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PubMed2004

TAGE (toxic AGEs) hypothesis in various chronic diseases.

The advanced stage of the glycation process (one of the post-translational modifications of proteins) leads to the formation of advanced glycation end-products (AGEs) and plays an important role in the pathogenesis of angiopathy in diabetic patients, in aging, and in neurodegenerative diseases. However, it is still not clear which AGEs subtypes play a pathogenetic role and which of several AGEs receptor mediate AGEs effects on cells. We have provided direct immunochemical evidence for the existence of six distinct AGEs structures (AGEs-1 to -6) within the AGEs-modified proteins and peptides that circulate in the serum of diabetic patients. Recently we demonstrated for the first time that glyceraldehyde-derived AGEs (AGEs-2) and glycolaldehyde-derived AGEs (AGE-3) have diverse biological activities on vascular wall cells, mesangial cells, Schwann cells, malignant melanoma cells and cortical neurons. We also demonstrated for the first time that acetaldehyde (AA)-derived AGEs (AA-AGE) have cytotoxic activity on cortical neurons and the AA-AGE epitope was detected in human brain of alcoholics. These results indicate that of the various types of AGEs structures that can form in vivo, the toxic AGEs (TAGE) structures (AGEs 2, 3, and AA-AGE), but not non-toxic AGEs (N-carboxymethyllysine, pentosidine, pyrraline etc.) are likely to play an important role in the pathophysiological processes associated with AGEs formation.

Aging↗
PubMed2004

Alternative routes for the formation of glyceraldehyde-derived AGEs (TAGE) in vivo.

The advanced stage of the glycation process (one of the post-translational modifications of proteins) leads to the formation of advanced glycation end-products (AGEs) and plays an important role in the pathogenesis of angiopathy in diabetic patients, and in Alzheimer's disease (AD). Recently we have provided direct immunochemical evidence for the existence of six distinct AGEs structures, designated AGEs-1 to -6, within the AGEs-modified proteins and peptides that circulate in the serum of diabetic patients. We found for the first time that glyceraldehyde-derived AGEs (AGE-2), which comprise main structure of TAGE (toxic AGEs), in the serum of diabetic patients have diverse biological activities on vascular wall cells and cortical neurons. These results suggest a causal role for AGE-2 in the pathogenesis of diabetic complications and AD in vivo. In AD brains, AGE-2 epitope was mainly present in the cytosol of neurons in the hippocampus and para-hipocampal gyrus. We propose three pathways for the in vivo formation of AGE-2 precursor, glyceraldehyde, by: (i) glycolytic pathway, (ii) polyol pathway, and (iii) fructose metabolic pathway. Glyceraldehyde can be transported or can leak passively across the plasma membrane. It can react non-enzymatically with proteins to lead to accelerated formation of AGE-2 at both intracellular and extracellular region.

Aging↗
PubMed2006

epsilon-Glycation, APP and Abeta in ageing and Alzheimer disease: a hypothesis.

The post-translational modifications of protein molecules include glycation, which may not only occur enzymatically controlled in N and O position, but also wherever proteins meet reducing sugars non-enzymatically in epsilon position at lysines (non-enzymatic (epsilon) glycation (NEG)). The formation of keto-amines from the amine-sugar compounds (Amadori re-arrangement) and further processing of the largely undigestible Amadori compounds eventually results in insoluble advanced glycation end products (AGEs). The latter can induce or favour disease including mental disorders. Preferential targets of NEG include large cell surface proteins. Ample evidence has been provided that NEG also occurs in the brain where cross-linking of epsilon-glycated proteins, induction of oxidative stress and signalling of AGEs through their specific receptor (RAGE) likely play a role in (brain) ageing and Alzheimer disease (AD). This is underscored by the demonstration of particular interactions between AGE/RAGE and amyloid-beta (Abeta) that favour the aggregation and deposition of Abeta and, perhaps, the formation of Abeta itself. The close relationship between NEG and Abeta, as well as other facts foster the hypothesis that NEG of the large trans-membrane amyloid precursor protein (APP) might be a significant factor in the induction of aberrant APP cleavage with production of Abeta, not only in normal ageing, but also in AD. Blockade of lysine cleavage sites on APP by sugar chains or marker effects induced by NEG akin to ubiquitination of proteins for degradation at lysines could be expected to contribute to altered processing of APP. The hypothesis of epsilon-glycation in APP proposed here and the review of evidences for the significance of NEG in brain ageing and AD are aimed at the stimulation of investigations into the still open question which role NEG plays with respect to APP and its abnormal processing in AD. It can be rendered likely that such research might open new avenues towards decreasing the risk of AD and/or slowing its progression through the prevention of NEG in APP with aberrant APP processing, increased generation of Abeta and the formation of AGEs from epsilon-glycated APP.

Aging↗
PubMed2006

The role of tissue transglutaminase in 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells.

Tissue transglutaminase (TG2) can induce post-translational modification of proteins, resulting in protein cross-linking or incorporation of polyamines into substrates, and can also function as a signal transducing G protein. The role of TG2 in the formation of insoluble cross-links has led to its implication in some neurodegenerative conditions. Exposure of pre-differentiated SH-SY5Y cells to the Parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP(+)) resulted in significant dose-dependent reductions in TG2 protein levels, measured by probing Western blots with a TG2-specific antibody. Transglutaminase (TG) transamidating activity, on the other hand, monitored by incorporation of a polyamine pseudo-substrate into cellular proteins, was increased. Inhibitors of TG (putrescine) and TG2 (R283) exacerbated MPP(+) toxicity, suggesting that activation of TG2 may promote a survival response in this toxicity paradigm.

1-Methyl-4-phenylpyridinium↗
PubMed2004

Understanding global changes in histone H1 phosphorylation using mass spectrometry.

Histone post-translational modifications have been implicated in a variety of biological processes such as gene expression, DNA replication, and chromatin assembly. The modifications include methylation, acetylation, phosphorylation, ubiquitination, glycosylation, and ADP-ribosylation. For several years, we have been investigating the role of histone H1 phosphorylation in transcription using the hormone inducible mouse mammary tumor virus (MMTV) promoter. When mouse cells were exposed to prolonged treatment with dexamethasone, a significant decrease in the level of histone H1 phosphorylation was observed. Traditionally, Western analyses with anti-histone H1 and phospho-specific H1 antibodies were performed to observe changes in phosphorylation levels of the bulk H1 histones. More recently, we have applied electrospray ionization mass spectrometry to the analysis of histone H1 isoforms. Utilizing this approach, we have investigated the phosphorylation state of the specific H1 isoforms before and after prolonged treatment with dexamethasone. Specifically, we could determine that the relative phosphorylation levels of the histone H1.3, H1.4, and H1.5 isoforms decrease after prolonged hormone exposure. Recent advancements in mass spectrometry have proven invaluable toward the analysis of post-translational modifications on proteins. The continued developments in the area of mass spectrometry should provide new insights into not only the function of proteins but also into the basic regulatory mechanisms that control cellular functions.

Animals↗
PubMed1999

From genes to channels: normal mechanisms.

Electrophysiologic remodeling is a process whereby heart disease alters the electrophysiologic properties of cardiac tissue. These alterations, in turn, can cause or exacerbate disease-related arrhythmias. Ion channels are the fundamental molecular units underlying cardiac electrophysiology, and it therefore follows that electrophysiologic remodeling represents alterations in the function or expression of genes encoding ion channels or other proteins crucial for cardiac electrophysiologic activity. This review will describe the mechanisms whereby normal function of these proteins arises from the processes of gene transcription, mRNA processing, and protein transport, post-translational modification, assembly with other proteins, and degradation. Identification of entirely novel targets for drug intervention should result from further understanding of the fundamental mechanisms underlying remodeling.

Animals↗
PubMed2002

Collection of alpha1-acid glycoprotein molecular species by capillary electrophoresis and the analysis of their molecular masses and carbohydrate chains. Basic studies on the analysis of glycoprotein glycoforms.

A highly heterogeneous glycoprotein, alpha1-acid glycoprotein, was resolved into their glycoforms by capillary electrophoresis using a surface-modified capillary in 20 mM acetate buffer (pH 4.2) containing 0.5% (w/v) hydroxypropylmethylcellulose. We collected the fractions containing each glycoform as nearly pure state by capillary electrophoresis, and examined the molecular masses of these glycoforms by matrix assisted laser desorption time-of-flight mass spectrometry. We also analyzed carbohydrate chains after releasing them with N-glycosidase F followed by fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonate. We found that the separation of glycoforms was mostly due to the presence of multiantennary carbohydrate chains. We propose that the present technique is useful for the analysis of post translational modification of proteins with carbohydrate chains.

Carbohydrate Sequence↗
PubMed2005

Preparation of myristoylated Arf1 and Arf6.

Arf proteins are members of the Arf family of small Ras-like GTP binding proteins. Six Arfs, grouped into three classes, have been identified in mammalian cells and three members have been identified in yeasts. Arf1 and Arf6, more extensively studied than other Arfs, have been found to affect membrane traffic and actin remodeling. A structural feature that distinguishes Arfs from other Ras superfamily members is an N-terminal alpha-helix, extending from the basic G-protein fold, which is cotranslationally myristoylated. Both the helix and the myristate affect biochemical properties of Arfs, including nucleotide exchange, membrane association, and interaction with some effector proteins. Preparation of myristoylated Arf for in vitro studies of Arf function requires consideration of both the reaction yielding myristoylated protein and the properties of the modified Arfs. Here, we describe methods that yield homogeneous preparations of myristoylated Arf1 and Arf6.

ADP-Ribosylation Factor 1↗
PubMed2003

Glycation--a sweet tempter for neuronal death.

Glycation, one of the post-translational modifications of proteins, is a nonenzymatic reaction initiated by the primary addition of a sugar aldehyde or ketone to the amino groups of proteins. In the early stage of glycation, the synthesis of intermediates leading to the formation of Amadori compounds occurs. In the late stage, advanced glycation end products (AGE) are irreversibly formed after a complex cascade of reactions. Several AGEs have been characterized chemically, while other new compounds remain to be identified. To date, studies of the contribution of glycation to diseases have been primarily focused on its relationship to diabetes and diabetes-related complications. However, glucose-induced damage is not limited to diabetic patients. Although it does not cause rapid or remarkable cell damage, glycation advances slowly and accompanies every fundamental process of cellular metabolism. It has recently become clear that glycation also affects physiological aging and neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Glycation alters the biological activity of proteins and their degradation processes. Protein cross-linking by AGE results in the formation of detergent-insoluble and protease-resistant aggregates. Such aggregates may interfere with both axonal transport and intracellular protein traffic in neurons. In addition, glycation reactions lead to the production of reactive oxygen species. Conversely, glycation is promoted by oxidative stress. We speculate on the presence of synergism between glycation and oxidative stress. In this review, we provide an outline of glycation and propose some possible mechanisms of its cytotoxicity and defense systems against it.

Animals↗
PubMed1997

Inhibition by compactin demonstrates a requirement of isoprenoid metabolism for long-term potentiation in rat hippocampal slices.

Hippocampal long-term potentiation of synaptic transmission is the primary experimental model of learning and memory in the vertebrate brain. However, the detailed intracellular mechanisms giving rise to this persistent increase in synaptic efficacy remain incompletely understood. Mevalonic acid constitutes the basic precursor not only for cholesterol, dolichol and ubichinone but also for farnesyl-pyrophosphate and geranylgeranylpyrophosphate, which are required for post-translational modification of proteins. We have used the specific 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, compactin, to examine the role of isoprenoid metabolism for long-term potentiation in rat hippocampal slices. Compactin was applied at a concentration of 25 microM for 70 min before and during tetanization and the orthodromic population spike amplitude and field excitatory postsynaptic potentials were recorded from CA1 pyramidal cells. Compactin had no effect on the initial tetanization. However, compactin-treated slices were not able to maintain long-term potentiation for more than 60 min and population spike as well as field excitatory postsynaptic potentiation returned to basal levels after 120 min. When the slices were retetanized after 180 min, an almost full potentiation of the population spike and an only partial potentiation of the field excitatory postsynaptic potentials were observed. These results suggest an essential role of isoprenoid intermediates for maintenance of hippocampal long-term potentiation.

Animals↗
PubMedDate not supplied

Tissue-transglutaminase in rat and human brain: light and electron immunocytochemical analysis and in situ hybridization study.

Tissue-type transglutaminases constitute a family of enzymes having a dual role. They catalyze the post-translational modification of proteins and play a role in signal transduction pathways, several isoforms have been cloned in the brain. Many in vitro experiments and post-mortem studies have claimed that the enzyme plays a central role in the development of neurodegenerative disorders, especially in CAG-triplet diseases. In the present investigation, we conducted an immunocytochemical study using two different antibodies raised against tissue-type transglutaminase. To confirm the enzyme expression, non-radioactive in situ hybridization was performed on adjacent sections. The study was completed by analyzing the ultrastructural localization of the enzyme by electron microscopy. Tissue-type transglutaminase was widely expressed in both the human and rat brain. Many positive cells exhibiting neuronal features were found in the brain and cerebellum. There was a preferential expression in elements of pyramidal and extrapyramidal pathways with less expression in the somatosensory system. The mRNA detection confirmed the distribution of the enzyme. The ultrastructural approach revealed the presence of the enzyme in all neuronal compartments. Light and electron microscopy studies showed the ubiquitous nature of the enzyme and its putative role in functional as well as putative pathological processes.

Animals↗
PubMed1998

Structure of the gene encoding the ubiquitin-conjugating enzyme Ubcm4, characterization of its promoter, and chromosomal location.

Ubiquitin-conjugating enzymes (E2 or Ubc) play a key role in the post-translational modification of proteins by ubiquitylation. They are encoded by a large family of genes that are closely related to each other. In this paper we present the first complete structural analysis, including the promoter and the chromosomal location, of a member of this family, the mouse Ubcm4 gene. At the genomic level the Ubcm4 gene spans approx. 50kb and is composed of four exons. Only about 1% of the total gene codes for amino acids. The four different Ubcm4 specific RNAs encode the same protein and differ only in the length of the 3' untranslated region. The polyadenylation signals used by the four different RNAs are all within the 3' terminal exon. At the 5' end of the gene, multiple transcriptional start sites were mapped within a region of 25bp. The region proximal to the initiation sites does not contain a TATA box and is not GC-rich. Transient chloramphenicol acetyltransferase assays, however, showed that this region can promote the expression of a reporter gene and that 15bp upstream of the first initiation site were sufficient for basal expression. The Ubcm4 gene was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 16.

Amino Acid Sequence↗
PubMed2004

Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer.

Post-translational modification of proteins is an important regulatory event. Numerous biologically active peptides that play an essential role in cancerogenesis contain an evolutionary conserved proline residue as a proteolytic-processing regulatory element. Proline-specific proteases could therefore be viewed as important "check-points". Limited proteolysis of such peptides may lead to quantitative but, importantly, due to the change of receptor preference, also qualitative changes of their signaling potential. Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with CD26) was for many years believed to be a unique cell membrane protease cleaving X-Pro dipeptides from the N-terminal end of peptides and proteins. Subsequently, a number of other molecules were discovered, exhibiting various degree of structural homology and DPP-IV-like enzyme activity, capable of cleaving similar set of substrates. These comprise for example, seprase, fibroblast activation protein alpha, DPP6, DPP8, DPP9, attractin, N-acetylated-alpha-linked-acidic dipeptidases I, II and L, quiescent cell proline dipeptidase, thymus-specific serine protease and DPP IV-beta. It is tempting to speculate their potential participation on DPP-IV biological function(s). Disrupted expression and enzymatic activity of "DPP-IV activity and/or structure homologues" (DASH) might corrupt the message carried by their substrates, promoting abnormal cell behavior. Consequently, modulation of particular enzyme activity using e.g. DASH inhibitors, specific antibodies or DASH expression modification may be an attractive therapeutic concept in cancer treatment. This review summarizes recent information on the interactions between DASH members and their substrates with respect to their possible role in cancer biology.

Animals↗
PubMed2006

Zirconium phosphonate-modified porous silicon for highly specific capture of phosphopeptides and MALDI-TOF MS analysis.

Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activity of proteins. The analysis of phosphopeptides is still one of the most challenging tasks in proteomics research by mass spectrometry. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of zirconium phosphonate (ZrP) modified surface with phosphopeptides has been developed. ZrP modified porous silicon (ZrP-pSi) wafer was prepared to specifically capture the phosphopeptides from complex peptide mixtures, and then the captured phosphopeptides were analyzed by MALDI-TOF MS by directly placing the wafer on a MALDI target. The phosphopeptide enrichment and MALDI analysis were both performed on the ZrP-pSi wafer which significantly reduced the sample loss and simplified the analytical procedures. The prepared ZrP-pSi wafer has been successfully applied for the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins beta-casein and alpha-casein. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of beta-casein and bovine serum albumin with molar ratio of 1:100. High detection sensitivity has been achieved for the analysis of the phosphopeptides from tryptic digestion of 2 fmol beta-casein on the ZrP-pSi surface.

Amino Acid Sequence↗
PubMed2002

Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update.

Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. The best known poly(ADP-ribosylating) enzyme, PARP-1, is a DNA nick sensor and uses betaNAD(+) to form polymers of ADP-ribose which are further bound to nuclear protein acceptors. To strictly regulate poly(ADP-ribose) turnover, its degradation is assured by the enzyme poly(ADP-ribose) glycohydrolase (PARG). During apoptosis, PARP-1 plays two opposite roles: its stimulation leads to poly(ADP-ribose) synthesis, whereas caspases cause PARP-1 cleavage and inactivation. PARP-1 proteolysis produces an 89 kDa C-terminal fragment, with a reduced catalytic activity, and a 24 kDa N-terminal peptide, which retains the DNA binding domains. The fate and the possible role of these fragments during apoptosis will be discussed.

Animals↗
PubMed2004

Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase.

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

Animals↗
PubMed2003

cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type I.

Peptidylarginine deiminases (PADs) catalyse a post-translational modification of proteins through the conversion of arginine residues into citrullines. The existence of four isoforms of PAD (types I, II, III and IV) encoded by four different genes, which are distinct in their substrate specificities and tissue-specific expression, was reported in rodents. In the present study, starting from epidermis polyadenylated RNA, we cloned by reverse transcriptase-PCR a full-length cDNA encoding human PAD type I. The cDNA was 2711 bp in length and encoded a 663-amino-acid sequence. The predicted protein shares 75% identity with the rat PAD type I sequence, but displays only 50-57% identity with the three other known human isoforms. We have described the organization of the human PAD type I gene on chromosome 1p36. A recombinant PAD type I was produced in Escherichia coli and shown to be enzymically active. Human PAD type I mRNAs were detected by reverse transcriptase-PCR not only in the epidermis, but also in various organs, including prostate, testis, placenta, spleen and thymus. In human epidermis extracts analysed by Western blotting, PAD type I was detected as a 70 kDa polypeptide, in agreement with its predicted molecular mass. As shown by immunohistochemistry, the enzyme was expressed in all the living layers of human epidermis, with the labelling being increased in the granular layer. This is the first description of the human PAD type I gene and the first demonstration of its expression in epidermis.

Amino Acid Sequence↗
PubMed2004

Redox signalling: from nitric oxide to oxidized lipids.

Cellular redox signalling is mediated by the post-translational modification of proteins in signal-transduction pathways by ROS/RNS (reactive oxygen species/reactive nitrogen species) or the products derived from their reactions. NO is perhaps the best understood in this regard with two important modifications of proteins known to induce conformational changes leading to modulation of function. The first is the addition of NO to haem groups as shown for soluble guanylate cyclase and the newly discovered NO/cytochrome c oxidase signalling pathway in mitochondria. The second mechanism is through the modification of thiols by NO to form an S-nitrosated species. Other ROS/RNS can also modify signalling proteins although the mechanisms are not as clearly defined. For example, electrophilic lipids, formed as the reaction products of oxidation reactions, orchestrate adaptive responses in the vasculature by reacting with nucleophilic cysteine residues. In modifying signalling proteins ROS/RNS appear to change the overall activity of signalling pathways in a process that we have termed 'redox tone'. In this review, we discuss these different mechanisms of redox cell signalling, and give specific examples of ROS/RNS participation in signal transduction.

Lipid Metabolism↗