Roberto Colombo

Protein carbonyl groups as biomarkers of oxidative stress

Oxidative stress, an imbalance toward the pro-oxidant side of the pro-oxidant/antioxidant homeostasis, occurs in several human diseases. Among these diseases are those in which high levels of protein carbonyl (CO) groups have been observed, including Alzheimers disease (AD), rheumatoid arthritis, diabetes, sepsis, chronic renal failure, and respiratory distress syndrome. What relationships might be among high level of protein CO groups, oxidative stress, and diseases remain uncertain.The usage of protein CO groups as biomarkers of oxidative stress has some advantages in comparison with the measurement of other oxidation products because of the relative early formation and the relative stability of carbonylated proteins. Most of the assays for detection of protein CO groups involve derivatisation of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl (DNP) hydrazone product. This then can be detected by various means, such as spectrophotometric assay, enzyme-linked immunosorbent assay (ELISA), and one-dimensional or two-dimensional electrophoresis followed by Western blot immunoassay. At present, the measurement of protein CO groups after their derivatisation with DNPH is the most widely utilized measure of protein oxidation.

Protein carbonylation in human diseases

Oxidative modifications of enzymes and structural proteins play a significant role in the aetiology and/or progression of several human diseases. Protein carbonyl content is the most general and well-used biomarker of severe oxidative protein damage. Human diseases associated with protein carbonylation include Alzheimers disease, chronic lung disease, chronic renal failure, diabetes and sepsis. Rapid recent progress in the identification of carbonylated proteins should provide new diagnostic (possibly pre-symptomatic) biomarkers for oxidative damage, and yield basic information to aid the establishment an efficacious antioxidant therapy.

Protein carbonylation, cellular dysfunction, and disease progression

Carbonylation of proteins is an irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease‐derived protein dysfunction. Whereas moderately carbonylated proteins are degraded by the proteasomal system, heavily carbonylated proteins tend to form high‐molecular‐weight aggregates that are resistant to degradation and accumulate as damaged or unfolded proteins. Such aggregates of carbonylated proteins can inhibit proteasome activity. A large number of neurodegenerative diseases are directly associated with the accumulation of proteolysis‐resistant aggregates of carbonylated proteins in tissues. Identification of specific carbonylated protein(s) functionally impaired and development of selective carbonyl blockers should lead to the definitive assessment of the causative, correlative or consequential role of protein carbonylation in disease onset and/or progression, possibly providing new therapeutic aproaches.

The actin cytoskeleton response to oxidants: from small heat shock protein phosphorylation to changes in the redox state of actin itself

Actin is the major constituent of the cytoskeleton of almost all the eukaryotic cells. In vitro experiments have indicated that oxidant-stressed nonmuscle mammalian cells undergo remarkable changes in their morphology and in the structure of the actin cytoskeleton, often resulting in plasma membrane blebbing. Although the microfilament network is one of the earliest targets of oxidative stress, the mechanism by which oxidants change both the structure and the spatial organization of actin filaments is still a matter of debate and far from being fully elucidated. Starting from the 2-fold role of oxidants as injurious by-products of cellular metabolism and essential participants in cell signaling and regulation, this review attempts to gather the most relevant information related to (i) the activation of mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38) which, via MAP kinase-activated protein (MAPKAP) kinase 2/3, leads to the phosphorylation of the actin polymerization (F-actin) modulator 25/27 kDa heat shock protein (HSP25/27), whose phosphorylation is causally related to the regulation of microfilament dynamics following oxidative stress; (ii) the alteration of the redox state of actin or some actin regulatory proteins. The actin cytoskeleton response to oxidants is discussed on the basis of the growing body of evidence indicating the actin system as the most sensitive constituent of the cytoskeleton to the oxidant attack.

Retrieval of leaf area index in different vegetation types using high resolution satellite data

With the successful launch of the IKONOS satellite, very high geometric resolution imagery is within reach of civilian users. In the 1-m spatial resolution images acquired by the IKONOS satellite, details of buildings, individual trees, and vegetation structural variations are detectable. The visibility of such details opens up many new applications, which require the use of geometrical information contained in the images. This paper presents an application in which spectral and textural information is used for mapping the leaf area index (LAI) of different vegetation types. This study includes the estimation of LAI by different spectral vegetation indices (SVIs) combined with image textural information and geostatistical parameters derived from high resolution satellite data. It is shown that the relationships between spectral vegetation indices and biophysical parameters should be developed separately for each vegetation type, and that the combination of the texture indices and vegetation indices results in an improved fit of the regression equation for most vegetation types when compared with one derived from SVIs alone. High within-field spatial variability was found in LAI, suggesting that high resolution mapping of LAI may be relevant to the introduction of precision farming techniques in the agricultural management strategies of the investigated area.

S‐Glutathionylation: from redox regulation of protein functions to human diseases

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an integral role in the modulation of several physiological functions but can also be potentially destructive if produced in excessive amounts. Protein cysteinyl thiols appear especially sensitive to ROS/RNS attack. Experimental evidence started to accumulate recently, documenting that S‐glutathionylation occurs in a number of physiologically relevant situations, where it can produce discrete modulatory effects on protein function. The increasing evidence of functional changes resulting from this modification, and the growing number of proteins shown to be S‐glutathionylated both in vitro and in vivo support this contention, and confirm this as an attractive area of research. S‐glutathionylated proteins are now actively investigated with reference to problems of biological interest and as possible biomarkers of human diseases associated with oxidative/nitrosative stress.

Integration of remote sensing data and GIS for accurate mapping of flooded areas

This paper describes a synergetic use of satellite radar images and ancillary information to detect flooded areas at their peak and evaluates its potential with mapping. The procedure was tested on the catastrophic flood that occurred in Regione Piemonte in Italy in November 1994. Two ERS-1 synthetic aperture radar (SAR) images were processed, one acquired one month before the flood and the other acquired three days after the event. Visual interpretation and two different thresholding techniques were performed. The flood map derived shows only a small fraction (20%) of the actually flooded lands because of the time delay between the flood peak and the satellite overpass. To overcome this limitation, the authors developed a new procedure to estimate the flooded area at the peak time by integrating the flooded area from SAR imagery with digital topographic data from a GIS technique. This method allowed inundated areas covering 96.7% of the flooded area officially recorded by the local government to be mapped. The proposed procedure is suitable for mapping flooded areas even when satellite data are acquired some days after the event, thus overcoming the constraint of temporal resolution in the application of SAR imagery in hydrology.

Reversible S-glutathionylation of Cys374 regulates actin filament formation by inducing structural changes in the actin molecule

S-glutathionylation, the reversible formation of mixed disulphides of cysteinyl residues in target proteins with glutathione, occurs under conditions of oxidative stress; this could be a posttranslational mechanism through which protein function is regulated by the cellular redox status. A novel physiological relevance of actin polymerization regulated by glutathionylation of Cys(374) has been recently suggested. In the present study we showed that glutathionylated actin (GS-actin) has a decreased capacity to polymerize compared to native actin, filament elongation being the polymerization step actually inhibited. Actin polymerizability recovers completely after dethiolation, indicating that S-glutathionylation does not induce any protein denaturation and is therefore a reversible oxidative modification. The increased exposure of hydrophobic regions of protein surface observed upon S-glutathionylation indicates changes in actin conformation. Structural alterations are confirmed by the increased rate of ATP exchange as well as by the decreased susceptibility to proteolysis of the subtilisin cleavage site between Met(47) and Gly(48), in the DNase-I-binding loop of the actin subdomain 2. Structural changes in the surface loop 39-51 induced by S-glutathionylation could influence actin polymerization in view of the involvement of the N-terminal portion of this loop in intermonomer interactions, as predicted by the atomic models of F-actin.

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimers disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimers disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.