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 Conversion of spent CHO cell culture media waste to new fermentation feed efficiently supports production of recombinant protein by Escherichia coli

2021 ◽  
Author(s):  
Ciara Lynch ◽  
David J O'Connell
Keyword(s):  
Cell Culture ◽  
Recombinant Protein ◽  
Culture Media ◽  
Mass Spectrometry Analysis ◽  
Microbial Fermentation ◽  
Differential Protein Expression ◽  
Cell Culture Media ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Rich Media

Deriving new value from waste streams is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be effectively recycled and used as a feed in microbial fermentation to produce new recombinant protein products. Our results show that 1) CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich media (LB) used as a baseline reference. 2) The amount of recombinant protein produced following induction in an expression screen was approximately two-fold higher in the CDSM fed cultures than that of baseline. 3) Mass spectrometry analysis of the proteome of E. coli cultures fed in CDSM revealed a greater or lesser differential protein expression pattern depending on supplementation conditions. Further, in a 16 hr fermentation the optimised CDSM-fed culture delivered a protein yield of more than double that achieved by the baseline media. We conclude that spent cell culture media, which represents millions of litres of waste generated by the bioprocessing industry annually, has the potential to be a valuable feed resource for the production of recombinant proteins in secondary microbial fermentation.

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 Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows

BioTechniques ◽  
2021 ◽  
Author(s):  
David Kotol ◽  
Andreas Hober ◽  
Linnéa Strandberg ◽  
Anne-Sophie Svensson ◽  
Mathias Uhlén ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Plasma Proteins ◽  
Mass Spectrometry Analysis ◽  
Targeted Proteomics ◽  
Plasma Samples ◽  
Blood Proteins ◽  
Proteomics Analysis ◽  
Protein Targets ◽  
Spectrometry Analysis ◽  
Absolute Concentrations

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.

scholarly journals Neuronal Proteomic Analysis of the Ubiquitinated Substrates of the Disease-Linked E3 Ligases Parkin and Ube3a

2018 ◽  
Vol 2018 ◽  
pp. 1-14 ◽  
Author(s):  
Aitor Martinez ◽  
Juanma Ramirez ◽  
Nerea Osinalde ◽  
Jesus M. Arizmendi ◽  
Ugo Mayor
Keyword(s):  
Proteasome Inhibitors ◽  
Brain Dysfunction ◽  
Mass Spectrometry Analysis ◽  
E3 Ligases ◽  
Spectrometry Analysis ◽  
Ubiquitinated Proteins ◽  
Neuronal Proteins ◽  
Culture Models ◽  
First Time ◽  
Cell Culture Models

Both Parkin and UBE3A are E3 ubiquitin ligases whose mutations result in severe brain dysfunction. Several of their substrates have been identified using cell culture models in combination with proteasome inhibitors, but not in more physiological settings. We recently developed theUbbiostrategy to isolate ubiquitinated proteins in flies and have now identified by mass spectrometry analysis the neuronal proteins differentially ubiquitinated by those ligases. This is an example of how flies can be used to provide biological material in order to reveal steady state substrates of disease causing genes. Collectively our results provide new leads to the possible physiological functions of the activity of those two disease causing E3 ligases. Particularly, in the case of Parkin the novelty of our data originates from the experimental setup, which is not overtly biased by acute mitochondrial depolarisation. In the case of UBE3A, it is the first time that a nonbiased screen for its neuronal substrates has been reported.

scholarly journals Specific Roles of the iroBCDEN Genes in Virulence of an Avian Pathogenic Escherichia coli O78 Strain and in Production of Salmochelins

2008 ◽  
Vol 76 (8) ◽  
pp. 3539-3549 ◽  
Author(s):  
Mélissa Caza ◽  
François Lépine ◽  
Sylvain Milot ◽  
Charles M. Dozois
Keyword(s):  
Escherichia Coli ◽  
Respiratory Infections ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Avian Pathogenic Escherichia Coli ◽  
Prevalence Studies ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Negative Mutant

ABSTRACT Avian pathogenic Escherichia coli (APEC) strains are a subset of extraintestinal pathogenic E. coli (ExPEC) strains associated with respiratory infections and septicemia in poultry. The iroBCDEN genes encode the salmochelin siderophore system present in Salmonella enterica and some ExPEC strains. Roles of the iro genes for virulence in chickens and production of salmochelins were assessed by introducing plasmids carrying different combinations of iro genes into an attenuated salmochelin- and aerobactin-negative mutant of O78 strain χ7122. Complementation with the iroBCDEN genes resulted in a regaining of virulence, whereas the absence of iroC, iroDE, or iroN abrogated restoration of virulence. The iroE gene was not required for virulence, since introduction of iroBCDN restored the capacity to cause lesions and colonize extraintestinal tissues. Prevalence studies indicated that iro sequences were associated with virulent APEC strains. Liquid chromatography-mass spectrometry analysis of supernatants of APEC χ7122 and the complemented mutants indicated that (i) for χ7122, salmochelins comprised 14 to 27% of the siderophores present in iron-limited medium or infected tissues; (ii) complementation of the mutant with the iro locus increased levels of glucosylated dimers (S1 and S5) and monomer (SX) compared to APEC strain χ7122; (iii) the iroDE genes were important for generation of S1, S5, and SX; (iv) iroC was required for export of salmochelin trimers and dimers; and (v) iroB was required for generation of salmochelins. Overall, efficient glucosylation (IroB), transport (IroC and IroN), and processing (IroD and IroE) of salmochelins are required for APEC virulence, although IroE appears to serve an ancillary role.

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.

Altered levels of circulating GABAergic 5α/β-reduced pregnane and androstane steroids in schizophrenic men

2011 ◽  
Vol 6 (2) ◽  
Author(s):  
Marie Bicikova ◽  
Martin Hill ◽  
Daniela Ripova ◽  
Pavel Mohr
Keyword(s):  
Atypical Antipsychotics ◽  
Well Being ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Adult Men ◽  
Drug Naïve ◽  
First Time ◽  
Circulating Levels

AbstractThe role of GABAergic pathways in the pathophysiology of schizophrenia is generally accepted. Therefore, the information concerning alterations of the steroid metabolome associated with the disease and/or its treatment is of interest with regard to the pathophysiology of the disease. Hence, we assessed 18 serum steroids and steroid polar conjugates in a group of drug-naive patients (13 adult men) and after 6-months therapy by atypical antipsychotics and age-matched controls (19 men) using gas chromatography-mass spectrometry analysis. This study, for the first time, demonstrates the altered circulating GABAergic steroids in schizophrenic men as well as the effect of the therapy with two types of atypical antipsychotics. The GABAergic androsterone (3α5α) and etiocholanolone (3α5β) are reduced in schizophrenic men but the therapy with atypical antipsychotics reinstates their levels. This reinstatement could be of importance when considering that the GABAergic substances generally improve the well-being of patients. In addition to the unconjugated androsterone, being the most abundant GABAergic steroid in men, most of the other GABAergic steroids also tended to decrease in the patients. By contrast, the conjugated 5β-pregnanolone isomers were elevated in the patients. In conclusion, although schizophrenia status in adult men is associated with unfavorable alterations in neuroactive steroids, the treatment with antipsychotics could at least partly reinstate their circulating levels.

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 Refining the composition of the Arabidopsis thaliana 80S cytosolic ribosome

2019 ◽  
Author(s):  
Karzan Jalal Salih ◽  
Owen Duncan ◽  
Lei Li ◽  
Josua Troesch ◽  
A. Harvey Millar
Keyword(s):  
Arabidopsis Thaliana ◽  
Ribosome Biogenesis ◽  
Cell Function ◽  
Protein A ◽  
Mass Spectrometry Analysis ◽  
Turnover Rates ◽  
80S Ribosome ◽  
Spectrometry Analysis ◽  
Peptide Mass

AbstractThe cytosolic 80S ribosome is composed of protein and RNA molecules and its function in protein synthesis is modulated through interaction with other cytosolic components. Defining the role of each of the proteins associated with ribosomes in plants is a major challenge which is hampered by difficulties in attribution of different proteins to roles in ribosome biogenesis, the mature cytosolic ribosome (r-proteins) or to the broader translatome associated with functioning ribosomes. Here we refined the core r-protein composition in plants by determining the abundance of proteins in low, partially and highly purified ribosomal samples from Arabidopsis thaliana cell cultures. To characterise this list of proteins further we determined their degradation (KD) and synthesis (KS) rate by progressive labelling with 15N combined with peptide mass spectrometry analysis. The turnover rates of 55 r-proteins, including 26 r-proteins from the 40S subunit and 29 r-proteins from the 60S subunit could be determined. Overall, ribosome proteins showed very similar KD and KS rates suggesting that half of the ribosome population is replaced every 3-4 days. Three proteins showed significantly shorter half-lives; ribosomal protein P0D (RPP0D) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The ribosomal RPP0D protein is a homolog of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role as a 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins in Arabidopsis. Of this set, 26 were enriched more than 10-fold during ribosome purification. A number have known roles in translation or ribosome-association while others are newly proposed ribosome-associated factors in plants. This analysis provides a more robust understanding of Arabidopsis ribosome content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and reveals how protein turnover can identify r-proteins involved in ribosome biogenesis or regulation in plants. Data are available via ProteomeXchange with identifier PXD012839.

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