PubMed Health⌕ Search

Biomedical subjects

M Conconi

Publications and source records attributed to M Conconi.

10 recordsLinked to original sources
PubMed2000

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.

Adolescent↗
PubMed2000

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.

Aging↗
PubMed1999

Conformational changes in the 20S proteasome upon macromolecular ligand binding analyzed with monoclonal antibodies.

Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).

Animals↗
PubMed1999

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.

Cells, Cultured↗
PubMed1998

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.

Animals↗
PubMed1998

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.

Aging↗
PubMed1997

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.

Animals↗
PubMed1997

Proteasome inactivation upon aging and on oxidation-effect of HSP 90.

Increases of oxidatively modified protein in the cell have been associated with the aging process. Such an accumulation of damaged protein may be the result of increase in the rate of protein oxidation and/or decrease in the rate of degradation of oxidized protein. The multicatalytic proteinase or proteasome is known to be the major proteolytic system involved in the removal of oxidized protein. We have reported that, after isolation of the 20S proteasome from the liver of young and old male Fischer 344 rat, out of the three peptidase activities (chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolase) we assayed with fluorogenic peptides, the peptidyl-glutamyl peptide hydrolase activity was declining with age to a value approximately 50% of that observed for protease purified from young rats. The proteasome was subjected to metal catalyzed oxidation to determine the susceptibility of the different peptidase activities to oxidative inactivation. Both trypsin-like and peptidyl-glutamyl peptide hydrolase activities were found sensitive to oxidation. Treatment of the proteasome with 4-hydroxy-2-nonenal, a major lipid peroxidation product, was also found to inactivate the trypsin-like activity. However, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation in proteasome preparations contaminated with HSP 90, a protein that often copurifies with the proteasome. Upon addition of HSP 90 to pure 20S active proteasome, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation and from inactivation by treatment with 4-hydroxy-2-nonenal. These results suggest a possible intervention of HSP 90 in response to oxidative stress in preventing the inactivation of the proteasome by oxidative damage.

Aging↗
PubMed1996

Age-related decline of rat liver multicatalytic proteinase activity and protection from oxidative inactivation by heat-shock protein 90.

To test whether an observed age-related increase in the level of oxidized protein in rat liver is due to a decrease in the activity of the multicatalytic proteinase (MCP), this protease was isolated from liver of young (8-month-old) and old (24-month-old) male Fischer 344 rats. Three peptidase activities of the MCP were assayed using fluorogenic peptides: trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolase. Only peptidylglutamyl-peptide hydrolase activity declined with age, with protease from old animals exhibiting approximately 50% of the activity of that from young animals. Bidimensional gel electrophoresis and thermostability studies did not reveal age-related structural modifications of the MCP subunits. Peptidylglutamyl-peptide hydrolase activity and trypsin-like activity were sensitive to metal-catalyzed oxidation. In some preparations, a 95-kDa protein that has been identified as the heat shock protein 90 copurified with the MCP. In the presence of HSP 90, trypsin-like activity is protected from oxidative inactivation and chymotrypsin-like activity is slightly activated. Peptidylglutamyl-peptide hydrolase activity remained sensitive to oxidation in protease isolated from young rats, but that from old rats was resistant to oxidative inactivation. Furthermore, addition of rat HSP 90 to rat liver MCP (purified from 8-month-old animals and free of contaminating HSP 90) was found to protect trypsin-like activity from oxidative inactivation.

Aging↗