Circadian rhythmicity of the thioredoxin system in cultured murine peritoneal macrophages.

Macrophages play a pivotal role in atherosclerosis through a variety of events related to cellular oxidative stress. This process is mainly due to an excessive production of reactive oxygen species whose elimination occurs through antioxidant systems including the thioredoxin (Trx) system. In this paper, we investigated whether the Trx system would exhibit circadian rhythmicity in dexamethasone synchronized cultured macrophages and monitored the impact of the rhythmicity of Trx-1 on markers of atherosclerosis. We found that the clock-related genes BMAL-1, PER-2, CRY-1 and REV ERB α exhibited a robust circadian expression. However, the Trx genes family (Trx-1, Trx-2, TrxR1 and TXNIP) did not exhibit a circadian expression at the mRNA level in spite of the presence of E-box elements within the promoter regions of TrxR1 and TXNIP genes. Nevertheless, both Trx-1 and TXNIP exhibited a circadian expression at the protein level and proteasome inhibition abolished the rhythmicity of Trx-1. Moreover, we found a link between low Trx-1 level and elevated atherogenic markers such as 4-HNE, TNF-α and cholesterol accumulation in macrophages. Our results indicate that the Trx gene family does not exhibit the same circadian regulation and that the presence of E-box elements in the TXNIP promoter is not sufficient to ensure a circadian rhythmicity at the transcriptional level. In addition, since a link was found between a low level of Trx-1 protein during circadian rhythm and high levels of atherogenic markers, administration of Trx-1 at certain time points could be an interesting approach to protect against atherosclerosis development.

How best to halt and/or revert UV-induced skin ageing: strategies, facts and fiction.

Once considered mainly a cosmetic issue, photoageing research has long moved to the forefront of investigative dermatology. Besides obvious market pressures, increasing insight into the mechanistic overlap between UV-induced skin cancer and UV-induced skin ageing has contributed to this development. Also, as strategies that work to antagonize intrinsic skin ageing/senescence may also be exploited against photoageing (and vice versa!), it has become an important skin research challenge to dissect both the differences and the overlap mechanisms between these interwined, yet distinct phenomena. Finally, the current surge in putative 'antiageing' products, devices, and strategies - too many of which boldly promise to fight and/or repair the perils that come along with a lifetime spent in the sun in the absence of convincing evidence of efficacy - makes it particularly pertinent to critically review the available evidence to support often made antiageing claims. The current CONTROVERSIES feature, therefore, aimed to provide both guidance through, and critical voices in, the antiageing circus. Here, a panel of experts defines relevant key problems, points the uninaugurated to intriguing aspects of photoageing that one may not have considered before, highlights promising strategies for how best to halt and/or revert it, and spiritedly debates some controversially discussed approaches.

Identification of proteins undergoing expression level modifications in WI-38 SV40 fibroblasts overexpressing methionine sulfoxide reductase A.

Methionine sulfoxide reductase A overexpressing WI-38 SV40 human fibroblasts have been previously shown to exhibit higher resistance to oxidative stress by decreasing intracellular reactive oxygen species content and oxidative damage to proteins [C.R. Picot, I. Petropoulos, M. Perichon, M. Moreau, C. Nizard, B. Friguet, Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H(2)O(2)-mediated oxidative stress, Free Radic Biol Med 39 (2005) 1332-1341]. In order to get further insight into the molecular mechanisms underlying this resistance to oxidative stress, proteins that are differentially expressed in methionine sulfoxide reductase A overexpressing cells were identified by 2D gel and Western blot quantitative analyses. Five proteins were shown to be differentially expressed and were identified by mass spectrometry, some of them were related to either cellular protection against oxidative stress, apoptosis or premature ageing.

The carboxy-terminus of I kappaB alpha determines susceptibility to degradation by the catalytic core of the proteasome.

The Rel/NF-kappaB family of transcription factors controls the expression of a wide variety of genes that are implicated in immune and inflammatory responses and cellular proliferation. Disregulation of NF-kappaB is associated with cellular transformation and the maintenance of a high anti-apoptotic threshold in transformed cells. NF-kappaB activity is in turn regulated by its sequestration in the cytoplasm by the inhibitor I kappaB. I kappaB alpha, the most abundant and well-characterized member of the I kappaB multiprotein family, is rapidly degraded in response to multiple physiologic stimuli. In the present study we show that not only the amino-terminus, but also the carboxy-terminus of I kappaB alpha contain transferable signals that must be simultaneously present in an unrelated protein to render it susceptible to activation-induced, proteasome-mediated degradation. We show here that I kappaB alpha amino-terminal modifications occur independently of the carboxy-terminus. Moreover, we present evidence indicating a critical role for the carboxy-terminal region in facilitating proteolysis by the catalytic core of the proteasome. When incubated with 20S proteasome extracted from rat liver, I kappaB alpha was quickly degraded while a deletion mutant lacking the carboxy-terminus was resistant to proteolysis. Likewise, chimeric proteins of beta-galactosidase with the I kappaB alpha carboxy-terminus were degraded in vitro independently of the presence of the I kappaB alpha amino-terminus, whereas chimeric proteins lacking the I kappaB alpha carboxy-terminus were stable. Our results identify the carboxy-terminus of I kappaB alpha as a domain critical for degradation through interaction with an as yet unidentified component of the proteasome.

Inhibition of nitric oxide synthase activity by early and advanced glycation end products in cultured rabbit proximal tubular epithelial cells.

Nitric oxide (NO) is important in the regulation of renal tubular function. We have investigated whether glycated proteins could impair the NO production by examining the effects of Amadori products (AP-BSA) and advanced glycation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by measurement of the conversion of L-arginine to L-citrulline and by production of NO, after short-term (30 min) or long-term (1 or 3 days) incubation. Short incubations of PTE cells with either 200 microg/ml AP-BSA or 40 microg/ml AGE-BSA significantly decreased NO production. AP-BSA (3000 microg/ml) inhibited the Ca(2+)-dependent NOS activity even though above 50 microg/ml it increased Ca(2+)-independent NOS activity. In contrast, 40 microg/ml AGE-BSA inhibited both isoforms of NOS. Longer incubations with 200 microg/ml AP-BSA or 250 microg/ml AGE-BSA decreased NO release and inhibited Ca(2+)-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso-N-acetyl-penicillamine (SNAP), while 250 microg/ml AGEs decreased it. After 3 days incubation, glycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, suggesting that the fall in NO production was not due to there being fewer cells. These data indicate that APs and AGEs directly inhibit NOS activity, and additionally that AGEs quench released NO. Thus, both types of glycated proteins alter the production of NO by PTE cells and could participate in the renal tubule dysfunction associated with aging and diabetes.

Folding on the ribosome of Escherichia coli tryptophan synthase beta subunit nascent chains probed with a conformation-dependent monoclonal antibody.

Experimental analysis of protein folding during protein synthesis on the ribosome is rendered very difficult by the low concentration of nascent polypeptides and the heterogeneity of the translation mixture. In this study, an original approach is developed for analysing nascent polypeptide structures still carried by the ribosome. Folding on the ribosome of nascent chains of the beta subunit of Escherichia coli tryptophan synthase was investigated using a monoclonal antibody (mAb 19) recognizing a conformation-dependent antigenic determinant. Upon synthesis of beta subunits in an E. coli coupled transcription-translation system, it is shown that ribosome-bound nascent polypeptides can react with the monoclonal antibody provided their size is above 11.5 kDa, which is smaller than that of both the N-terminal proteolytic and crystallographic domains (29 and 21 kDa, respectively). The gene fragments coding only for the 11.5 kDa polypeptide, with and without stop codon at the end of the corresponding mRNAs, were constructed and expressed in a cell-free wheat germ translation system. It is shown that antibody 19 reacts with this polypeptide either bound to the ribosome or free in solution. That the 11.5 kDa polypeptide acquires a condensed structure is shown by gel filtration in native conditions and by urea gradient gel electrophoresis. Moreover, it is demonstrated that this condensed structure resembles that of native beta 2 in the vicinity of the epitope for antibody 19. Indeed, the affinity of antibody 19 for the 11.5 kDa fragment, either free or bound to the ribosome, was measured (6 x 10(8) M-1) and shown to be close to that for native beta 2. It is therefore proposed that the polypeptide chain may start to fold during its biosynthesis and that, even before the appearance of an entire domain, a folded intermediate is formed that already exhibits some local structural features of the native state and of an immunoreactive intermediate previously detected during the in vitro refolding of denatured complete beta chains.

The secondary fungal metabolite gliotoxin targets proteolytic activities of the proteasome.

BACKGROUND: The fungal epipolythiodioxopiperazine metabolite gliotoxin has a variety of toxic effects such as suppression of antigen processing, induction of macrophagocytic apoptosis and inhibition of transcription factor NF-kappaB activation. How gliotoxin acts remains poorly understood except that the molecule's characteristic disulfide bridge is important for immunomodulation. As this fungal metabolite stabilizes the NF-kappaB inhibitor IkappaBalpha in the cytoplasm, we decided to investigate its molecular mechanism of action. RESULTS: We show that gliotoxin is an efficient, noncompetitive inhibitor of the chymotrypsin-like activity of the 20S proteasome in vitro. Proteasome inhibition can be reversed by dithiothreitol, which reduces gliotoxin to the dithiol compound. In intact cells, gliotoxin inhibits NF-kappaB induction through inhibition of proteasome-mediated degradation of IkappaBalpha. CONCLUSIONS: Gliotoxin targets catalytic activities of the proteasome efficiently. Inhibition by gliotoxin may be countered by reducing agents, which are able to inactivate the disulfide bridge responsible for the inhibitory capacity of gliotoxin.

Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen.

Recent studies with monoclonal antibodies directed against different epitopes of the beta 2-subunit of Escherichia coli tryptophan synthase have shown that some of these antibodies bind rapidly in solution to the native protein; others bind very slowly to native beta 2 in solution while they recognize quite rapidly this antigen adsorbed on a microtitration plate. In the present work, an enzyme-linked immunosorbent assay competition test with either the native or a denatured form of the antigen has been developed. It allowed us to show that the rapidly binding antibodies recognize epitopes present on the native protein while those which react very slowly in solution bind preferentially to the denatured form of the protein. These results prompted us to emphasize how important it is, in the characterization of antibodies, to ascertain that the antigen they recognize remains native in the specificity test.

Peptide/antibody recognition: synthetic peptides derived from the E. coli tryptophan synthase beta 2 subunit interact with high affinity with an anti-beta 2 monoclonal antibody.

Recent studies have shown that the antigenic determinant recognized by a monoclonal antibody (mAb 164-2) elicited against the beta 2 subunit of E. coli tryptophan synthase is localized between residues 276 and 297 of this protein. In order to delineate more precisely the epitope recognized by this antibody, peptides ranging in length from 11 to 29 amino acids and belonging to this region were synthesized, and their interactions with the antibody are described in this paper. The smallest peptide recognized with a high affinity by antibody 164-2 contains 11 residues (273-283). This peptide is recognized by antibody 164-2 with an affinity (KD = 7.5 x 10(-9) M) very close to that of the native beta 2 subunit, suggesting a high structural similarity of the epitope inside the protein and in the isolated peptide. The corresponding sequence of beta 2 is located in a region protruding from the protein surface that contains a beta-turn as unique element of secondary structure in the crystallographic model. The absence of interaction between antibody 164-2 and the octapeptide lacking the three residues at the C-terminal end of peptide 11 suggests that the beta-turn is important in the recognition by the antibody. Kinetic studies were performed to find out whether or not the binding of the antibody to the peptide involves any conformational adaptation. The dissociation equilibrium constant (KD), the dissociation rate constant (koff) and the association rate constant (kon) were measured for eight peptide/antibody complexes. The values obtained were compatible with a one-step reaction, suggesting that no important conformational adaptation is involved in the formation of the peptide/antibody complexes. Furthermore, it has been shown that differences in affinity of antibody 164-2 for the various peptides were mainly due to differences in the dissociation rate constants (koff) and not in the association rate constants (kon). The exceptional location of the epitope in the native protein and the unusually high affinity of the 11-residue peptide for mAb 164-2, makes this peptide a good model for studying the interaction between an antibody and a continuous epitope of a protein.

Inhibition of the multicatalytic proteinase (proteasome) by 4-hydroxy-2-nonenal cross-linked protein.

Oxidative modification of glucose-6-phosphate dehydrogenase (Glu-6-PDH), as observed for other proteins, increases the susceptibility of the protein to degradation by the multicatalytic proteinase/proteasome (MCP). Oxidized Glu-6-PDH is, however, more prone to cross-linking reactions by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE), processes which render the protein resistant to proteolysis. In addition, HNE cross-linked protein inhibits the degradation of oxidatively modified glutamine synthetase by the MCP. In contrast to oxidized Glu-6-PDH, which inhibits the proteolysis of GS in a competitive manner, HNE cross-linked protein acts as a noncompetitive inhibitor. As judged by binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, a common structural feature of both macromolecular substrates and inhibitors of the MCP is an increased accessibility of hydrophobic regions on the protein.

Renaturation of guanidine-unfolded tryptophan synthase by multi-mixing stopped-flow dilution in D2O.

Guanidine hydrochloride (GdnHCl) at high concentrations, e.g. 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins. The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 50-100-fold in a 'renaturation buffer'. Fast kinetic studies, using a stopped-flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing. Secondly, the density differences existing between the concentrated GdnHCl solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped-flows. Here, it is shown that these difficulties can be overcome by using a multi-mixing stopped-flow to achieve 2 successive 7-fold dilutions and by using heavy water (D2O) to adjust the density of the renaturation buffer. This enabled us to study the appearance of a short-lived intermediate during the folding of the beta 2-subunit of Escherichia coli tryptophan synthase.

An early immunoreactive folding intermediate of the tryptophan synthease beta 2 subunit is a 'molten globule'.

The refolding kinetics of the tryptophan synthase beta 2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured beta 2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded beta 2, the fluorescence of ANS passes through a maximum after about 1 s and then 'slowly' decreases. These results show the accumulation, in the 1-10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the 'molten globule' state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t1/2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early immunoreactive intermediate recognized by a monoclonal antibody (t1/2 12 s), it is shown that this native-like epitope forms within the 'molten globule', before the tight packing of the protein side chains.

In vitro gene expression for the localization of antigenic determinants: application to the E. coli tryptophan synthase beta 2 subunit.

Gene expression of the beta subunit of E. coli tryptophan synthase in an E. coli cell-free transcription-translation system proceeds by pauses and produces a discrete but quite continuous pattern of nascent chains starting from the N terminus and ranging in size up to the 44 kDa end product corresponding to the completed beta chains. Using specific immunoadsorption of [35S]Met radiolabelled nascent chains by different monoclonal antibodies directed against the beta 2 subunit of E. coli tryptophan synthase, the size of the smallest N-terminal fragment reacting with each antibody has been determined by SDS electrophoretic analysis of the immunoadsorbed polypeptides. The immunoadsorption assay is performed in solution under conditions avoiding the usual drawbacks of solid phase immunoassay. This approach, in combination with the results obtained with a DNA fragment library permitted us to localize the antigenic determinants recognized by the monoclonal antibodies. The proposed method could help to localize rapidly the C-terminal boundary of an epitope, before starting systematic and precise mapping by other approaches. Moreover, the method described may be of general interest for the rapid production of a large set of C-terminal truncated polypeptides for studies of antigen-antibody recognition.

A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase.

Seven hybridoma clones, producing antibodies directed against the beta 2-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on beta 2, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to beta-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes. Using this test, it is shown that, of the 7 anti-beta 2 monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.

Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.

A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.

Age-related alterations of proteasome structure and function in aging epidermis.

Recent studies on the effect of aging in epidermal cells have evidenced a decrease of proteasome activity and content, suggesting that proteasome is down-regulated in aged cells. The 20S proteasome is the major proteolytic system that has been implicated in removal of abnormal and oxidatively damaged proteins. Therefore, a decreased proteasome content may explain, at least in part, the well-documented age-related accumulation of oxidized proteins. To gain further insight in other mechanisms that may be implicated in a decreased activity of the proteasome with age, 20S proteasome has been purified from the epidermis from donors of different ages: young, middle-aged and old. The patterns of proteasome subunits have been analyzed by 2D gel electrophoresis to determine whether its structure is also affected with age. The 2D gel pattern of proteasome subunits was found to be modified for four subunits, indicating that the observed decline in proteasome activity with age may also be related to alterations of its subunits. These subunit alterations are likely to be involved in the age-related decrease of proteasome activity since the specific peptidase activities of the purified proteasome were found to be decreased with age.

Fibroblast cultures from healthy centenarians have an active proteasome.

Healthy centenarians represent the best example of successful ageing. Various studies have shown that centenarians have escaped the major age-associated diseases, they have several well-conserved immune parameters and at least one gene allele has been identified and linked with their increased longevity. During ageing there is an accumulation of oxidised proteins, a phenomenon that has been related to an impaired function of the 20S proteasome in aged cells. We have, therefore, analysed the expression and the proteolytic activity of the proteasome in centenarian cells. Four fibroblast cultures derived from healthy centenarians were studied and compared with cultures derived from adult donors of different ages. Analysis of several proteasome subunits RNA expression levels, determination of one peptidase activity and identification of oxidised proteins in these samples revealed that centenarian cultures have a functional proteasome. In addition, it was found that the centenarian cultures exhibit characteristics similar to the younger rather than the older control donors derived cultures in all three assays. These data indicate that centenarian cells may be different from elderly donors cells, thus opening up new dimensions for the identification and characterisation of factors that are linked with longevity.

Protein degradation by the proteasome and its implications in aging.

Free radical damage to cellular components is believed to contribute to the aging process. Studies on proteins have shown both an age-related decline in several enzyme activities and an age-related accumulation of oxidized forms of protein. Oxidized forms of protein are generally degraded more rapidly than their native counterparts. Indeed, the normal functions of the cell involve the regular elimination of these altered molecules. The proteasome, a multienzymatic proteolytic complex, is the major enzymatic system in charge of cellular "cleansing" and plays a key role in the degradation of damaged proteins. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Because the steady-state level of oxidized protein reflects the balance between the rate of protein oxidation and the rate of protein degradation, age-related accumulation of altered protein can be due to an increase of free radical-mediated damage, a loss of protease activity, or the combination of both mechanisms. One of the hypotheses put forward to explain the accumulation of altered proteins is the decrease of proteasome activity with age. In this paper, the importance of oxidative damage to proteins and that of their elimination by the proteasome are first described. Then, evidence for a decline of proteasome activity upon aging and upon oxidative stress is provided by studies from our and other laboratories.

Dietary self-selection can compensate an age-related decrease of rat liver 20 S proteasome activity observed with standard diet.

Aged Lou female rats (33 months) submitted to a self-selection regimen showed a decrease in protein intake (down to 11% of the total intake), whereas mature rats (18 months) selected a high percentage of protein (20% of the total intake) similar to the protein content of the standard diet. To find out if this decrease in protein intake would prevent an observed age-related decrease in proteasome activity, four peptidase activities and oxidized protein degradation were tested with proteasome purified from the liver of 18- and 33-month-old rats. The peptidylglutamyl-peptide hydrolase activity, which is decreased with age for rats fed the standard diet, was restored in the self-selecting old rats to the level observed for the mature rats. Degradation of oxidized glutamine synthetase, which is also decreased with age for rats fed the standard diet, was partly restored. Proteasome from self-selecting old rats showed a slight increase in trypsin-like and chymotrypsin-like activities as compared to proteasome from old rats fed the standard diet. Two-dimensional gel electrophoresis followed by quantitative analysis of the pattern of proteasome subunits revealed an increase in the intensity of two protein spots for proteasome from old rats fed the standard diet as compared with proteasome from either mature rats or self-selecting old rats. These findings may have important implications in aging for proteasome-mediated proteolysis and subsequent accumulation of oxidatively damaged protein.

Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.