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Results for “Protein Modification, Translational”

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PubMed2002

Intracellular glycosylation and development.

O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification of cytoplasmic and nuclear proteins. Although the function of this abundant modification is yet to be definitively elucidated, all O-GlcNAc proteins are phosphoproteins. Further, the serine and threonine residues substituted with O-GlcNAc are often sites of, or close to sites of, protein phosphorylation. This implies that there may be a dynamic interplay between these two post-translational modifications to regulate protein function. In this review, the functions of some of the proteins that are modified by O-GlcNAc will be considered in the context of the potential role of the O-GlcNAc modification. Furthermore, predictions will be made as to how cellular function and developmental regulation might be affected by changes in O-GlcNAc levels.

Animals↗

PubMed1995

Induction of UV-damage recognition protein by cisplatin treatment.

The biological functions of DNA damage recognition proteins are not well understood. Using the band shift assay, we detected in nuclear extracts from human carcinoma cell lines damage recognition protein which bound selectively to UV-damaged double-stranded DNA. No consistent correlation was found between steady-state levels of the UV-damage recognition protein and either cisplatin cytotoxicity or DNA repair activity. However, cisplatin treatment caused accumulation of the UV-damage recognition protein. The cisplatin-responsive induction of UV-damage recognition protein in the nucleus was higher in cisplatin-resistant cell lines than in their parental counterparts. These results imply that the level of inducibility in response to treatment, but not the constitutive binding activity, of UV-damage recognition protein correlates with cisplatin resistance. Inhibition of UV-damage recognition protein expression by actinomycin and cycloheximide suggests that induction of UV-damage recognition protein requires de novo RNA and protein synthesis, rather than post-translational modification of pre-existing protein. The increased level of UV-damage recognition protein after cisplatin treatment could be a direct response to adduct formation, since it correlated with the number of Pt-DNA adducts. However, it could also be a secondary effect of DNA replication inhibition following DNA damage, since inhibition of DNA synthesis by aphidicolin and hydroxyurea caused the same induction of UV-damage recognition protein. Inducibility of UV-damage recognition protein binding activity by Pt drug treatment suggests involvement of this protein in drug resistance, although a direct link between its function and DNA repair or damage tolerance has not been demonstrated.

Cell Cycle↗

PubMed2000

The molecular basis of lysosomal storage diseases and their treatment.

The lysosomal system is the main intracellular mechanism for the catabolism of naturally occurring endogenous and exogenous macromolecules and the subsequent recycling of their constituent monomeric components. It also plays an important part in processing essential metabolites. A genetic defect in a protein responsible for maintaining the lysosomal system results in the accumulation within lysosomes of partially degraded molecules, the initial step in the process leading to a lysosomal storage disease. The defective protein can be a luminal lysosomal enzyme or protein cofactor, a lysosomal membrane protein or a protein involved in the post-translational modification or transport of lysosomal proteins. Over 40 lysosomal storage diseases are known and they have a collective incidence of approximately 1 in 7000-8000 live births. Most of the genes for the lysosomal proteins have been cloned, permitting mutation analysis in individual cases. This information can be used for genotype/phenotype correlation, genetic counselling and the selection of patients for novel forms of therapy, such as substrate deprivation or dispersal, enzyme replacement, bone-marrow transplantation and gene transfer.

1-Deoxynojirimycin↗

PubMed2004

Characterization and quantitation of membrane proteomes using multidimensional MS-based proteomic technologies.

A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.

Cell Adhesion↗

PubMedDate not supplied

[Inhibitors of HMG CoA reductase: new modes of action, new indications?].

3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitors, or statins, are hypocholesterolaemic agents used in the treatment of hypercholesterolaemia and cardiovascular diseases. Their hypocholesterolaemic action results from the inhibition of the intracellular synthesis of cholesterol via the mevalonate pathway. Recent studies have demonstrated that cholesterol is not the only intracellular target of statins. Indeed, statins have been shown to inhibit protein isoprenylation, a post-translational modification involved in membrane localization and protein activity. By inhibiting isoprenylation of Ras protein, statins induce the inhibition of the Ras-AP1 pathway and the decrease in cell proliferation. Similarly, inhibition of Rho protein insoprenylation leads to the disorganization of the cytoskeleton and the induction of the fibrinolytic system. All these effects need to be confirmed in vivo and in the human, and open new areas for these therapeutics agents.

Anticholesteremic Agents↗

PubMed2000

P53 regulation and function in normal cells and tumors.

Mechanisms to protect organisms from the consequences of DNA damage include the tumor suppressor p53 pathway. p53 protein binds specifically to a DNA consensus sequence to induce growth inhibitory genes or nonspecifically to damaged sites leading to DNA repair or apoptosis. While p53 protein is susceptible to post-translational modifications and binding to other proteins, few of the modifications or associations have been demonstrated in the context of the cell. We used a novel, sensitive DNA binding assay to examine p53 proteins in lysates prepared from cells responding to DNA damage. Non-transformed progenitor keratinocytes exhibited rapid and sustained induction of activated p53 protein binding to the consensus sequence, correlated with sustained induction of a downstream target gene, the cyclin-dependent kinase inhibitor p21. Binding to a mismatched DNA probe was transient and correlated with total p53 protein in the lysate, suggesting that most or all of the endogenous p53 proteins induced after damage were capable of mismatched DNA binding. Squamous cell carcinoma (SCC) derivates showed defects in induction p53 protein after DNA damage and disproportional losses in DNA binding, compared to total p53 protein steady state levels, along with increased NDN2/p53 association. Maximum induction of endogenous p53 protein binding to the mismatched probe correlated with transcription-independent induction of apoptosis. We suggest a model in which activated forms of p53 carry out transcription-dependent functions in growth arrest and DNA repair which may vary by cell type, while most or all wild type forms of p53 are capable of binding to damaged DNA. p53 protein loss of binding to damaged DNA causes failure of cells to trigger apoptosis and may lead to resistance to chemotherapy. Implications for new directions in therapeutics include functional molecular profiling of individual patient tumors for p53 DNA binding to predict treatment response better than mutational analysis alone and targeted activation of p53 DNA damage binding in tumor cells for increasing their sensitivity to chemotherapy.

Apoptosis↗

PubMedDate not supplied

Polypeptide ligation occurs during post-translational modification of concanavalin A.

Lectins are proteins with multivalent carbohydrate-binding sites, which confer the ability to agglutinate. The seeds of legumes are particularly rich in lectins, for example, concanavalin A (Con A) comprises up to 15% of the protein in the cotyledons of jack bean (Canavalia ensiformis) seeds. The amino acid sequences of Con A and several other legume lectins have been partially or fully determined, and comparison of these sequences from different species reveals a circular homology (Fig. 1A); rearrangements within the genome have been suggested to explain this. We report here that the circular homology displayed by Con A is due to a post-translational transposition and ligation within the initial polypeptide. This type of modification has not been reported previously for eukaryotes, although it has been suggested to occur in bacteriophage lambda.

Amino Acid Sequence↗

PubMed1989

Intracellular localisation and expression of mammalian CDC2 protein during myogenic differentiation.

Myogenic differentiation involves withdrawal of myoblasts from the cell cycle and fusion to form multinucleate myotubes. To examine the role that cell cycle control genes may play in this process, we investigated the steady state levels of CDC2 protein and RNA during myogenesis of L6E9 rat myoblasts. Indirect immunofluorescence using a CDC2 affinity-purified antibody showed that this protein is localised exclusively in the cytoplasm with a higher concentration perinuclearly. Both protein and RNA levels were down-regulated to similar extents early in the differentiation process, as cells became quiescent. There was a further down-regulation of protein after fusion to form myotubes. Autonomous expression of CDC2 protein in L6E9 cells, after stable transfection with a metallothionein: CDC2 gene construct, failed to inhibit the differentiation process. This suggests that, although there is down-regulation in levels of CDC2 RNA and protein during myogenesis, this phenomenon per se does not play a primary role in controlling the differentiation process. If CDC2 is involved in control of differentiation, this must depend on post-translational modification of the protein.

Animals↗

PubMed2003

Oxidative post-translational modification of tryptophan residues in cardiac mitochondrial proteins.

We examined the distribution of N-formylkynurenine, a product of the dioxidation of tryptophan residues in proteins, throughout the human heart mitochondrial proteome. This oxidized amino acid is associated with a distinct subset of proteins, including an over-representation of complex I subunits as well as complex V subunits and enzymes involved in redox metabolism. No relationship was observed between the tryptophan modification and methionine oxidation, a known artifact of sample handling. As the mitochondria were isolated from normal human heart tissue and not subject to any artificially induced oxidative stress, we suggest that the susceptible tryptophan residues in this group of proteins are "hot spots" for oxidation in close proximity to a source of reactive oxygen species in respiring mitochondria.

Amino Acid Sequence↗

PubMed1997

Characterization of native and recombinant bone sialoprotein: delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain.

Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106-01 BSP) culture media is shown to have a hydroxyapatite Kd approximately 2.6 x 10(-9) M, perhaps the strongest affinity for this mineral of any of the matrix proteins. Both native BSP and a 47 kD fragment of UMR-BSP (Fragment 1 approximately 133A- approximately 265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post-translational modifications. The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral-binding properties are encoded directly within the protein sequence itself. BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence. Nuclear magnetic resonance (NMR) analysis of a 15N-enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium. Previous work has also shown that an endogenous fragment of UMR-BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence. In this report, non-RGD cell adhesion sites are localized within conserved amino- and carboxy-terminal tyrosine-rich domains of recombinant human BSP. Given the proximity of the latter non-RGD cell adhesion site to the RGD tripeptide, a model of BSP-receptor interactions is presented.

Amino Acid Sequence↗

PubMed1985

1 alpha,25-Dihydroxyvitamin D-induced modification of a cytosolic protein in embryonic chick intestine.

Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3.

Animals↗

PubMed2005

ATM-mediated phosphorylations inhibit Mdmx/Mdm2 stabilization by HAUSP in favor of p53 activation.

The p53 tumor suppressor protein has a major role in protecting genome integrity. Under normal circumstances Mdmx and Mdm2 control the activity of p53. Both proteins inhibit the transcriptional regulation by p53, while Mdm2 also functions as an E3 ubiquitin ligase to target both p53 and Mdmx for proteasomal degradation. HAUSP counteracts the destabilizing effect of Mdm2 by direct deubiquitination of p53. Subsequently, HAUSP was shown to deubiquitinate Mdm2 and Mdmx, thereby stabilizing these proteins. The ATM protein kinase is a key regulator of the p53 pathway in response to double strand breaks (DSBs) in the DNA. ATM fine-tunes p53's response to DNA damage by directly phosphorylating it, by regulating additional post-translational modifications of this protein, and by affecting two p53 regulators: Mdm2 and Mdmx. ATM directly and indirectly induces Mdm2 and Mdmx phosphorylation, resulting in decreased activity and stability of these proteins. We recently provided a mechanism for the reduced stability of Mdm2 and Mdmx by showing that ATM-dependent phosphorylation lowers their affinity for the deubiquitinating enzyme HAUSP. Altogether, the emerging picture portrays an elaborate, but fine-tuned, ATM-mediated control of p53 activation and stabilization following DNA damage. Further insight into the mechanism by which ATM switches the interactions between HAUSP, Mdmx, Mdm2 and p53, to favor p53 activation may offer new tools for therapeutic intervention in the p53 pathway for cancer treatment.

Adenosine Triphosphate↗

PubMed1999

Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins.

The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90-104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp(93) to Leu(94) occurs through the beta-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiological conditions. These post-translationally modified peptides may serve as an early immunologic stimulus in autoimmune disease.

Amino Acid Sequence↗

PubMed1985

Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells.

Newly synthesized G protein of vesicular stomatitis virus is not transported to the surface of cultured mammalian cells during mitosis (Warren et al., 1983, J. Cell Biol. 97:1623-1628). To determine where intracellular transport is inhibited, we have examined the post-translational modifications of G protein, which are indicators of specific compartments on the transport pathway. G protein in mitotic cells had only endo H-sensitive oligosaccharides containing seven or eight mannose residues, but no terminal glucose, and was not fatty acylated. These modifications were indicative of processing only by enzymes of the endoplasmic reticulum (ER). Quantitative immunocytochemistry was used as an independent method to confirm that transport of G protein out of the ER was inhibited. The density of G protein in the ER cisternae was 2.5 times greater than in infected G1 cells treated similarly. Incubation of infected mitotic cells with cycloheximide, which inhibits protein synthesis without affecting transport, did not result in a decrease in the density of G protein in the ER cisternae, demonstrating that G protein cannot be chased out of the ER. These results suggest that intracellular transport stops at or before the first vesicle-mediated step on the pathway.

Animals↗

PubMed1996

Hormone-induced changes in cell shape: role of cytoskeletal proteins.

Shape is a characteristic phenotype for a given cell. It is affected by various physiological and pathological factors. Hormones, which are the chemical messengers affecting a wide range of biological phenomena, also alter cell morphology by influencing the expression and post-translational modifications of cytoskeletal proteins. The assembly/disassembly of cytoskeletal proteins and their interactions with intracellular components, the plasma membrane and extracellular matrix, is mainly responsible for determining cell shape. The role of hormones in the induction of altered cell shape due to changes in the cytoarchitecture in varied biological tissues under both in vivo and in vitro conditions, are discussed in this review.

Animals↗

PubMed2006

Ageing is associated with increased expression but decreased activity of CYP2E1 in male Wistar rats.

The effect of ageing on CYP2E1 activity and its protein and mRNA contents was investigated in both adult (9 months) and senescent (24 months) male Wistar rats. The CYP2E1 activity (as measured by chlorzoxazone hydroxylation) was significantly decreased by 36% in senescent rats as compared to adult rats. However, this decrease of activity was not associated with a loss of protein content because the amount of both CYP2E1 protein and CYP2E1 mRNA did not decrease in senescent rats but rather increased, by 79% and 64% respectively, as compared to adult rats. Lipid peroxidation was increased significantly by 140% with ageing. The decrease in CYP2E1 activity could be explained by post-translational modification of CYP2E1 proteins, due to an increase in oxidative stress in senescent animals, leading to a loss of their functionality. However, no changes in the extent of protein carbonyls were observed in the adult versus senescent rats (16.2 +/- 9.6 vs. 12.7 +/- 7.3 nmol/mg prot) and the major proteasome activity remained unchanged. With regards to the increase of CYP2E1 expression, our results showed that the amount of hepatocyte nuclear factor 1alpha mRNA, a transcription factor that positively regulates CYP2E1, was strongly increased (154%) in senescent rats.

Aging↗

PubMed1991

DBcAMP effect on the expression of GFAP and of its encoding mRNA in astroglial primary cultures.

Short term and chronic dBcAMP effects on the expression of glial fibrillary acidic protein (GFAP) in astroglial primary cultures are investigated. Short (48 h) and long (more than 7 days) treatments with the cAMP derivative induce both cell shape changes and an increase in GFAP immunolabelling. Such effects are only associated with an increase in GFAP and in GFAP-mRNA levels in the long term treatment. These results suggest that the short term effect of dBcAMP induces post-translational modifications of the protein whereas the long term effect is associated with an increase in GFAP mRNA transcription and/or stability.

Animals↗

PubMed1999

Selective beta1-adrenoceptor blockade enhances the activity of the stimulatory G-protein in human atrial myocardium.

1. Chronic selective beta1-adrenoceptor (beta1AR) blocker treatment enhances the sensitivity of beta2-adrenoceptor (beta2AR) in human heart (Hall et al., 1990; 1991). To clarify the mechanism of the cross-sensitization between beta1AR and beta2AR, we determined whether the stimulatory G-protein (G(s)alpha) function is increased in atria from beta1AR-blocker treated patients compared with non-beta-blocked patients, and investigated whether this change is caused by an alteration of post-translational modification of Gsalpha protein. 2. G(s)alpha function was determined by reconstitution of human atrial G(s)alpha into S49 cyc- cell membranes. In the reconstitution system, GTPgammaS stimulated cyclic AMP generation in a dose-dependent manner. Upon 10(-4) M GTPgammaS stimulation, G(s)alpha activity in the beta1AR-blocker, atenolol, treated group (78.2+/-10. 3 pmol cyclic AMP mg(-1) min(-1) 10(-3)) was 65% higher than that in non-beta-blocked patients (47.3+/-6.3 pmol cyclic AMP mg(-1) min(-1) 10(-3), n=15, P=0.02). 3. Isoelectric point (pI) valu G(s)alpha were measured by two dimensional gel electrophoresis (2D-E) and the amount of each isoform quantified by image analysis of a Western blot of the gel using specific antibody. Multiple isoforms of G(s)alpha were detected by 2D-E with different pI values. There were no significant differences between the groups of patients in either pI values or the proportions of the acidic isoforms of G(s)alpha to the main basic form (n=12, P>0.05). 4. The results suggest that chronic beta1AR-blockade enhances Gsalpha function in human atrium, and this may account in part for the hypersensitivity of beta2AR and other Gs-coupled receptors during beta1AR-blockade. The increased G(s)alpha function is unlikely to be caused directly by blockade of protein kinase A phosphorylation of G(s)alpha protein.

Adenylyl Cyclases↗