Roberta Moschini

Chaperone-like features of bovine serum albumin: a comparison with α-crystallin

Abstract.The chaperone behaviour of bovine serum albumin was compared with that of α-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with α-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than α-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family.

A New Approach to Control the Enigmatic Activity of Aldose Reductase

Aldose reductase (AR) is an NADPH-dependent reductase, which acts on a variety of hydrophilic as well as hydrophobic aldehydes. It is currently defined as the first enzyme in the so-called polyol pathway, in which glucose is transformed into sorbitol by AR and then to fructose by an NAD+-dependent dehydrogenase. An exaggerated flux of glucose through the polyol pathway (as can occur in diabetes) with the subsequent accumulation of sorbitol, was originally proposed as the basic event in the aethiology of secondary diabetic complications. For decades this has meant targeting the enzyme for a specific and strong inhibition. However, the ability of AR to reduce toxic alkenals and alkanals, which are products of oxidative stress, poses the question of whether AR might be better classified as a detoxifying enzyme, thus raising doubts as to the unequivocal advantages of inhibiting the enzyme. This paper provides evidence of the possibility for an effective intervention on AR activity through an intra-site differential inhibition. Examples of a new generation of aldose reductase “differential” inhibitors (ARDIs) are presented, which can preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. Some selected inhibitors are shown to preferentially inhibit enzyme activity on glucose or glyceraldehyde and 3-glutathionyl-4-hydroxy-nonanal, but are less effective in reducing 4-hydroxy-2-nonenal. We question the efficacy of D, L-glyceraldehyde, the substrate commonly used in in vitro inhibition AR studies, as an in vitro reference AR substrate when the aim of the investigation is to impair glucose reduction.

In vitro evaluation of 5-arylidene-2-thioxo-4-thiazolidinones active as aldose reductase inhibitors

2-Thioxo-4-thiazolidinone derivatives were evaluated as aldose reductase inhibitors (ARIs) and most of them exhibited good or excellent in vitro efficacy. Out of the tested compounds, most N-unsubstituted analogues were found to possess inhibitory effects at low micromolar doses and two of them exhibited higher potency than sorbinil, used as a reference drug. The insertion of an acetic chain on N-3 of the thiazolidinone scaffold led to analogues with submicromolar affinity for ALR2 and IC(50) values very similar to that of epalrestat, the only ARI currently used in therapy.

Complete protection by α-crystallin of lens sorbitol dehydrogenase undergoing thermal stress

Sorbitol dehydrogenase (l-iditol:NAD+ 2-oxidoreductase, E.C. 1.1.1.14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens α-crystallin. An α-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 °C. Moreover, an α-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 °C for at least 40 min. The protection by α-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the α-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by α-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of α-crystallin against the inactivation of SDH induced at 55 °C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of α-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with α-crystallin. Two different adducts between α-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a highM r of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or lowM r aggregates of α-crystallin. Even though it had a reduced efficiency with respect to α-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of α-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of α-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.

Identification of new non-carboxylic acid containing inhibitors of aldose reductase

Non-carboxylic acid containing bioisosteres of (5-arylidene-2,4-dioxothiazolidin-3-yl)acetic acids, which are active as aldose reductase (ALR2) inhibitors, were designed by replacing the carboxylic group with the trifluoromethyl ketone moiety. The in vitro evaluation of the ALR2 inhibitory effects of these trifluoromethyl substituted derivatives led to the identification of two inhibitors effective at low micromolar doses. It was further confirmed that a carboxylic chain on N-3 of the thiazolidinedione scaffold is a determining requisite to obtain the highest efficacy levels; however, it is not essential for the interaction with the target enzyme and it can be replaced by different polar groups, thus obtaining less ionised or unionised inhibitors.

Alpha-crystallin: an ATP-independent complete molecular chaperone toward sorbitol dehydrogenase

Abstract.α-Crystallin, the major component of the vertebrate lens, is known to interact with proteins undergoing denaturation and to protect them from aggregation phenomena. Bovine lens sorbitol dehydrogenase (SDH) was previously shown to be completely protected by α-crystallin from thermally induced aggregation and inactivation. Here we report that α-crystallin, in the presence of the SDH pyridine cofactor NAD(H), can exert a remarkable chaperone action by favoring the recovery of the enzyme activity from chemically denaturated SDH up to 77%. Indeed, even in the absence of the cofactor, α-crystallin present at a ratio with SDH of 20:1 (w:w) allows a recovery of 35% of the enzyme activity. The effect of ATP in enhancing α-crystallin-promoted SDH renaturation appears to be both nonspecific and to not involve hydrolysis phenomena, thus confirming that the chaperone action of α-crystallin is not dependent on ATP as energy donor.

NADP(+)-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification.

An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

AbstractA simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG. FigColorimetric method for the specific measurement of glutathione. γ-glutamyltransferase (γ-GT) transfers the γ-glutamyl moiety from glutathione to an acceptor (Gly-Gly), with the formation of γ-glutamyl-Gly-Gly and Cys-Gly. The latter dipeptide is hydrolized by leucyl-aminopeptidase (LAP) to form cysteine, which can be easily measured using a colorimetric assay at 560 nm

Modulation of aldose reductase activity by aldose hemiacetals

BACKGROUND Glucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate. METHODS The reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR. RESULTS The apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l-idose. CONCLUSIONS Due to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates. GENERAL SIGNIFICANCE The discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications.

l-Idose: an attractive substrate alternative to d-glucose for measuring aldose reductase activity

Although glucose is one of the most important physio-pathological substrates of aldose reductase, it is not an easy molecule for in vitro investigation into the enzyme. In many cases alternative aldoses have been used for kinetic characterization and inhibition studies. However these molecules do not completely match the structural features of glucose, thus possibly leading to results that are not fully applicable to glucose. We show how aldose reductase is able to act efficiently on L-idose, the C-5 epimer of D-glucose. This is verified using both the bovine lens and the human recombinant enzymes. While the kcat values obtained are essentially identical to those measured for D-glucose, a significant decrease in KM was observed. This can be due to the significantly higher level of the free aldehyde form present in L-idose compared to D-glucose. We believe that L-idose is the best alternative to D-glucose in studies on aldose reductase.