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Bertrand Friguet

Publications and source records attributed to Bertrand Friguet.

At least 37 records · Page 2Linked to original sources
PubMed2004

Age-related impairment of mitochondrial matrix aconitase and ATP-stimulated protease in rat liver and heart.

Mitochondrial matrix proteins are sensitive to oxidative inactivation, and oxidized proteins are known to accumulate during ageing. The Lon protease is believed to play an important role in the degradation of oxidized matrix proteins such as oxidized aconitase. We reported previously that an age-related accumulation of altered proteins occurs in the liver matrix of rats and that the ATP-stimulated proteolytic activity, referred as to Lon-like protease activity, decreases considerably in 27 month-old rats, whereas no concomitant changes in the levels of Lon protein expression occur in the liver. Here, we report that this decline is associated with a decrease in the activity of aconitase, an essential Krebs' cycle enzyme. Contrary to what we observed in the liver, the ATP-stimulated protease activity was found to remain constant in the heart mitochondrial matrix during ageing, and the levels of expression of the Lon protease increased in the older animals in comparison with the younger ones. Although the ATP-stimulated protease activity remained practically the same in older animals as in younger ones, a decrease in the level of aconitase activity was still observed. Altogether, these results indicate that matrix proteins, such as the critical enzymes aconitase and Lon protease, are inactivated with ageing and that the effects of ageing vary from one organ to another.

Aconitate Hydratase↗
PubMed2004

Evidence of preferential protein targets for age-related modifications in peripheral blood lymphocytes.

Oxidatively modified proteins have been analyzed in aging human peripheral blood lymphocytes since protein modification by oxidation and other related pathways are believed to contribute to the intracellular age-related accumulation of damaged proteins, a process that has been associated with the cellular functional deficits that occur with age. Advanced glycation end products (AGE) were quantified and the pattern of glycated proteins analyzed by two-dimensional gel electrophoresis followed by Western blotting using an anti-AGE antibody raised against glycated RNAse. The protein silver stain and the immunoblot patterns were not superimposable, indicating that glycoxidative modifications are targeting only a restricted set of proteins. Modification of proteins with the lipid peroxidation product 4-hydroxy-2-nonenal has also been studied. The patterns of modified proteins have been analyzed using two- dimensional gel electrophoresis followed by Western blotting with an antibody recognizing 4-hydroxy-2-nonenal protein adducts using the same proteomic approach as for glycoxidative modifications. Specific protein targets for these modifications, that might serve as biomarkers of aging lymphocytes, are currently characterized and identified by mass spectrometry.

Adult↗
PubMed2004

Algae extract protection effect on oxidized protein level in human stratum corneum.

Modification of proteins by reactive oxygen species is implicated in different disorders. The proteasome is a multicatalytic proteinase in charge of intracellular protein turnover and of oxidized proteins degradation. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Evidence for a decline in proteasome activity during skin photo-aging has been provided in Bulteau et al. in 2002. The ability of a lipid algae extract (Phaeodactylum tricornutum) to stimulate 20S proteasome peptidase activities was described by Nizard et al. in 2001. Furthermore, keratinocytes treated with Phaeodactylum tricornutum extract and then UVA and UVB irradiated, exhibited a sustained level of proteasome activity comparable to the one of nonirradiated cells. The level of modified proteins can be quantified by measurement of protein carbonyl content (Oxyblot technique), which has been shown to increase with aging and other disorders. In this paper, it is described that, in the presence of this lipid algae extract, the level of oxidized proteins is reduced, as assessed by the Oxyblot technique. These results are obtained both with culture of human keratinocytes and stratum corneum skin cells (obtained by stripping) from human volunteers. Altogether, these results argue for the presence of compounds in this algae extract that have a stimulating and/or protective effect on proteasome activity, resulting in a decreased level of protein oxidation.

Adult↗
PubMed2003

Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells.

Proteins are sensitive to reactive oxygen species, and the accumulation of oxidized proteins has been implicated in the aging process and in other age-related pathologies. In proteins, methionine residues are especially sensitive to oxidation, leading to S - and R -methionine sulphoxide diastereoisomers, the reversion of which is achieved by the peptide methionine sulphoxide reductases MsrA and MsrB respectively. The MsrA enzyme, in addition to its role in repair, forms part of the reactive oxygen species scavenging systems that are important in cellular antioxidant defence. MsrA is present in most living organisms, and the mammalian enzyme has been detected in all tissues investigated. In the present study, we investigated the subcellular distribution of MsrA in rat liver cells. Since it seemed likely that MsrA may be localized in areas where reactive oxygen species are produced, rat liver mitochondrial matrix and cytosolic extracts were prepared. The presence of MsrA was assayed in these subcellular compartments by monitoring peptide methionine sulphoxide reductase enzymic activity, by Western blotting and by in situ immunolocalization by electron microscopy using a specific antibody. Moreover, MsrA was identified by MS in a partially purified cytosolic fraction and in a mitochondrial matrix crude extract. Rat MsrA isoforms are encoded by a single gene, and it is suggested that the precursor of the mitochondrial form contains an N-terminal cleavable signal sequence that localizes the MsrA to this organelle. Finally, two-dimensional gel electrophoresis followed by Western-blot analysis of partially purified MsrA from the cytosol and mitochondria, and comparison with the two-dimensional patterns of oxidized recombinant MsrA, revealed oxidative modifications of cysteine residues.

Amino Acid Sequence↗
PubMed2003

Central role of the proteasome in senescence and survival of human fibroblasts: induction of a senescence-like phenotype upon its inhibition and resistance to stress upon its activation.

Normal human fibroblasts undergo a limited number of divisions in culture and progressively they reach a state of irreversible growth arrest, a process termed as replicative senescence. The proteasome is the major cellular proteolytic machinery, the function of which is impaired during replicative senescence. However, the exact causes of its malfunction in these conditions are unknown. Using WI38 fibroblasts as a model for cellular senescence we have observed reduced levels of proteasomal peptidase activities coupled with increased levels of both oxidized and ubiquitinated proteins in senescent cells. We have found the catalytic subunits of the 20 S complex and subunits of the 19 S regulatory complex to be down-regulated in senescent cells. This is accompanied by a decrease in the level of both 20 S and 26 S complexes. Partial inhibition of proteasomes in young cells caused by treatment with specific inhibitors induced a senescence-like phenotype, thus demonstrating the fundamental importance of the proteasome for retaining cellular maintenance and homeostasis. Stable overexpression of beta1 and beta5 subunits in WI38 established cell lines was shown to induce elevated expression levels of beta1 subunit in beta5 transfectants and vice versa. Transfectants possess increased proteasome activities and most importantly, increased capacity to cope better with various stresses. In summary these data demonstrate the central role of the proteasome during cellular senescence and survival as well as provide insights toward a better understanding of proteasome regulation.

Cell Division↗
PubMed2003

Increased level of glycoxidation product N(epsilon)-(carboxymethyl)lysine in rat serum and urine proteins with aging: link with glycoxidative damage accumulation in kidney.

Accumulation of carboxymethylated proteins (CML-proteins) is taken as a biomarker of glycoxidative stress which is thought to contribute to the age-related impairment in tissue and cell function. To investigate the occurrence and extent of glycoxidative damage with aging in rat kidney, serum and urine, we have prepared a polyclonal antibody against CML-modified bovine serum albumin. We subsequently used it for immunolocalization and in enzyme-linked immunosorbent assays to evaluate CML-protein content. In the serum, CML-protein level was 1.43+/-0.14 pmol CML/micrograms protein at 3 months and significantly increased by 50% from 10 to 27 months (1.50+/-0.14 pmol CML/micrograms protein vs 2.27+/-0.26 pmol CML/micrograms protein), albumin and transferrin being the main modified proteins. In the urine, CML-protein level was 2.50+/-0.14 pmol CML/micrograms protein at 3 months and markedly increased from 10 months (2.99+/-0.24 pmol CML/micrograms protein) to 27 months (3.76+/-0.25 pmol CML/micrograms protein), with albumin as the main excreted modified protein. Immunolocalization of CML-proteins in kidney provided evidence for an age-dependent increased accumulation in extracellular matrices. Intense staining of the glomerular basement membrane (GBM), Bowman's capsule, and the tubular basement membrane was found. Additionally, the CML content for collagen from GBM was 195.85+/-28.95 pmol CML/microgrms OHPro at 3 months and significantly increased from 10 months (187.61+/-21.99 pmol CML/micrograms OHPro) to 27 months (334.55+/-62.21 pmol CML/micrograms OHPro). These data show that circulating CML-protein level in serum and urine and CML accumulation in nephron extracellular matrices with aging are increasing in parallel. The CML-protein measurement in serum and urine may thus be used as an index for the assessment of age-associated glycoxidative kidney damage.

Aging↗
PubMed2003

Cellular senescence in human keratinocytes: unchanged proteolytic capacity and increased protein load.

In order to assess the activity of cellular proteasome, we developed a method to permeabilize keratinocyte monolayers and measure proteasome activities intracellularly, using fluorogenic peptide substrates. The observed K(m) did not differ significantly in situ and in soluble extracts, and the K(i) of proteasome inhibitor MG132 was slightly higher in situ (34nM instead of 4nM). Inhibition studies in permeabilized cells showed that MG132 followed competitive inhibition patterns, and clasto-lactacystin beta-lactone non-competitive patterns, as expected. The observed velocities in situ (500pmoles/min/mg protein) were comparable to the best values of proteasome activity in crude cellular extracts. These features altogether allowed to identify the in situ activity as that of proteasome. To characterize proteasome complexes present in human keratinocytes, we analyzed cellular lysates by ultracentrifugation and gel filtration: most proteasome activity was associated with PA700-bound, presumably 26S, particles. PA28 activator was detected only when cells were treated by gamma interferon. Proteasome activities were determined using the in situ method in keratinocytes at different stages of replicative senescence. Only a slight decrease of proteasome activity per cell was seen at intermediate passages, followed by a slight increase in senescent cells. In the same time, the amount of total proteins increased notably with cellular ageing. Thus, proteasome activity decreased relatively to total proteins, but not relatively to cell numbers. Flow cytometry confirmed that the size of aged keratinocytes increased with the ageing marker beta-galactosidase.

Cell Division↗
PubMed2003

Impact of ageing on proteasome structure and function in human lymphocytes.

Key actors of the immune response, lymphocytes exhibit functional deficits with advancing age. For instance, the age-related decline in lymphocyte proliferation may be related to alteration in the degradation of crucial proteins such as cell-cycle regulators. Degradation of these proteins is mediated by the ubiquitin-26S proteasome system. The proteasome is also the major "housekeeping" proteolytic complex responsible for eliminating intracellular damaged proteins. To investigate the occurrence of proteasome structural and functional age-related alterations, 26S proteasome was purified from peripheral blood lymphocytes of 20-63-year-old donors. Changes in peptidase activity were measured and modifications in the proteasome particle structure were analysed using bi-dimensional electrophoresis. We found the age-related decline of 26S proteasome-specific activity to be associated with an increased yield of post-translational modifications of proteasome subunits, while proteasome content and subunit composition were unchanged. In particular, some catalytic and assembly subunits of the 20S proteasome were preferentially modified with age. Western blotting of proteasome subunits resolved by bi-dimensional electrophoresis showed some of these modified subunits to be glycated, conjugated with a lipid peroxidation product and/or ubiquitinated. In conclusion, it is suggested that structural alterations of proteasome subunits may contribute to the observed decline of proteasome activity with age and could play a major role in immune senescence.

Adult↗
PubMed2003

Characterization and role of protozoan parasite proteasomes.

The proteasome, a large non-lysosomal multi-subunit protease complex, is ubiquitous in eukaryotic cells. In protozoan parasites, the proteasome is involved in cell differentiation and replication, and could therefore be a promising therapeutic target. This article reviews the present knowledge of proteasomes in protozoan parasites of medical importance such as Giardia, Entamoeba, Leishmania, Trypanosoma, Plasmodium and Toxoplasma spp.

Animals↗
PubMed2003

Changes in rat liver mitochondria with aging. Lon protease-like reactivity and N(epsilon)-carboxymethyllysine accumulation in the matrix.

Aging is accompanied by a gradual deterioration of cell functions. Mitochondrial dysfunction and accumulation of protein damage have been proposed to contribute to this process. The present study was carried out to examine the effects of aging in mitochondrial matrix isolated from rat liver. The activity of Lon protease, an enzyme implicated in the degradation of abnormal matrix proteins, was measured and the accumulation of oxidation and glycoxidation (Nepsilon-carboxymethyllysine, CML) products was monitored using immunochemical assays. The function of isolated mitochondria was assessed by measuring respiratory chain activity. Mitochondria from aged (27 months) rats exhibited the same rate of oxygen consumption as those from adult (10 months) rats without any change in coupling efficiency. At the same time, the ATP-stimulated Lon protease activity, measured as fluorescent peptides released, markedly decreased from 10-month-old rats (1.15 +/- 0.15 FU x micro g protein-1 x h-1) to 27-month-old-rats (0.59 +/- 0.08 FU x micro g protein-1 x h-1). In parallel with this decrease in activity, oxidized proteins accumulated in the matrix upon aging while the CML-modified protein content assessed by ELISA significantly increased by 52% from 10 months (11.71 +/- 0.61 pmol CML x micro g protein-1) to 27 months (17.81 +/- 1.83 pmol CML x micro g protein-1). These results indicate that the accumulation of deleterious oxidized and carboxymethylated proteins in the matrix concomitant with loss of the Lon protease activity may affect the ability of aging mitochondria to respond to additional stress.

Adenosine Triphosphate↗
PubMed2003

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease.

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.

1-Methyl-4-phenylpyridinium↗
PubMed2003

Potentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha.

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappa B. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-kappa B-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact kappa B sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-kappa B-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-kappa B in the nucleus. We showed that the p65 subunit of NF-kappa B was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-kappa B after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the I kappa B alpha inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-kappa B. This delay was due neither to a defect in I kappa B alpha mRNA production nor to a nuclear retention of I kappa B alpha but was rather due to a persistent proteasome-mediated degradation of I kappa B alpha. A prolongation of I kappa B kinase activity could explain, at least partially, the delayed I kappa B alpha cytoplasmic reappearance observed in presence of TNF plus TSA.

Animals↗
PubMed2002

Proteolysis, free radicals, and aging.

Aging is accompanied by declines in cellular proteolytic capacity. Proteolytic processing is an important step in numerous cellular processes required for normal metabolic function. These include regulation of protein turnover, degradation of altered forms of protein, signal transduction, protein sorting/trafficking, receptor-mediated endo- and exocytosis, stress/immune responses, and activation of gene transcription. Thus, loss of cellular proteolytic function is likely to contribute to the enhanced fragility of cells from senescent relative to young and adult organisms. Free radicals have been implicated as contributing factors to observed age-dependent declines in proteolytic capacity. The current review offers an overview of the evidence linking free radical events to functional alterations in the lysosomal system and the proteasome, two major pathways by which proteins are degraded within cells. Implications for future investigations in the field are discussed in light of these findings.

Aging↗
PubMed2002

Impairment of proteasome function upon UVA- and UVB-irradiation of human keratinocytes.

The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratinocytes were irradiated with 10 J/cm(2) of UVA and 0.05 J/cm(2) of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation.

Aldehydes↗
PubMed2002

Protein repair and degradation during aging.

Cellular aging is characterized by a build-up of oxidatively modified proteins. The steady-state level of oxidized proteins depends on the balance between the rate of protein oxidative damage and the rates of protein degradation and repair. Therefore, the accumulation of oxidized protein with age can be due to increased protein damage, decreased oxidized protein degradation and repair, or the combination of both mechanisms. The proteasomal system is the major intracellular proteolytic pathway implicated in the degradation of oxidized protein, and the peptide methionine sulfoxide reductase catalyzes the reduction of methionine sulfoxide (i.e., oxidized methionine) to methionine within proteins. A short summary on protein oxidative damage and oxidized protein degradation is given, and evidence for a decline of proteasome function with age is presented. Arguments for the implication of peptide methionine sulfoxide reductase in the age-related accumulation of oxidized protein are also discussed.

Aging↗
PubMed2002

Age-dependent declines in proteasome activity in the heart.

The proteasome is a major intracellular proteolytic system involved in the removal of oxidized and ubiquitinated protein and the induction of certain stress response pathways. In this study, age-dependent alterations in proteasome function were investigated to gain insight into potential factors which contribute to increased susceptibility to various forms of heart disease during aging. Proteasome activity in cellular extracts prepared from Fisher 344 rat hearts was found to decrease with age. These declines in activity were associated with a decreased 20S proteasome content and loss of specific activities. As determined by two-dimensional gel electrophoresis of purified 20S proteasome, the distribution and silver staining intensities of enzyme subunits were found to vary with age, suggesting that alterations in proteasome subunit composition and/or structure are involved in age-related declines in proteasome activity. In addition, age-dependent increases in the levels of oxidized and ubiquitinated proteins, known substrates of the proteasome, were observed. Thus, loss in proteasome function may impair the ability of myocytes to mount an appropriate response to stress, thereby enhancing the susceptibility of the aging heart to cardiovascular disease.

Aging↗