Biomedical subjects

I Petropoulos

Publications and source records attributed to I Petropoulos.

12 recordsLinked to original sources

PubMed2001

Rat peptide methionine sulphoxide reductase: cloning of the cDNA, and down-regulation of gene expression and enzyme activity during aging.

Peptide methionine sulphoxide reductase (PMSR, EC 1.8.4.6), the msrA or pmsR gene product, is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins. Decreased expression and/or activity of the PMSR with age could explain, at least in part, the accumulation of oxidized protein observed upon aging. To test this hypothesis, the rat pmsR cDNA was cloned and sequenced. The recombinant protein was expressed, its catalytic activity checked with a synthetic substrate and polyclonal antibodies were raised against recombinant PMSR. The expression of the pmsR gene and protein as well as its catalytic activity were then analysed as a function of age in the rat brain and in two organs that express the most PMSR, liver and kidney. It appears that pmsR gene expression decreases with age in liver and kidney as early as 18 months, whereas protein level and protein activity are reduced in the three organs at the very end of the life of the rat (26 months). These results suggest that the down-regulation of PMSR can contribute to the accumulation of oxidized protein that has been associated with the aging process.

Aging↗

PubMed2000

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.

Adolescent↗

PubMed2000

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.

Adult↗

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↗

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↗

PubMed1995

NOR-2 (neuron-derived orphan receptor), a brain zinc finger protein, is highly induced during liver regeneration.

Zinc-finger proteins are involved in several cellular processes. Some of these proteins are implicated in the primary cellular response in regenerating liver and mitogen-stimulated cells. Using a rat cDNA brain library, we have isolated a clone designated NOR-2, encoding a protein containing two zinc-finger motifs and whose expression is highly induced during G0/G1 transition. We analysed the expression of NOR-2 mRNAs during early growth in regenerating liver and in both insulin-stimulated H4-II cells and pheochromocytoma-derived cell line PC12 treated by NGF. In these systems, there is an early, rapid and transient accumulation of NOR-2 mRNAs. The induction of NOR-2 mRNAs does not require de novo protein synthesis, since it is not prevented by cycloheximide treatment. Mobility shift assays show that NOR-2 protein binds to NBRE, a target sequence for r-NGFI-B family. Structurally, NOR-2 is closely related to the recently identified NOR-1 factor. Therefore, like NOR-1, NOR-2 belongs to the r-NGFI-B sub-family of nuclear receptors superfamily.

Amino Acid Sequence↗

PubMed1995

Interaction of DNA binding domain of HNF-3 alpha with its transferrin enhancer DNA specific target site.

Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3 alpha transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA-protein and protein-protein interactions. As a first step to understand the mechanisms responsible for its organization and activity, we have analyzed the interaction of the DNA binding domain of HNF-3 alpha (HDBD) with a specific DNA segment present in the transferrin enhancer by different biophysical techniques. The kinetic constants of this interaction were measured using surface plasmon resonance. The HDBD-DNA interaction was also characterized by circular dichroism and fluorescence spectroscopy. HDBD binds to its specific DNA site with high affinity (Kd approximately equal to 10(-8) M). The affinity is reduced after sequence modification of the target DNA. Size exclusion chromatography and binding stoichiometry determined by fluorescence measurements indicate that the protein is present in a monomeric form before and after interaction with the DNA. The secondary structure of the protein was not significantly altered upon binding to specific DNA. By contrast, a structural change of DNA by interaction with HDBD seems to occur.

Base Sequence↗

PubMed1995

Rev-erb beta 2, a novel isoform of the Rev-erb family of orphan nuclear receptors.

We have isolated a rat complementary DNA clone corresponding to a novel isoform of the hormone nuclear receptor superfamily. This clone encodes a 383 amino acid residue protein designated as Rev-erb beta 2. This protein is identical until residue 382 to the Rev-erb beta 1 protein, which is 195 amino acids longer. Several arguments pointed out that the Rev-erb beta 2 cDNA may originate from the same gene as Rev-erb beta 1 by alternative splicing and using a different polyadenylation site. Our results indicate that Rev-erb beta 2 is a new isoform of the Rev-erb family of orphan nuclear receptors.

Amino Acid Sequence↗

PubMed1993

Liver-enriched HNF-3 alpha and ubiquitous factors interact with the human transferrin gene enhancer.

The human transferrin gene enhancer is organized in two domains. Domain A contains a single enhanson designated Ia. Domain B contains four enhansons named Ib, II, III and IV. We demonstrate here that the liver-enriched transcription factor HNF-3 alpha interacts with enhanson Ia and that enhansons Ib and IV are binding sites for members of the NF1 family. In addition, enhansons II and III seem to be respectively the targets for the AP4 protein and for EIII, a factor not yet completely identified. Analysis of mutated enhancer regions establishes that each enhanson is required for full enhancer activity and that the proteins binding to enhansons II, III and IV may interact within a multiprotein complex. This enhancer region presents no activity in the Sertoli cells of testis, where transferrin is also synthesized. We demonstrate that in Sertoli cells, the members of the HNF-3 family are not expressed; this fact may account for the inactivity of the enhancer in these cells.

Base Sequence↗

PubMed1993

Transferrin gene as a model for liver-specific gene expression.

The results obtained in the recent years have led to an understanding of the mechanisms controlling liver-specific gene expression. They indicate that this expression needs an appropriate combination of hepatocyte and ubiquitous transcriptional factors, interacting with cis-acting DNA elements, in a characteristic manner for each gene. However, a number of important questions about liver organogenesis and specific cell differentiation remain to be answered.

Animals↗

PubMed1991

Characterization of the active part of the human transferrin gene enhancer and purification of two liver nuclear factors interacting with the TGTTTGC motif present in this region.

The human transferrin gene enhancer is composed of two functional domains (A and B). We have previously shown that domain A is able to mediate enhancer activity in transient expression experiments. Here, we show that multimers of the domain A single enhanson coupled to a canonical TATA box are sufficient to promote transcription in vitro with liver nuclear extracts. Gel mobility shift assays reveal the binding of two liver nuclear factors to this enhanson, and methylation interference experiments show that the motif 5'-TGTTTGCTTT-3' is the target site for these factors. This was confirmed by the use of mutants in gel retardation assays and transient expression experiments. The two proteins interacting with the decanucleotide have been purified from rat liver nuclear extracts by DNA affinity chromatography. The purified proteins named enhancer-binding protein (EBP)-45 and EBP-40 appear as single polypeptide bands with respective molecular masses of 45 and 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The TGTTTGC motif was found to be important for the hepatocyte-specific expression of several genes; and in some cases, it was demonstrated that the transcription factor CCAAT/enhancer-binding protein (C/EBP) is able to bind to this sequence. In vitro experiments show that EBP-45 and EBP-40 are different from C/EBP; they also show that the two proteins interact with the TGTTTGC motif present in control regions of other hepatic genes, such as the mouse albumin enhancer eH and hepatitis B virus enhancer E elements.

Animals↗