Reactive oxygen species (ROS) are generated as the result of a number of physiological and pathological processes. Once formed ROS can promote multiple forms of oxidative damage, including protein oxidation, and thereby influence the function of a diverse array of cellular processes. This review summarizes the mechanisms by which ROS are generated in a variety of cell types, outlines the mechanisms which control the levels of ROS, and describes specific proteins which are common targets of ROS. Additionally, this review outlines cellular processes which can degrade or repair oxidized proteins, and ultimately describes the potential outcomes of protein oxidation on cellular homeostasis. In particular, this review focuses on the relationship between elevations in protein oxidation and multiple aspects of cellular metabolism. Together, this review describes a potential role for elevated levels of protein oxidation contributing to cellular dysfunction and oxidative stress via impacts on cellular metabolism.

The purpose of the work reported here is to look at 0; production during autoxidation df the isolated a and fi chains of human hemoglobin. The formation of superoxide by isolated chains, in fact, may be involved in pathological phenomena observed in red blood cell diseases where excess of either a or fi chains is produced because of inherited defects of synthesis (thalassemus).The autoxidation of the oxygenated chains of human hemoglobin is followed by the transformation of the oxidized molecule (high-spin Fe3+) into a species absorbing as a low-spin Fe3+ compound, that is a hemichrome, which tends to precipitate. The overall process may be described by the following reactions :Oxychains -% Ferric chains % Hemichrome % Precipitate.Depending on conditions the different steps proceed with different relative rates and therefore may overlap to a greater or lesser extent.The results obtained indicate that superoxide is produced during autoxidation of the isolated a and B chains of human hemoglobin. This conclusion has been reached on the basis of an experimental approach based on cooxidation of epinephrine to adrenochrome during 0; production and inhibition of this effects by superoxide dismutase. In addition the rate of precipitation is, in all cases, very much reduced upon addition of either superoxide dismutase or catalase. These facts seem to imply very strongly that oxygen radicals are involved also in the precipitation following oxidation and hemichrome formation in the chains. In particular the effect of superoxide dismutase implies the involvement of 0,. Thus production of 0; ,could well be one of the important events leading to alteration of the red cell membrane and consequently to reduced life span of thalassemic erythrocytes.

The thermodynamic parameters affecting proteinprotein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.H-NS is a nucleoid-associated protein (1-3) involved in the transcriptional regulation of a fairly large number of genes, including some virulence genes (for reviews, see Refs. 4 -6). H-NS affects transcription by stimulating and, more often, repressing the activity of different promoters, by a variety of mechanisms (7-10) which underlie the general properties of H-NS. These properties include the sequence-independent recognition of intrinsic DNA curvatures, the active bending of its DNA targets, constraining negative DNA supercoils in vitro (11), and causing compaction of the genetic material both in vitro (3) and in vivo (12). H-NS is composed of 137 amino acids (M r 15.4 kDa), and its binding to DNA occurs, at least in part, via the C-terminal fragment of the molecule (last 47 residues) which is able to interact with DNA as an isolated domain, albeit very weakly (13). H-NS is believed to interact with DNA in an oligomeric form because it is known that the monomers of this protein undergo self-association to form oligomers (1), a process which was shown to influence the interaction of this protein with DNA and its ability to induce DNA-bending (14). Indeed, several proteins modulate their interaction with DNA through association/dissociation equilibria among oligomer subunits, and it has been suggested that this could be a common strategy utilized by these proteins to exert their regulatory functions (15, 16). Thus, a better knowledge of the nature of the self-assembly equilibria of H-NS might be particularly relevant in understanding the mechanism of action of this protein and its effects on the structure and functional organization of the bacterial nucleoid.In the present study, we have undertaken a quantitative thermodynamic analysis of the process of H-NS oligomerization using "large zone" gel permeation chromatography (17) of in vivo 35 S-labeled protein. Our approach allows quantification of the assembly equilibria to be determined down to the nanomolar concentration range. Our results show that oligomerization of the protein is strictly dependent on protein concentration (in the range of 10 Ϫ8 to 10 Ϫ6 M) and strongly affected by physical (temperature) and chemical (cation) effectors. From the results obtained, we propose a structural model for the H-NS association equilibria and discuss it in light of the in vivo functional role of this protein. EXPERIMENTAL PROCEDURES...

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The effect of several polyphenols on the 20S proteasomes, both the constitutive and the LMP proteasomes, isolated from bovine tissues, has been investigated. Polyphenolic compounds show many biological activities such as antiviral, antibacterial, antifungal, anti-inflammatory, antimutagenic, and antiallergic activities. However, the molecular mechanism underlying these effects has not been identified. It is well established that polyphenols possess inhibitory activities on several enzymes and among them the 20S proteasome. In the present work, the ChT-L, BrAAP, PGPH, and T-L activities of the isolated constitutive and immuno-proteasomes were assayed in order to get an overall information on the polyphenols binding to the complexes. The effects of the polyphenols on the proteasomal activities were analyzed, taking into account the different subunits composition of the two complexes. Furthermore the same activities were measured on whole extracts from cancer cells exposed to EGCG and gallic acid, evaluating, also, their antioxidant action under oxidative stress. EGCG and gallic acid are able to affect the 20S proteasomes functionality, depending on the complex subunit composition and, in cell extracts, they behave both as antioxidants and proteasome effectors.

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The proteasome and heat shock proteins have been found in the centrosome. The evidence of their copurification reported by several studies suggests that they form stable complex. In addition, Hsp90 is involved in the loading of proteasome-generated antigenic peptides to the class I major histocompatibility complex. In this article, we report a detailed thermodynamic and kinetic characterization of the Hsp90-20S proteasome interaction, using a surface plasmon resonance technique. The modulation exerted by protons in solution has been investigated, and the results have been discussed, taking into account structural motifs characterizing the binding interface between the two macromolecules.

BACKGROUND: Owing to the demand for 'health foods', commercial development of wild vegetables may find a sizable market niche if adequate agricultural methods are used to domesticate such species. Available techniques of cultivation (even in traditional farming) may provide many advantages, such as enhancement of the content of active principles in plants and improvement in the quality of the raw material to be processed on an industrial scale. In this context, the flavonoid composition and content of roots and leaves from five varieties of wild herbs (Cichorium intybus, Portulaca oleracea, Tragopogon porrifolius, Urtica dioica and Valerianella eriocarpa) and their cultivated relatives in central Italy were compared. The aims of the study were (i) to reveal the metabolic profile of particular bioactive metabolites present in some unusual food species by using a simple method of analysis, (ii) to quantify and compare the amount of polyphenolic metabolites in wild and cultivated plants and (iii) to evaluate the effect of growing conditions on polyphenolic variability. Data were subjected to statistical analysis