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Results for “Protein Modification, Translational”

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PubMed2003

Proteomic and transcriptomic analysis of interleukin-1beta treated lung carcinoma cell line.

Mucin hypersecretion is one of the main symptoms of inflammatory disease in the respiratory tract. We previously reported that the pleiotypic pro-inflammatory cytokine, interleukin (IL)-1beta, plays a significant role in respiratory tract inflammation by inducing mucins. However, the molecular mechanism for mucin hypersecretion in the respiratory tract remains still unclear. In order to understand the mechanisms of mucin hypersecretion in the airway epithelium, the differentially expressed proteins and genes in the lung mucoepidermoid carcinoma cell line (NCI-H292 cells), which were treated for 6 and 24 hours with IL-1beta (10 ng/mL) were identified using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) proteomics and cDNA microarray analysis (8.6K). In the 2-D PAGE, eight differentially expressed proteins and 14 post-translational modification proteins were identified at 6 and 24 hours after the IL-1beta-treatment. Four hundred and thirteen genes (6.6%) and 115 genes (2.0%) were differentially expressed, respectively, at 6 and 24 hours after the IL-1beta-treatment by microarray analysis. The differentially expressed genes and proteins that were regulated by the IL-1beta-treatment were mostly in the metabolic pathway rather than in the regulatory pathway. These results clearly show that the transcript levels have little value in predicting the extent of protein expression.

Carcinoma↗
PubMed2001

Proteome analysis of Helicobacter pylori: major proteins of type strain NCTC 11637.

Proteome analysis involves the simultaneous resolution and display of proteins produced by an organism, followed by the quantitation, characterisation and identification of these proteins. As part of an ongoing study mapping and comparing the proteins expressed by various strains of the pathogenic bacterium Helicobacter pylori, we have resolved and identified 93 of the most abundant proteins expressed by type reference strain NCTC 11637. Proteins were separated by two-dimensional gel electrophoresis and stained with Coomassie G250. Intensely-stained spots were excised and digested with trypsin, and the resulting peptides were characterised by mass spectrometry. Proteins were then identified by correlating actual peptide profiles with theoretical profiles generated from published nucleotide sequences. Ninety-three of the most intensely-stained protein spots were identified as the products of 35 genes, giving a ratio of 2.7 protein gene-products per gene. The products of the tsaA, pfr, ureA and ureB genes were amongst several proteins present in multiple isoforms. Peptide mass fingerprinting data were used to identify probable post-translational protein modifications. These results suggest that H. pylori proteins are subject to a high degree of post-translational modification. Comparative proteomics of H. pylori strains should greatly assist in investigating the pathogenic properties of this bacterium.

Bacterial Proteins↗
PubMed1996

The evolutionary conservation of a novel protein modification, the conversion of cysteine to serinesemialdehyde in arylsulfatase from Volvox carteri.

A novel post-translational protein modification has recently been described in two human sulfatases, by which a cysteine is replaced by a serinesemialdehyde (2-amino-3-oxopropionic acid) residue [Schmidt, B., Selmer, T., Ingendoh, A. & von Figura, K. (1995) Cell 82, 271-278]. This cysteine is conserved among all known eukaryotic sulfatases. Here we report the presence of this modification in arylsulfatase from the green alga Volvox carteri. The evolutionary conservation of this novel protein modification between sulfatases of V. carteri and man lends further support to the assumption that this modification is required for the catalytic activity of sulfatases and may be present in all sulfatases of eukaryotic origin.

Alanine↗
PubMed2005

Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions.

Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach. Our results suggest that large libraries of functional human protein fragments can be efficiently displayed on the yeast surface. In addition to the analysis of post-translational modifications, yeast surface-displayed human cDNA libraries have many potential applications, including identifying targets and defining potential cross-reactive proteins for small molecules or drugs.

Adult↗
PubMed2026

Lysine methylation is an endogenous post-translational modifications of tau protein in human brain and a modulator of aggregation propensity.

Tau protein undergoes a broad range of post-translational modifications in the brain, influencing its structure, solubility, and propensity to aggregate. This chapter presents an integrated methodological framework for characterizing tau methylation and evaluating its impact on tau biology. We describe procedures for isolating soluble and filamentous tau from post-mortem human brain tissue while preserving modifications for proteomic analysis. These approaches support precise mapping of methylation sites alongside other co-occurring modifications. To model methylation under controlled conditions, we outline protocols for recombinant tau expression, purification, and chemical reductive methylation, including radiolabeled assays for determining modification stoichiometry. We then detail biophysical assays used to assess how methylation alters tau conformation and aggregation propensity. This methodological framework supports experimentation seeking insight into mechanisms relevant to Alzheimer's disease and related tauopathies.

Humans↗
PubMed2004

Post-translational modifications of tau protein in Alzheimer's disease.

Microtubule-associated protein tau undergoes several post-translational modifications and aggregates into paired helical filaments (PHFs) in Alzheimer's disease (AD) and other tauopathies. These modifications of tau include hyperphosphorylation, glycosylation, ubiquitination, glycation, polyamination, nitration, and proteolysis. Hyperphosphorylation and glycosylation are crucial to the molecular pathogenesis of neurofibrillary degeneration of AD. The others appear to represent failed mechanisms for neurons to remove damaged, misfolded, and aggregated proteins. This review summarizes the abnormal post-translational modifications of tau and discusses the pathophysiological relevance of hyperphosphorylation and glycosylation of tau. Total tau and phosphorylated tau levels in cerebrospinal fluid as a diagnostic biomarkers are also reviewed. Analyses of the current advances in tau modifications suggest that intervention addressing these abnormalities may offer promising therapeutic opportunities to prevent and treat neurofibrillary degeneration of AD and other tauopathies.

Alzheimer Disease↗
PubMed2004

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells.

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.

3T3 Cells↗
PubMed2001

[Protein databank for several tissues derived from five instar of silkworm].

We attempted to construct protein databank of silkworm (Bombyx mori L.) for clarifying the gene expression and post-translational modification. Proteins from silkworm body wall, fat body and middle intestines were separated by two-dimensional electrophoresis. The N-terminal amino acid sequences of 40 proteins and their homology to proteins from other organisms were determined. 58.5% proteins were high homologous to the already reported proteins in D. melanogaster and 36.5% to those in other organisms. Only 5% of the proteins were homologous to the known protein in silkworm. The N-terminal sequences of 27 proteins were first found in silkworm, and all these data were registered in SWISS-PROT through the Internet. The results suggested that the information of gene expression and post-translational modification could be obtained by the information of protein databank.

Amino Acid Sequence↗
PubMed1991

Post-translational modification of lens proteins in cataract.

Evidence supporting the role of non-enzymic post-translational modification of lens proteins in cataract is reported as presented at a meeting in Bydgoszcz, Poland in August 1990. Glycation and carbamylation have been studied intensively recently. Both produce modified proteins with properties similar to those of 'molten-globule' intermediates of protein folding and unfolding pathways.

Cataract↗
PubMedDate not supplied

Quantification in proteomics through stable isotope coding: a review.

This review focuses on techniques for quantification and identification in proteomics by stable isotope coding. Methods are examined for analyzing expression, post-translational modifications, protein:protein interactions, single amino acid polymorphism, and absolute quantification. The bulk of the quantification literature in proteomics focuses on expression analysis, where a wide variety of methods targeting different features of proteins are described. Methods for the analysis of post-translational modification (PTM) focus primarily on phosphorylation and glycosylation, where quantification is achieved in two ways, either by substitution or tagging of the PTM with an isotopically coded derivatizing agent in a single process or by coding and selecting PTM modified peptides in separate operations. Absolute quantification has been achieved by age-old internal standard methods, in which an isotopically labeled isoform of an analyte is synthesized and added to a mixture at a known concentration. One of the surprises is that isotope coding can be a valuable aid in the examination of intermolecular association of proteins through stimulus:response studies. Preliminary efforts to recognize single amino acid polymorphism are also described. The review ends with the conclusion that (1) isotope ratio analysis of protein concentration between samples does not necessarily relate directly to protein expression and rate of PTM and (2) that multiple new methods must be developed and applied simultaneously to make existing stable isotope quantification methods more meaningful. Although stable isotope coding is a powerful, wonderful new technique, multiple analytical issues must be solved for the technique to reach its full potential as a tool to study biological systems.

Isotopes↗
PubMed2004

The multi-dimensional regulation of gene expression by fatty acids: polyunsaturated fats as nutrient sensors.

PURPOSE OF REVIEW: A diet that provides 2-5% of energy as highly unsaturated 20- and 22-carbon omega-6 or omega-3 fatty acids is associated with an inhibition of hepatic lipogenesis, a stimulation of hepatic fatty acid oxidation, and consequently a lowering of blood triglyceride levels. The purpose of this review is to demonstrate that highly unsaturated fatty acids regulate lipid metabolism by modulating protein expression at many levels including gene transcription, messenger RNA processing, mRNA decay, and post-translational protein modifications. Although the intracellular signaling mechanisms employed by highly unsaturated fatty acids are unknown, this review presents a summary of the emerging knowledge regarding highly unsaturated fatty acids as kinase cascade activators. RECENT FINDINGS: Highly unsaturated fatty acids suppress lipogenic gene transcription by reducing the DNA binding activity of several transcription factors, notably sterol regulatory-element binding protein 1 and nuclear factor Y. Highly unsaturated fatty acids inhibit the proteolytic release of sterol regulatory-element binding protein 1 from its membrane-anchored precursor through a ceramide-dependent signal, and impart a post-translational modification to nuclear factor Y. Highly unsaturated fatty acids accelerate sterol regulatory-element binding protein 1 mRNA decay and may function as antagonistic ligands for liver receptor X, thereby interfering with the liver receptor X stimulation of sterol regulatory-element binding protein 1 gene transcription. Highly unsaturated fatty acid activation of peroxisome proliferator-activated receptor alpha combined with their displacement of the oxysterol from liver receptor X may 'trap' liver receptor X as transcriptionally inactive peroxisome proliferator-activated receptor alpha/liver receptor X heterodimer. The gene expression consequences of liver receptor X 'trapping' may explain how dietary highly unsaturated fatty acids lead to a repartitioning of fatty acids away from storage and towards oxidation. SUMMARY: The liver appears to use the highly unsaturated fatty acid status as a nutrient sensor to determine whether fatty acids are to be stored or oxidized. In this way highly unsaturated fatty acids may function as nutritional factors that reduce the risk of developing hepatic lipotoxicity and insulin resistance.

DNA-Binding Proteins↗
PubMed1994

Nitric oxide: a signal for ADP-ribosylation of proteins.

Nitric oxide (NO), a highly reactive gas, is now established as a major messenger molecule regulating blood vessel dilation, immune functions and serving as a neurotransmitter in brain and peripheral nervous system. NO can also act as a tumoricidal and bactericidal molecule. The effect of NO to dilate blood vessels is largely explained by stimulation of soluble guanylate cyclase (a heme-iron containing protein) leading to formation of cGMP and protein phosphorylation. This is considered to be the main physiological signaling mechanism of NO. NO also binds to non-heme iron-containing proteins and this has been considered as a pathophysiological or cytotoxic action of NO. Furthermore, NO, more correctly nitrosonium (NO+) which can be formed by the removal of one electron, reacts with protein SH-groups to cause the S-nitrosylation of proteins. We have recently established a link between NO and the S-nitrosylation and mono-ADP-ribosylation of the enzyme glyceraldehyde 3-monophosphate dehydrogenase, which adds a further protein modification mechanism for NO action. This links the formation of the second messenger molecule NO to post-translational protein modification and adds a new dimension to NO in the communication of intracellular signals.

Adenosine Diphosphate Ribose↗
PubMedDate not supplied

Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots.

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.

Antibodies, Monoclonal↗
PubMed1986

Acute ACTH regulation of adrenal corticosteroid biosynthesis. Rapid accumulation of a phosphoprotein.

Two-dimensional gel electrophoresis was used to monitor proteins synthesized in unstimulated control and in adrenocorticotropic hormone (ACTH)- or cAMP-stimulated rat adrenal cells. Four proteins, which have similar proteolytic peptide maps, have been identified. The two found primarily in unstimulated cells are referred to as pb and pa, where pb is the protein with more basic isoelectric point. Similarly, proteins ib and ia were detected only in stimulated cells. The synthesis of pb occurs only in unstimulated cells and that of ib only in stimulated cells. Protein ib accumulates with the same lag time, rate, and stimulant dose response as the increase in steroid hormone synthesis. Pulse-chase studies showed that protein ib is not produced from pb by a post-translational modification. Proteins pb and ib thus seem identical with proteins p and i previously identified in rat adrenal cortex and corpus luteum (Krueger, R.J., and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167, and Pon, L.A., and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6594-6599). The acidic forms, pa and ia, appear after a longer lag time and are produced at a slower rate than the basic forms. Pulse-chase studies showed that the disappearance of the basic form of each protein occurs concurrently with the appearance of the corresponding acidic form. Addition of [32P]orthophosphate to stimulated adrenal cells allowed direct demonstration that proteins ib and ia are phosphorylated. Moreover, alkaline phosphatase treatment of [35S]methionine-labeled, cAMP-stimulated adrenal cells caused a large decrease in the amounts of ib and ia and the appearance of proteins with the same two-dimensional electrophoretic mobilities as pb and pa. These observations suggest that protein ib may mediate stimulation of steroidogenesis, be produced by an ACTH- or cAMP-dependent, cotranslational phosphorylation of protein pb, and be lost by a cycloheximide-insensitive, post-translational conversion to ia.

Adrenal Cortex Hormones↗
PubMed2006

A study of CD33 (SIGLEC-3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing.

The expression of CD33, a restricted leukocyte antigen considered specific for myeloid lineage, has been studied extensively on lymphoid cells. We demonstrated that wide subsets of mitogen- or alloantigen-activated human T and natural killer (NK) cells express CD33 at protein and nucleic acid levels. CD33+ and CD33- T and NK cell populations showed identical surface expression of activation markers such as CD25, CD28, CD38, CD45RO, or CD95. Myeloid and lymphoid CD33 cDNA were identical. However, lymphoid CD33 protein had lower molecular weight, suggesting cell type-specific, post-translational modifications. Additionally, reverse transcriptase-polymerase chain reaction and Northern blot analysis showed an unknown CD33 isoform (CD33m) expressed on all CD33+ cell lines or T cell clones tested. CD33m was identical to CD33 (CD33M) in the signal peptide, the immunoglobulin (Ig) domain C2, the transmembrane, and the cytoplasmic regions but lacked the extracellular ligand-binding variable Ig-like domain encoded by the second exon. CD33m mRNA was mostly detected on NKL and myeloid cell lines but poorly expressed on B cell lines and T lymphocytes. The CD33m extracellular portion was successfully expressed as a soluble fusion protein on transfected human cells, suggesting a functional role on cell membranes. Cross-linking of CD33 diminished the cytotoxic activity of NKL cells against K562 and P815 target cells, working as an inhibitory receptor on NK cells. These data demonstrate that CD33 expression is not restricted to the myeloid lineage and could exist as two different splicing variants, which could play an important role in the regulation of human lymphoid and myeloid cells.

Alternative Splicing↗
PubMed1998

Ceramide-induced apoptosis is mediated by caspase activation independently from retinoblastoma protein post-translational modification.

Recent evidence suggests that untimely retinoblastoma protein (RB) dephosphorylation and/or proteolytic degradation might provide key events down-stream cysteine protease (caspase) activation in apoptosis induction. We have dealt with this issue by studying apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells, maintained in complete growth medium. We report that C6-Cer-induced apoptosis occurred predominantly in G1/S phases of the cycle and was associated with RB dephosphorylation, in the setting of negligible Bcl-2 expression. Apoptosis was also associated with poly(ADP-ribose) polymerase (PARP) cleavage, thus indicating activation of CPP32/Yama/apopain (caspase-3); however, while the tripeptide caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone was able to prevent both C6-Cer-induced PARP cleavage and apoptosis, it was ineffective in preventing RB dephosphorylation. Moreover proteolytic RB cleavage occurred only to a marginal extent after C6-Cer treatment. These results indicate that apoptosis induced by ceramide in CHP-100 cells is caspase-mediated, but RB post-translational modification does not provide a key step, downstream caspase activation, in apoptosis execution.

Apoptosis↗
PubMed2006

O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks.

O-GlcNAc is an ubiquitous post-translational protein modification consisting of a single N-acetlyglucosamine moiety linked to serine or threonine residues on nuclear and cytoplasmic proteins. Recent work has begun to uncover the functional roles of O-GlcNAc in cellular processes. O-GlcNAc modified proteins are involved in sensing the nutrient status of the surrounding cellular environment and adjusting the activity of cellular proteins accordingly. O-GlcNAc regulates cellular responses to hormones such as insulin, initiates a protective response to stress, modulates a cell's capacity to grow and divide, and regulates gene transcription. This review will focus on recent work involving O-GlcNAc in sensing the environment and regulating signaling cascades.

Acetylglucosamine↗
PubMed2005

Proteomic analysis of Deinococcus radiodurans recovering from gamma-irradiation.

In order to reveal the mechanisms of the extreme radioresistance and DNA repair in Deinococcus radiodurans, we examined proteome changes in a wild-type strain following gamma-irradiation using two-dimensional polyacrylamide gel electrophoresis and Silver-staining. The expression levels of 26 protein spots showed significant changes under radiation stress. Of these spots, 21 were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry after tryptic in-gel digestion. These proteins exhibited various cellular functions, including (i) translation; (ii) transcription; (iii) signal transduction; (iv) post-translational modification, protein turnover, chaperones; (v) carbohydrate transport and metabolism; (vi) energy production and conversion; (vii) nucleotide transport and metabolism; (viii) inorganic ion transport and metabolism; (ix) DNA replication, recombination and repair; and (x) yet unknown. Most of the proteins have not previously been reported to be relevant to radioresistance.

Bacterial Proteins↗