterone, typhasterol, castasterone, 24-epicastasterone, brassinolide, and 28-homobrassinolide), seven free bases of cytokinins [trans-zeatin (tZ), cis-zeatin (cZ), dihydrozeatin (DHZ), N6-isopentenyladenine, N6-isopentenyladenosine, ortho-topolin, and meta-topolin], eight conjugates of cytokinins (tZ riboside, tZ-9-glucoside, tZ-7-glucoside, tZ-O-glucoside riboside, cZ-9-glucoside, DHZ riboside, DHZ-O-glucoside, and N6-isopentenyladenosine-7-glucoside) and gibberellic acid (GA3) in this duckweed. The level of phytohormones in plants treated with Cd has changed, e.g., the ABA level increased while GA3 decreased. Whereas the amount of BRs and CKs was different in Cd dose-dependent manner. Besides, it is worth noting that the distribution of 25 various phytohormones in the Wolffia arrhiza is reported for the first time.

scholarly journals The Mammalian Cytosolic Type 2 (R)-β-hydroxybutyrate Dehydrogenase (BDH2) is 4-oxo-L-proline Reductase (EC 1.1.1.104)

2021 ◽  
Author(s):  
Sebastian P. Kwiatkowski ◽  
Maria Bozko ◽  
Michal Zarod ◽  
Apolonia Witecka ◽  
Adam K. Jagielski ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Characterization ◽  
Mass Spectrometry Analysis ◽  
Protein Preparation ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Mammalian Tissues ◽  
Reversible Reduction

AbstractThe early studies on chicken embryos revealed that exposition to 4-oxo-L-proline resulted in the explicit increase in 4-hydroxy-L-proline content in their tissues. In 1962, 4-oxo-L-proline reductase, an enzyme responsible for the reduction of 4-oxo-L-proline, was partially purified from rabbit kidneys and characterized biochemically, but only recently the molecular identity of the enzyme has been unveiled in our laboratory. The present investigation reports the purification, identification as well as biochemical characterization of 4-oxo-L-proline reductase. The enzyme was purified from rat kidneys about 280-fold. Following mass spectrometry analysis of the purified protein preparation, the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only meaningful candidate for the reductase. Rat and human BDH2 were expressed in E. coli, purified, and shown to catalyze the reversible reduction of 4-oxo-L-proline to cis-4-hydroxy-L-proline, as confirmed by chromatographic and mass spectrometry analysis. Specificity studies carried out on both enzymes showed that 4-oxo-L-proline was the best substrate, particularly the human enzyme acted with 9400-fold higher catalytic efficiencies on 4-oxo-L-proline than on (R)-β-hydroxybutyrate. Finally, HEK293T cells efficiently metabolized 4-oxo-L-proline to cis-4-hydroxy-L-proline and simultaneously accumulated trans-4-hydroxy-L-proline in the culture medium, suggesting that 4-oxo-L-proline is most likely an inhibitor of trans-4-hydroxy-L-proline metabolism in human cells. We conclude that BDH2 is mammalian 4-oxo-L-proline reductase that converts 4-oxo-L-proline to cis-4-hydroxy-L-proline, and not to trans-4-hydroxy-L-proline as currently thought, and hypothesize that the enzyme may be considered as a potential source of cis-4-hydroxy-L-proline in mammalian tissues.

scholarly journals PTEN arginine methylation by PRMT6 suppresses PI3K–AKT signaling and modulates pre-mRNA splicing

2019 ◽  
Vol 116 (14) ◽  
pp. 6868-6877 ◽  
Author(s):  
Jiawen Feng ◽  
Yaping Dang ◽  
Weiqi Zhang ◽  
Xuyang Zhao ◽  
Cong Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Alternative Splicing ◽  
Posttranslational Modification ◽  
Arginine Methylation ◽  
Akt Signaling ◽  
Mrna Splicing ◽  
Mass Spectrometry Analysis ◽  
Arginine Methyltransferase ◽  
Spectrometry Analysis ◽  
Cellular Processes

Arginine methylation is a ubiquitous posttranslational modification that regulates critical cellular processes including signal transduction and pre-mRNA splicing. Here, we report that the tumor-suppressor PTEN is methylated by protein arginine methyltransferase 6 (PRMT6). Mass-spectrometry analysis reveals that PTEN is dimethylated at arginine 159 (R159). We found that PTEN is mutated at R159 in cancers, and the PTEN mutant R159K loses its capability to inhibit the PI3K–AKT cascade. Furthermore, PRMT6 is physically associated with PTEN, promotes asymmetrical dimethylation of PTEN, and regulates the PI3K–AKT cascade through PTEN R159 methylation. In addition, using transcriptome analyses, we found that PTEN R159 methylation is involved in modulation of pre-mRNA alternative splicing. Our results demonstrate that PTEN is functionally regulated by arginine methylation. We propose that PTEN arginine methylation modulates pre-mRNA alternative splicing and influences diverse physiologic processes.

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