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Results for “Protein Modification, Translational”

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PubMed2004

Detection and proteomic identification of S-nitrosylated proteins in endothelial cells.

Nitric oxide (NO) is a key signalling molecule in the vascular endothelium. It can induce different post-translational modifications in mammalian proteins that can alter their functionality, among which incorporation of a NO group in cysteine thiols, called S-nitrosation or S-nitrosylation, is one of the best characterized. Identification of the proteins that are susceptible to this modification would help us to determine the relevance of this modification in physiological and pathophysiological conditions. For this purpose, we have used a proteomic approach to identify S-nitrosylated proteins in endothelial cells, which includes replacing S-nitrosylation by a specific biotinylation and subsequent purification (called the "biotin switch" method). We have applied this methodology to identify proteins that are S-nitrosylated in endothelial cells acutely exposed to S-nitroso-l-cysteine, a physiological S-nitrosothiol. We describe the identified proteins and discuss the characterization of their S-nitrosylation, confirming the validity of the methodology for approaching the description of the "nitrosylome."

Animals↗

PubMed2004

Evidence for a post-translational modification, aspartyl aldehyde, in a photosynthetic membrane protein.

In oxygenic photosynthesis, photosystem II (PSII) carries out the oxidation of water and reduction of plastoquinone. Three PSII subunits contain reactive groups that covalently bind amines and phenylhydrazine. It has been proposed that these reactive groups are carbonyl-containing, co- or post-translationally modified amino acids. To identify modified amino acid residues in one of the PSII subunits (CP47), tandem mass spectrometry was performed. Modified residues were affinity-tagged with either biotin-LC-hydrazide or biocytin hydrazide, which are known to label carbonyl groups. The affinity-tagged subunit was isolated by denaturing gel electrophoresis, and tryptic peptides were then subjected to affinity purification and tandem mass spectrometry. This procedure identified a hydrazide-labeled peptide, which has the sequence XKEGR. This result is supported by quantitative results acquired from peptide mapping and methylamine labeling. The gene sequence and these tandem data predict that the first amino acid, X, which is labeled with the hydrazide reagent, is a modified form of aspartic acid. On the basis of these data, we propose that D348 of the CP47 subunit is post- or co-translationally modified to give a novel amino acid side chain, aspartyl aldehyde.

Amino Acids↗

PubMed2002

Chemical strategies for functional proteomics.

With complete genome sequences now available for several prokaryotic and eukaryotic organisms, biological researchers are charged with the task of assigning molecular and cellular functions to thousands of predicted gene products. To address this problem, the field of proteomics seeks to develop and apply methods for the global analysis of protein expression and protein function. Here we review a promising new class of proteomic strategies that utilizes synthetic chemistry to create tools and assays for the characterization of protein samples of high complexity. These approaches include the development of chemical affinity tags to measure the relative expression level and post-translational modification state of proteins in cell and tissue proteomes. Additionally, we discuss the emerging field of activity-based protein profiling, which aims to synthesize and apply small molecule probes that monitor dynamics in protein function in complex proteomes.

Affinity Labels↗

PubMed1994

High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.

Animals↗

PubMed1999

Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase.

P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2'-5'-linked oligoadenylate products containing up to 30 residues. 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.

2',5'-Oligoadenylate Synthetase↗

PubMed1993

A PCR-based method for the analysis of human CD44 splice products.

CD44 is a transmembrane glycoprotein involved in the interaction between cells and extracellular matrix. Several variant forms of CD44 exist, which differ from each other in the composition of both the intra- and extracellular domain of the protein. Post-translational modification and alternative RNA processing are responsible for this variation. Recently, it was found that certain variant CD44 proteins, containing extra sequences in the extracellular domain of the protein, are involved in metastatic spread of tumor cells. Variant CD44 proteins are also involved in immunological functions of T and B cells. A large variety of alternatively spliced CD44 mRNAs can be expressed by cells. We have developed a method for the analysis of CD44 mRNAs present in the cell. This reverse transcription-polymerase chain reaction (RT-PCR)-based method can be used to analyze the exon composition of each major CD44 mRNA species present in the cell. In this study we describe the analysis of CD44 mRNAs isolated from six different human cell lines.

Antigens, CD↗

PubMed1981

Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity.

Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase. The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity. Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h. However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts. This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells.

Antigen-Antibody Complex↗

PubMed1994

Alternative splicing governs sulfation of tyrosine or oligosaccharide on peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal alpha-amidation of neuro-endocrine peptides through the sequential action of monooxygenase and lyase domains contained within this bifunctional protein. Alternative splicing leads to the expression of soluble and integral membrane bifunctional PAM proteins as well as a soluble monofunctional monooxygenase. In order to determine how alternative splicing affects post-translational modification of PAM proteins, we investigated the sulfation of PAM proteins expressed in stably transfected hEK-293 cells. Metabolic labeling with [35S]SO4(2-) or [35S]methionine and immunoprecipitation demonstrated that [35S]SO4(2-) was efficiently incorporated into PAM proteins that have the noncatalytic exon A region following the monooxygenase domain (PAM-1 and PAM-4) and into a soluble bifunctional PAM protein (PAM-3). Alkaline hydrolysis, radiosequencing, and deglycosylation experiments demonstrated the presence of a sulfated tyrosine (Tyr965) in the COOH-terminal domain of PAM-3 and multiple sulfated O-glycans in the exon A region of PAM-1 and PAM-4. A mutant PAM-3 protein in which Tyr965 was changed to Ala965 (PAM-3/Y965A) was not sulfated and exhibited monooxygenase and lyase activities similar to those of wild type PAM-3. Pulse-chase and temperature block experiments showed that the PAM-3/Y965A protein exits the trans-Golgi network faster than wild type PAM-3. Thus inclusion of exon A results in the sulfation of O-glycans, while elimination of the transmembrane domain results in the sulfation of Tyr965.

Alternative Splicing↗

PubMed2026

Deciphering S-nitrosylation-regulated metabolic networks in postmortem beef based on label-free modificomics: Identification of ferroptosis as a novel quality-related pathway.

This study elucidated the molecular mechanisms of S-nitrosylation on postmortem beef metabolism and quality based on the label-free modificomics. Varying degrees of S-nitrosylation were exogenously induced in beef semimembranosus (SM) muscle. Results indicated that a high S-nitrosylation level significantly increased beef pH and Warner-Bratzler shear force (WBSF) while reducing centrifugal loss (P < 0.05). A total of 828 S-nitrosylated proteins and 1458 modification sites were identified, of which 114 sites on 81 proteins (DSNPs) exhibited differential modification abundance, representing an increase of 125% compared with previous proteomics studies. DSNPs were mainly involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, calcium signaling, cell structure, and ferroptosis. Notably, this study provides the first evidence in postmortem muscle that S-nitrosylation regulates key ferroptosis-related proteins, including ACSL, CP, and TF, offering new insights into the link between S-nitrosylation and the ferroptosis pathway in meat. Correlation analysis demonstrated that TF was significantly negatively correlated with pH and WBSF, but positively correlated with centrifugal loss (P < 0.05). Collectively, protein S-nitrosylation critically modulates postmortem beef quality through the coordinated regulation of multiple metabolic processes. More importantly, the identification of ferroptosis as a S-nitrosylation-sensitive pathway provides a new perspective for regulating meat quality through protein post-translational modifications.

Animals↗

PubMedDate not supplied

Comparison of normal and breast cancer cell lines using proteome, genome, and interactome data.

Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled identification of 2235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multistep search strategy was used to identify post-translational modifications, revealing several proteins that are preferentially modified in cancer cells. Information regarding both unmodified and modified protein forms was combined with publicly available gene expression and protein-protein interaction data. The resulting integrated dataset revealed several functionally related proteins that are differentially regulated between normal and cancer cell lines.

Amino Acid Sequence↗

PubMed2004

Proteomic characterization of herbicide safener-induced proteins in the coleoptile of Triticum tauschii seedlings.

Proteomic methods such as two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry, as well as immunoblotting, were used to identify herbicide safener-induced proteins in the coleoptile of Triticum tauschii, a diploid wheat containing the D genome also found in the cultivated, hexaploid wheat Triticum aestivum. The herbicide safener fluxofenim dramatically increased protein abundance in the molecular weight (M(r)) range of 24 to 30 kDa, as well as a few higher M(r) proteins, in the coleoptile of T. tauschii seedlings. In total, twenty proteins were identified in this study. Eleven proteins were highly safener induced and only weakly expressed in the control; seven proteins were new safener induced proteins that were not detected in the control. Two other proteins were constitutively expressed in both the control and safener-treated coleoptiles. Among the eighteen inducible proteins, fifteen were glutathione S-transferase (GST) subunits that fall into three subclasses: eight proteins were from the tau subclass, six proteins were from the phi subclass, and one protein was from the lambda class. Another three safener inducible proteins showed homology to the aldo/keto reductase family and with proteins that have roles in glycolysis and the Krebs cycle. Two constitutively expressed proteins were identified, one having highest homology to the dehydroascorbate reductase subclass of GSTs and one with an ascorbate peroxidase. Immunoblot analyses, using two different antisera raised against the same GST protein but differing in their specificity, were used to further characterize the GST proteins expressed in response to safener treatment. Results from immunoblotting, combined with mass spectral analysis, showed that post-translational modification of GST proteins in control and safener-treated coleoptiles may occur.

Chromatography, Liquid↗

PubMed2004

The AGE of the matrix: chemistry, consequence and cure.

Accumulation of advanced glycation endproducts (AGEs) plays a crucial part in the development of age-related diseases and diabetic complications. AGEs are formed in vivo via the so-called Maillard reaction: a reducing sugar reacts with a protein to form a labile Amadori product that is subsequently stabilized, producing an irreversible, non-enzymatic post-translational modification of the protein involved. Recently, it has become clear that, in addition to sugars, lipids play an important role in the initiation of AGE formation, and that genetic factors contribute to an individual's AGE levels. The highest AGE levels are found in tissues with slow turnover, such as tendon, skin, bone, amyloid plaques and cartilage. AGEs exert their effects by adversely affecting the mechanical properties of the matrix and by modulating tissue turnover. In cartilage, these detrimental effects result in tissue that is more prone to the development of osteoarthritis. As such, the accumulation of AGEs provides the first molecular mechanism explaining the age-related increase in the incidence of osteoarthritis. Ongoing research into anti-AGE-ing therapies, such as pyrodoxamine and thiazolium compounds, which are often developed to prevent AGE-induced diabetic complications, might also prove beneficial for the prevention of osteoarthritis.

Aging↗

PubMed1999

Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in synechocystis PCC 6803.

In Synechocystis PCC 6803 as in other cyanobacteria, involvement of protein PII in the co-regulation of inorganic carbon and nitrogen metabolism was established based on post-translational modifications of the protein resulting from changes in the carbon/nitrogen regimes. Uptake of bicarbonate and nitrate in response to changes of the carbon and/or nitrogen regimes is altered in a PII-null mutant, indicating that both processes are under control of PII. Modulation of electron flow by addition of methyl viologen with or without duroquinol, or in a NAD(P)H dehydrogenase-deficient mutant, affects the phosphorylation level of PII. The redox state of the cells would thus act as a trigger for PII phosphorylation.

Bacterial Proteins↗

PubMed2004

Molecular classification and molecular genetics of human lung cancers.

Recent advances in the molecular classification of lung carcinomas and the identification of causative genetic alterations will likely lead to improvements in the diagnosis and treatment of patients with lung cancer. It is now possible to identify gene expression profiles that associate with patient outcome in lung carcinomas, in particular adenocarcinoma. Furthermore, patient survival has been shown to correlate with lung cancer oligonucleotide microarray expression profiles. Large-scale microarray technology may allow for the identification of useful biomarkers for early cancer detection. Oligonucleotide microarray data can be optimized by relating them to protein expression levels in tissue microarrays, by annotation with mutational data, and with results of testing for post-translational modification of cellular proteins. These data may be useful in tailoring chemotherapeutic protocols to individual tumors and identifying new targets for therapeutic intervention.

Adenocarcinoma↗

PubMed1991

Escherichia coli exports previously folded and biotinated protein domains.

Biotination of proteins is a post-translational modification that requires a folded acceptor domain. We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo. We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli. Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product. This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein. The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease. Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes. These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo.

Alkaline Phosphatase↗

PubMed1996

Phosphorylation of myelin protein: recent advances.

Since it was first described 25 years ago, phosphorylation has come to be recognized as a widespread and dynamic post-translation modification of myelin proteins. In this review, the phosphorylation characteristics of myelin basic protein, protein zero (P0), myelin-associated glycoprotein and 2'3' cyclic nucleotide 3'-phosphodiesterase are summarized. Emphasis is placed on recent advances in our knowledge concerning the protein kinases involved and the sites of phosphorylation in the amino acid sequences, where known. The possible roles of myelin protein phosphorylation in modulating myelin structure, the process of myelin assembly and mediation of signal transduction events are discussed.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed2006

Proteomics of plant detergent-resistant membranes.

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains that play important roles in protein sorting, signal transduction, or infection by pathogens. Recent reports demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho- and glycerolipids as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed in silico. Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications, such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction, others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in these physiological functions.

Biological Transport↗

PubMed2006

Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics.

The small ubiquitin-like modifier (SUMO) family in vertebrates includes three different family members that are conjugated as post-translational modifications to target proteins. SUMO-2 and -3 are nearly identical but differ substantially from SUMO-1. We used quantitative proteomics to investigate the target protein preferences of SUMO-1 and SUMO-2. HeLa cells were established that stably express His6-SUMO-1 or His6-SUMO-2. These cell lines and control HeLa cells were labeled with stable arginine isotopes, and His6-SUMOs were enriched from lysates using immobilized metal affinity chromatography. 53 SUMO-conjugated proteins were identified, including 44 novel SUMO targets. 25 proteins were preferentially conjugated to SUMO-1, 19 were preferentially conjugated to SUMO-2, and nine proteins were conjugated to both SUMO-1 and SUMO-2. SART1 was confirmed by immunoblotting to have both SUMO-1- and SUMO-2-linked forms at similar levels. SUMO-1 and SUMO-2 are thus shown to have distinct and overlapping sets of target proteins, indicating that SUMO-1 and SUMO-2 may have both redundant and non-redundant cellular functions. Interestingly, 14 of the 25 SUMO-1-conjugated proteins contain zinc fingers. Although both SUMO family members play roles in many cellular processes, our data show that sumoylation is strongly associated with transcription because nearly one-third of the identified target proteins are putative transcriptional regulators.

Chromatography, Affinity↗