Martine Perichon

The peptide methionine sulfoxide reductases, MsrA and MsrB (hCBS-1), are downregulated during replicative senescence of human WI-38 fibroblasts

In contrast to other oxidative modifications of amino acids, methionine sulfoxide can be enzymatically reduced back to methionine in proteins by the peptide methionine sulfoxide reductase system, composed of MsrA and MsrB. The expression of MsrA and one member of the MsrB family, hCBS‐1, was analyzed during replicative senescence of WI‐38 human fibroblasts. Gene expression decreased for both enzymes in senescent cells compared to young cells, and this decline was associated with an alteration in catalytic activity and the accumulation of oxidized proteins during senescence. These results suggest that downregulation of MsrA and hCBS‐1 can alter the ability of senescent cells to cope with oxidative stress, hence contributing to the age‐related accumulation of oxidative damage.

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.

Accumulation of Advanced Glycation Endproducts in the Rat Nephron: Link with Circulating AGEs During Aging

The accumulation of advanced glycosylation end products (AGEs) is believed to be a factor in the development of aging nephropathy. We have attempted to establish a link between the formation of AGEs and the onset of renal impairment with aging, indicated by albuminuria, using a fluorescence assay and immunohistochemical detection of AGEs in the renal extracellular matrix in rats. The fluorescence of collagenase-digested Type IV collagen from GBM increased with age, from 1.65 ± 0.05 AU/mM OHPro (3 months) and 1.58 ± 0.04 (10 months) to 2.16 ± 0.06 (26 months) (p>0.001) and 2.53 ± 0.18 (30 months) (p>0.001). In contrast, the extent of early glycation products significantly decreased from 5.35 ± 0.25 nmol HCHO/nmol OHPro at 3 months to 3.14 ± 0.19 at 10 months (p>0.001), 3.42 ± 0.38 at 26 months, and 0.74 ± 0.08 at 30 months (p>0.001). The urinary fluorescence of circulating AGE rose from 2.42 ± 0.15 AU/mg protein (3 months), 1.69 ± 0.07 (10 months), to 4.63 ± 0.35 (26 months) (p>0.01) and 4.73 ± 0.72 (30 months), while the serum fluorescence increased from 0.39 ± 0.02 AU/mg protein at 3 months and 0.43 ± 0.02 at 10 months to 0.59 ± 0.04 at 26 months (p>0.001) and 0.54 ± 0.03 at 30 months (p>0.04). Polyclonal antibodies raised against AGE RNase showed faint areas of AGE immunoreactivity in mesangial areas in the nephrons of young rats. The immunolabeling of Bowmans capsule, the mesangial matrices, and the peripheral loops of glomerular and tubule basement membranes increased with rat age. The increase in circulating AGE peptides parallels the accumulation of AGEs in the nephron, and this parallels the pattern of extracellular matrix deposition, suggesting a close link between AGE accumulation and renal impairment in aging rats. (J Histochem Cytochem 45:1059–1068, 1997)

Alterations in mitochondrial and cytosolic methionine sulfoxide reductase activity during cardiac ischemia and reperfusion

During cardiac ischemia/reperfusion, proteins are targets of reactive oxygen species produced by the mitochondrial respiratory chain resulting in the accumulation of oxidatively modified protein. Sulfur-containing amino acids are among the most sensitive to oxidation. Certain cysteine and methionine oxidation products can be reversed back to their reduced form within proteins by specific repair enzymes. Oxidation of methionine in protein produces methionine-S-sulfoxide and methionine-R-sulfoxide that can be catalytically reduced by two stereospecific enzymes, methionine sulfoxide reductases A and B, respectively. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during conditions of oxidative stress, the fate of this system during ischemia/reperfusion was investigated. Mitochondrial and cytosolic methionine sulfoxide reductase activities are decreased during ischemia and at early times of reperfusion, respectively. Partial recovery of enzyme activity was observed upon extended periods of reperfusion. Evidence indicates that loss in activity is not due to a decrease in the level of MsrA but may involve structural modification of the enzyme.

Glycation of albumin with aging and diabetes in rats: changes in its renal handling

Albumin glycation was investigated in old rats to elucidate the link between the preferential excretion of glycated albumin and age-related microalbuminuria. Postprandial blood glucose and the glycated albumin in the serum and urine of 3-, 10- and 30-month-old Wistar rats and in streptozotocin diabetic rats were determined. Blood glucose increased from 1.46 +/- 0.046 g l-1 in 3-month-old rats to 2.08 +/- 0.06 (10 months) and 1.75 +/- 0.23 (30 months) (P < 0.05). Albumin glycation level in the serum increased from 0.79 +/- 0.07 nmol HCHO/nmol albumin (3 months) to 1.41 +/- 0.14 (10 months) and 1.73 +/- 0.21 (30 months) (P < 0.05); urinary level increased from 1.63 +/- 0.39 nmol HCHO/nmol albumin (3 months) to 2.92 +/- 0.57 (10 months) and 2.39 +/- 0.36 (30 months) (P < 0.01). The percent glycated albumin in serum rose from 3.33 +/- 0.64 to 6.81 +/- 0.63 and 6.99 +/- 1.79% of total albumin (P < 0.05), whereas the urine percentage decreased from 12.81 +/- 3.97 to 12.64 +/- 2.87 and 2.63 +/- 0.97% (P < 0.05) in 3-, 10- and 30-month-old rats, respectively. Editing decreased with aging from 4.28 +/- 0.83 (3 months) to 1.84 +/- 0.32 (10 months) and 0.52 +/- 0.14 (30 months) (P < 0.01). Editing in microproteinuric diabetic rats was lower (0.95 +/- 0.08) than in 3-month-old control rats (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc supplementation in the elderly subjects: Effect on oxidized protein degradation and repair systems in peripheral blood lymphocytes

Aging has been associated with zinc deficiency, leading to chronic inflammation and subsequent oxidative stress, especially in the immune system. The increased oxidative stress provokes the accumulation of oxidized proteins, raising the problem of the efficacy of intracellular protein maintenance systems responsible for the elimination of oxidatively modified proteins. Our objective was to analyse the effect of zinc supplementation in the elderly on protein maintenance in peripheral blood lymphocytes. The status of the proteasome, which is in charge of oxidized protein degradation and the repair enzymes peptide methionine sulfoxide reductases, which can reverse methionine oxidation in proteins, were analysed on peripheral blood lymphocytes collected from 20 elderly subjects (age range between 59 and 85 years old) before and after zinc supplementation (10mg of zinc per day for 48+/-2 days). A decrease of oxidized protein content in zinc supplemented subjects was observed and was associated with an increase of expression levels and/or activities of proteasome and methionine sulfoxide reductases. Our results indicate that zinc treatment could enhance the anti-oxidative defences of peripheral blood lymphocytes by increasing the activities of protein maintenance systems responsible for the elimination of oxidatively modified proteins.

Age-related changes in albumin binding by renal brush-border membrane vesicles

A selective proteinuria occurs with normal aging. We investigated the contribution of a defect in the receptor-mediated endocytosis to the age-related albuminuria by measuring albumin binding by renal brush-border membrane vesicles from young and old female Wistar rats using a filtration method. Old (24 months) rats had a significantly higher proteinuria (13.29 +/- 5.25 mg prot/24 h/100 g bw) than did young (3 months) rats (1.23 +/- 0.55 mg prot/24 h/100 g bw). Scatchard analysis of the kinetic parameters of 125I-albumin binding revealed a decrease in the binding capacity of brush-border membrane vesicles from old rats. The number of binding sites, N (pmol/mg protein/min) was 236.84 +/- 97.50 in old rat preparations and 380.27 +/- 178.36 in young rat vesicles (P < 0.05). By contrast, Km did not change significantly with age (478.86 +/- 259.29 nM in old rat vesicles and 498.00 +/- 220.36 nM in young rat preparations). Consequently the index of adsorptive endocytosis efficiency (the N/Km ratio) decreased drastically with age from 0.782 +/- 0.238 at 3 months to 0.547 +/- 0.199 at 24 months (P < 0.05). These data indicate that defective receptor-mediated endocytosis could, at least partly, explain the age-dependent rise in urinary albumin excretion.

Effect of glycation of albumin on its binding to renal brush-border membrane vesicles: influence of aging in rats.

Aging is associated with the loss of preferential urinary excretion of Amadori-product glycated albumin. We have measured the binding of 125I-labeled glycated albumin to the renal brush-border membrane vesicles from young and old rats to determine whether a specific receptor-mediated endocytosis system may be involved. 125I-Glycated albumin was specifically bound by renal brush-border membrane vesicles in a time- and temperature-dependent manner; the binding was concentration-dependent, saturable and reversible. Scatchard plots gave an apparent dissociation constant Km of 488 +/- 17 nM, and a number of binding sites N of 33.5 +/- 3.4 pmol/mg protein/min in membrane vesicles from young (3 months old) rats; the binding of native [125I]albumin, gave a Km of 1194 +/- 200 nM (P < 2%) and N of 82.4 +/- 16.3 pmol/mg protein/min (P < 3%). Vesicles from 10-month-old rats had a similar Km (619.6 +/- 135.3 nM) and N (21.91 +/- 2.98 pmol/mg protein/min), while those from older (30 months old) rats had significantly increased Km (1344 +/- 237 nM, P < 3%) and N (81.3 +/- 10.9 pmol/mg protein/min, P < 1%) for 125I-glycated albumin binding. 125I-Glycated HSA was not displaced by unlabeled native HSA in less than 100-fold excess and native [125I]HSA was only displaced by a 10-fold excess of unlabeled glycated HSA. The binding of native [125I]HSA was partly inhibited (85%) by unlabeled glycated HSA. Thus, there appear to be two different binding sites, one for glycated and the other for native albumin, lying close together; and the glycation site on albumin is the discriminatory recognition factor.

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the filtration method. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.