Red blood cells in the metabolism of nitric oxide-derived peroxynitrite

In this review we have analyzed the reactions of nitric oxide (.NO) with superoxide radical (O(2).-) at the vascular compartment which results in limitation of the bioavailability of .NO and the formation of peroxynitrite (ONOO-), a strong oxidant species. The intravascular formation of peroxynitrite can result in oxidative modifications of plasma and vessel wall proteins including the formation of protein-3-nitrotyrosine. The role of red blood cells (RBC) and oxyhemoglobin in the metabolism of intravascular peroxynitrite will be discussed. While RBC constitute an important 'sink' of both .NO and peroxynitrite, redox reactions of these species with oxyhemoglobin may in part contribute to erythrocyte aging. The intravascular formation, reactions and detoxification of peroxynitrite are revealed as important factors controlling vascular dysfunction and degeneration in a variety of pathophysiologically-relevant conditions.     

Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes. Formation of modified and oxidized particles.

Patients with diabetes mellitus suffer from an increased incidence of complications including cardiovascular disease and cataracts; the mechanisms responsible for this are not fully understood. One characteristic of such complications is an accumulation of advanced glycation end-products formed by the adduction of glucose or species derived from glucose, such as low-molecular mass aldehydes, to proteins. These reactions can be nonoxidative (glycation) or oxidative (glycoxidation) and result in the conversion of low-density lipoproteins (LDL) to a form that is recognized by the scavenger receptors of macrophages. This results in the accumulation of cholesterol and cholesteryl esters within macrophages and the formation of foam cells, a hallmark of atherosclerosis. The nature of the LDL modifications required for cellular recognition and unregulated uptake are poorly understood. We have therefore examined the nature, time course, and extent of LDL modifications induced by glucose and two aldehydes, methylglyoxal and glycolaldehyde. It has been shown that these agents modify Arg, Lys and Trp residues of the apoB protein of LDL, with the extent of modification induced by the two aldehydes being more rapid than with glucose. These processes are rapid and unaffected by low concentrations of copper ions. In contrast, lipid and protein oxidation are slow processes and occur to a limited extent in the absence of added copper ions. No evidence was obtained for the stimulation of lipid or protein oxidation by glucose or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions to a modest extent. These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification) of the protein component of LDL, whilst oxidative events (glycoxidation or direct oxidation of lipid or proteins) only occur to any significant extent at later time points. This 'carbonyl-stress' may facilitate the formation of foam cells and the vascular complications of diabetes.     

Evidence for non-enzymatic glycosylation in Escherichia coli

Non-enzymatic glycosylation (glycation) is a chain of chemical reactions affecting free amino groups in proteins of long-living eukaryotes. It proceeds in several steps leading to the consecutive formation of Schiff bases, Amadori products and advanced glycation end-products (AGEs). To our knowledge, this process has not been observed in prokaryotes so far. However, the present study provides clear-cut evidence that glycation takes place in bacteria despite their short life span. We have detected AGEs in recombinant human interferon gamma (rhIFN-gamma) produced in Escherichia coli as well as in total protein of the same bacterium using three different approaches: (i) Western blotting using two monoclonal antibodies raised against AGEs; (ii) fluorescent spectroscopy; and (iii) investigation of the effect of known AGE inhibitors (such as acetyl salicylic acid and thiamine) on the glycation reaction. Our study shows that non-enzymatic glycosylation is initiated during the normal growth of E. coli and results in AGE formation even after isolation of proteins. This process seems to be tightly associated with some post-translational modifications observed in the cysteineless rhIFN-gamma, such as covalent dimerization and truncation.     

Application of chemical selective cleavage methods to analyze post-translational modification in proteins

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.     

Maillard reaction products in tissue proteins: New products and new perspectives

Summary. The chemical modification of protein by nonenzymatic browning or Maillard reactions increases with age and in disease. Maillard products are formed by reactions of both carbohydrate- and lipid-derived intermediates with proteins, leading to formation of advanced glycation and lipoxidation end-products (AGE/ALEs). These modifications and other oxidative modifications of amino acids increase together in proteins and are indicators of tissue aging and pathology. In this review, we describe the major pathways and characteristic products of chemical modification of proteins by carbohydrates and lipids during the Maillard reactions and identify major intersections between these pathways. We also describe a new class of intracellular sulfhydryl modifications, Cys-AGE/ALEs, that may play an important role in regulatory biology and represent a primitive link between nonenzymatic and enzymatic chemistry in biological systems.     

Kinetic Memory Based on the Enzyme-Limited Competition

Cellular memory, which allows cells to retain information from their environment, is important for a variety of cellular functions, such as adaptation to external stimuli, cell differentiation, and synaptic plasticity. Although posttranslational modifications have received much attention as a source of cellular memory, the mechanisms directing such alterations have not been fully uncovered. It may be possible to embed memory in multiple stable states in dynamical systems governing modifications. However, several experiments on modifications of proteins suggest long-term relaxation depending on experienced external conditions, without explicit switches over multi-stable states. As an alternative to a multistability memory scheme, we propose “kinetic memory” for epigenetic cellular memory, in which memory is stored as a slow-relaxation process far from a stable fixed state. Information from previous environmental exposure is retained as the long-term maintenance of a cellular state, rather than switches over fixed states. To demonstrate this kinetic memory, we study several models in which multimeric proteins undergo catalytic modifications (e.g., phosphorylation and methylation), and find that a slow relaxation process of the modification state, logarithmic in time, appears when the concentration of a catalyst (enzyme) involved in the modification reactions is lower than that of the substrates. Sharp transitions from a normal fast-relaxation phase into this slow-relaxation phase are revealed, and explained by enzyme-limited competition among modification reactions. The slow-relaxation process is confirmed by simulations of several models of catalytic reactions of protein modifications, and it enables the memorization of external stimuli, as its time course depends crucially on the history of the stimuli. This kinetic memory provides novel insight into a broad class of cellular memory and functions. In particular, applications for long-term potentiation are discussed, including dynamic modifications of calcium-calmodulin kinase II and cAMP-response element-binding protein essential for synaptic plasticity.     

A close-up view of membrane contact sites between the endoplasmic reticulum and the endolysosomal system: From yeast to man

Abstract

Maintenance of organelle identity is crucial for the functionality of eukaryotic cells. Hence, transfer reactions between different compartments must be highly efficient and tightly regulated at the same time. Membrane contact sites (MCSs) represent an important route for inter-organelle transport and communication independent of vesicular trafficking. Due to extensive research, the mechanistic understanding of these sites increases constantly. However, how the formation and the versatile functions of MCSs are regulated is mainly unclear. Within this review, we focus on one well-known MCS, the nucleus–vacuole junction in yeast and discuss its analogy to endoplasmic reticulum-late endosome contacts in metazoan. Formation of the junction in yeast requires Vac8, a protein that is involved in various cellular processes at the yeast vacuole and a target of multiple posttranslational modifications. We discuss the possibility that dual functionality of proteins involved in contact formation is a common principle to coordinate inter-organelle transfer with organellar biogenesis.     

Using a multi-faceted approach to determine the changes in bacterial cell surface properties influenced by a biofilm lifestyle

Biofilm formation is a developmental process in which initial reversible adhesion is governed by physico-chemical forces, whilst irreversible adhesion is mediated by biological changes within a cell, such as the production of extracellular polymeric substances. Using two bacteria, E. coli MG1655 and B. cereus ATCC 10987, this study establishes that the surface of the bacterial cell also undergoes specific modifications, which result in biofilm formation and maintenance. Using various surface characterisation techniques and proteomics, an increase in the surface exposed proteins on E. coli cells during biofilm formation was demonstrated, along with an increase in hydrophobicity and a decrease in surface charge. For B. cereus, an increase in the surface polysaccharides during biofilm formation was found as well as a decrease in hydrophobicity and surface charge. This work therefore shows that surface modifications during biofilm formation occur and understanding these specific changes may lead to the formulation of effective biofilm control strategies in the future.     

Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein

Oxidation of low density lipoproteins (LDL) induced by incubation with Cu(2+) ions results in the formation of a heterogeneous group of aldehydic adducts on lysyl residues (Lys) of apolipoprotein B (apoB) that are thought to be responsible for the uptake of oxidized LDL (oxLDL) by macrophages. To define the structural and chemical criteria governing such cell recognition, we induced two modifications of lysines in LDL that mimic prototypic adducts present in oxLDL; namely, epsilon-amino charge-neutralizing pyrrolation by treatment with 2,5-hexanedione (hdLDL), and epsilon-amino charge-retaining pyridinium formation via treatment with 2,4,6-trimethylpyrylium (tmpLDL). Both modifications led to recognition by receptors on mouse peritoneal macrophages (MPM). To assess whether the murine scavenger receptor class A-I (mSR-A) was responsible for recognition of hdLDL or tmpLDL in MPM, we measured binding at 4 degrees C and degradation at 37 degrees C of these modified forms of (125)I-labeled LDL by mSR-A-transfected CHO cells. Although uptake and degradation of hdLDL by mSR-A-transfected CHO cells was quantitatively similar to that of the positive control, acLDL, tmpLDL was not recognized by these cells. However, both tmpLDL and hdLDL were recognized by 293 cells that had been transfected with CD36. In the human monocytic cell line THP-1 that had been activated with PMA, uptake of tmpLDL was significantly inhibited by blocking monoclonal antibodies to CD36, further suggesting recognition of tmpLDL by this receptor. Macrophage uptake and degradation of LDL oxidized by brief exposure to Cu(2+) was inhibited more effectively by excess tmpLDL and hdLDL than was more extensively oxidized LDL, consistent with the recognition of the former by CD36 and the latter primarily by SR-A.Collectively, these studies suggest that formation of specific pyrrole adducts on LDL leads to recognition by both the mSR-A and mouse homolog of CD36 expressed on MPM, while formation of specific pyridinium adducts on LDL leads to recognition by the mouse homolog of CD 36 but not by mSR-A. As such, these two modifications of LDL may represent useful models for dissecting the relative contributions of specific modifications on LDL produced during oxidation, to the cellular uptake of this heterogeneous ligand.     

Sperm proteomics: potential impact on male infertility treatment

Spermatozoa are unique cells that have highly compact DNA, motility (and hypermotility) patterns, a specific morphology, localized mitochondria and an apical acrosome. They are the end product of a dynamic process termed spermatogenesis. Sperm are therefore produced with specific proteins in order to effect different traits, such as the presence of cysteine-rich protamines in DNA, which effectively compacts DNA. Moreover, specific proteins are transferred during epididymal maturation and after ejaculation in order to render sperm capable of undergoing post-ejaculatory alterations, generally termed capacitation, which confers capacity to fertilize a mature oocyte. In addition, sperm exhibit several post-translational modifications, which are fundamental to their function, such as SUMOylation and ubiquitination. Discussed in this review is the current knowledge of the sperm proteome in terms of its composition and the function that these proteins determine, as well as their post-translational modifications and how these alter sperm functional integrity. Studies are emphasized that focus on shotgun proteomics – untargeted determination of the protein constituent of a cell in a given biological condition – and technologies currently applied toward that end are reviewed.     

Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications.

Given the complexity of the biosynthetic machinery and the delicate chemical composition of proteins, it is remarkable that cells manage to produce and maintain normally functioning proteins under most conditions. However, it is now well known that proteins are susceptible to various non-enzymatic covalent modifications (NECM) under physiological conditions. Such modifications can be of no or little importance to the protein or they can be absolutely detrimental. Often NECM are difficult to study due to the complex and technically demanding methods required to identify many of these modifications. Thus, the role of NECM has not yet been adequately resolved but recent research has allowed a better understanding of such modifications. The present review outlines the various forms of NECM that involve covalent modifications of proteins, and discusses their relevance, biological impact and potential applications in the study of protein turnover and diagnosis of disease.     

3-hydroxykynurenine-mediated modification of human lens proteins: structure determination of a major modification using a monoclonal antibody

Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.     

Carbonylation of milk powder proteins as a consequence of processing conditions

During industrial treatments, milk proteins could be oxidatively modified, thus leading to the formation of modified/oxidised amino acid residues. The apparition of such modified residues may contribute to the formation of new immunologically reactive structures. Some of these adducts could, in an advanced stage, lead to cross-linked protein species whose proteolytic susceptibility would be drastically decreased. Such protein species, that are resistant to digestion, could also constitute major food allergens. Therefore, these oxidative protein modifications tend to increase the natural allergenicity of milk proteins. For these reasons, monitoring milk protein oxidative modifications could be very useful regarding both product quality and allergenicity issues. In the present paper, we highlight, using different analytical approaches, the preferential carbonylation of beta-lactoglobulin (beta-Lg) during industrial treatments of milk. This result is particularly interesting since native beta-Lg represents one of the major milk allergens.     

Bacteriocins of gram-positive bacteria.

In recent years, a group of antibacterial proteins produced by gram-positive bacteria have attracted great interest in their potential use as food preservatives and as antibacterial agents to combat certain infections due to gram-positive pathogenic bacteria. They are ribosomally synthesized peptides of 30 to less than 60 amino acids, with a narrow to wide antibacterial spectrum against gram-positive bacteria; the antibacterial property is heat stable, and a producer strain displays a degree of specific self-protection against its own antibacterial peptide. In many respects, these proteins are quite different from the colicins and other bacteriocins produced by gram-negative bacteria, yet customarily they also are grouped as bacteriocins. Although a large number of these bacteriocins (or bacteriocin-like inhibitory substances) have been reported, only a few have been studied in detail for their mode of action, amino acid sequence, genetic characteristics, and biosynthesis mechanisms. Nevertheless, in general, they appear to be translated as inactive prepeptides containing an N-terminal leader sequence and a C-terminal propeptide component. During posttranslational modifications, the leader peptide is removed. In addition, depending on the particular type, some amino acids in the propeptide components may undergo either dehydration and thioether ring formation to produce lanthionine and beta-methyl lanthionine (as in lantibiotics) or thio ester ring formation to form cystine (as in thiolbiotics). Some of these steps, as well as the translocation of the molecules through the cytoplasmic membrane and producer self-protection against the homologous bacteriocin, are mediated through specific proteins (enzymes). Limited genetic studies have shown that the structural gene for such a bacteriocin and the genes encoding proteins associated with immunity, translocation, and processing are present in a cluster in either a plasmid, the chromosome, or a transposon. Following posttranslational modification and depending on the pH, the molecules may either be released into the environment or remain bound to the cell wall. The antibacterial action against a sensitive cell of a gram-positive strain is produced principally by destabilization of membrane functions. Under certain conditions, gram-negative bacterial cells can also be sensitive to some of these molecules. By application of site-specific mutagenesis, bacteriocin variants which may differ in their antimicrobial spectrum and physicochemical characteristics can be produced. Research activity in this field has grown remarkably but sometimes with an undisciplined regard for conformity in the definition, naming, and categorization of these molecules and their genetic effectors. Some suggestions for improved standardization of nomenclature are offered.     

Advanced glycation end-product pentosidine accumulates in various tissues of rats with high fructose intake

The slowly metabolized proteins of the extracellular matrix, typically collagen and elastin, accumulate reactive metabolites through uncontrolled non-enzymatic reactions such as glycation or the products arising from the reaction of unsaturated long chain fatty acid metabolites (possessing aldehydic groups). A typical example of these non-enzymatic changes is the formation of advanced glycation end-products (AGEs), resulting from the reaction of carbohydrates with the free amino group of proteins. The accumulation of AGEs and the resulting structural alterations cause altered tissue properties (increased stiffness, reduced elasticity) that contribute to their reduced catabolism and to their aging. Posttranslational nonenzymatic modifications of the proteins of the extracellular matrix (the formation of a typical AGE product - pentosidine) were studied in three types of tissue of three rat strains subjected to a high-fructose diet. Chronic (three-week) hyperglycemia (resulting from fructose loading) caused a significant increase in pentosidine concentration mainly in the aorta and skin of the three rat strains (Lewis, Wistar and hereditary hypertriglyceridemic rats).     

Crossed immunoelectrophoretic analysis of ARDS lavage proteins

Serum proteins in normal and ARDS bronchoalveolar lavages were analyzed using crossed immunoelectrophoresis. Normal lavages demonstrated relatively few proteins (albumin, alpha 1-antitrypsin, transferrin, and haptoglobin) in low concentrations. In contrast, substantial amounts of all identifiable serum proteins were detected in ARDS lavages. IgA was apparently locally produced. Two of the largest proteins, beta-lipoprotein (mol wt greater than 2 million) and IgM (mol wt approximately 900,000) were found to be complexed as evidenced by their coprecipitation in a single spike in ARDS lavage. Electrophoretic modifications of ARDS albumin and alpha 1-antitrypsin precipitation peaks and partial identity spurring of the alpha 1-lipoprotein peak with other precipitation loops indicated possible complex formation between these proteins and other possibly pathogenic lung fluid constituents. Similarly, modifications of orosomucoid and Gc-globulin peaks indicated possible molecular alterations resulting from interactions with other components. The relatively few protein modifications exhibited in ARDS lavages together with alpha 1-antitrypsin-protease complex formation confirm the relative absence of substantial proteolytic activity in ARDS edema fluids obtained within 12 hr of the onset of the syndrome demonstrated in previous studies.     

Peptides and the origin of life.

Considering the state-of-the-art views of the geochemical conditions of the primitive earth, it seems most likely that peptides were produced ahead of all other oligomer precursors of biomolecules. Among all the reactions proposed so far for the formation of peptides under primordial earth conditions, the salt-induced peptide formation reaction in connection with adsorption processes on clay minerals would appear to be the simplest and most universal mechanism known to date. The properties of this reaction greatly favor the formation of biologically relevant peptides within a wide variation of environmental conditions such as temperature, pH, and the presence of inorganic compounds. The reaction-inherent preferences of certain peptide linkages make the argument of 'statistical impossibility' of the evolutionary formation of the 'right' peptides and proteins rather insignificant. Indeed, the fact that these sequences are reflected in the preferential sequences of membrane proteins of archaebacteria and prokaryonta distinctly indicates the relevance of this reaction for chemical peptide evolution. On the basis of these results and the recent findings of self-replicating peptides, some ideas have been developed as to the first steps leading to life on earth.     

Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis

The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.