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The circadian system orchestrates the timing of physiological processes of an organism living in daily environmental changes. Disruption of circadian rhythmicity has been shown to result in increased oxidative stress and accelerated aging. The circadian regulation of antioxidant defenses suggests that other redox homeostasis elements such as oxidized protein degradation by the proteasome, could also be modulated by the circadian clock. Hence, we have investigated whether proteasome activities and oxidized protein levels would exhibit circadian rhythmicity in synchronized cultured mammalian cells and addressed the mechanisms underlying this process. Using synchronized human embryonic kidney HEK 293 cells and primary dermal fibroblasts, we have shown that the levels of carbonylated protein and proteasome activity vary rhythmically following a 24h period. Such a modulation of proteasome activity is explained, at least in part, by the circadian expression of both Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the proteasome activator PA28αβ. HEK 293 cells showed an increased susceptibility to oxidative stress coincident with the circadian-dependent lower activity of the proteasome. Finally, in contrast to young fibroblasts, no circadian modulation of the proteasome activity and carbonylated protein levels was evidenced in senescent fibroblasts. This paper reports a novel role of the circadian system for regulating proteasome function. In addition, the observation that proteasome activity is modulated by the circadian clock opens new avenues for both the cancer and the aging fields, as exemplified by the rhythmic resistance of immortalized cells to oxidative stress and loss of rhythmicity of proteasome activity in senescent fibroblasts., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Lon protease is an ATP-dependent protease of the mitochondrial matrix that contributes to the degradation of abnormal and oxidized proteins in this compartment. It is also involved in the stability and regulation of the mitochondrial genome. The effects of a depletion of this protease on the mitochondrial function and the identification of oxidized target proteins of Lon have been performed using as cellular model HeLa cells in which Lon level expression can be down-regulated. The expression level of proteins playing a role in the stress response was first determined. The amount of ClpP, another protease in charge of protein degradation of the mitochondrial matrix, and the amount of several chaperones have been evaluated. The expression level of respiratory chain subunits was also measured with or without Lon depletion. The mitochondrial compartment morphology was monitored in different stress conditions, and measured using a parameter devoted to the evaluation of the mitochondrial dynamics. None of these investigations showed a significant phenotype resulting from Lon down-regulation A possible impact of Lon depletion on oxidized mitochondrial proteins level was then sought. 1D gel electrophoresis after the derivatization of protein carbonyl groups with 2,4-dinitrophenyl hydrazine (DNPH) revealed an increase in carbonylated proteins more important in mitochondrial extracts than in total cellular extracts. 2D difference gel electrophoresis (DIGE) experiments provide results consistent with these observations with some enlightenments. Performed with fluorescent dyes labelling either proteins or their carbonyl groups, these experiments indicated proteome modifications in cells with Lon down-regulation both at the level of protein expression and at the level of protein oxidation. These variations are noted in proteins acting in different cellular activities, i.e. metabolism, protein quality control and cytoskeleton organization., (Copyright © 2014. Published by Elsevier Inc.)

ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Lon protease is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Lon, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to loose integrity of mitochondrial genome and to be respiratory deficient. In order to address the role of Lon in mitochondrial functionality in human cells, we have set up a HeLa cell line stably transfected with a vector expressing a shRNA under the control of a promoter which is inducible with doxycycline. We have demonstrated that reduction of Lon protease results in a mild phenotype in this cell line in contrast with what have been observed in other cell types such as WI-38 fibroblasts. Nevertheless, deficiency in Lon protease led to an increase in ROS production and to an accumulation of carbonylated protein in the mitochondria. Our study suggests that Lon protease has a wide variety of targets and is likely to play different roles depending of the cell type., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Methionine sulfoxide reductases (Msr's) are key enzymes proficient in catalyzing the reduction of oxidized methionines. This reductive trait is essential to maintaining cellular redox homeostasis from bacteria to mammals and is also regarded as a potential mechanism to regulate protein activities and signaling pathways, considering the inactivating effects that can be induced by methionine oxidation. In this study, we have generated stable human embryonic kidney HEK293 clones with an altered Msr system by silencing the expression of the main Msr elements-MsrA, MsrB1, or MsrB2. The isolated clones--the single mutants MsrA, MsrB1, and MsrB2 and double mutant MsrA/B1-show a reduced Msr activity and an exacerbated sensitivity toward oxidative stress. A two-dimensional difference in-gel electrophoresis analysis was performed on the Msr-silenced cells grown under basal conditions or submitted to oxidative stress. This proteomic analysis revealed that the disruption of the Msr system mainly affects proteins with redox, cytoskeletal or protein synthesis, and maintenance roles. Interestingly, most of the proteins found altered in the Msr mutants were also identified as potential Msr substrates and have been associated with redox or aging processes in previous studies. This study, through an extensive analysis of Msr-inhibited mutants, offers valuable input on the cellular network of a crucial maintenance system such as methionine sulfoxide reductases., (© 2013 Elsevier Inc. All rights reserved.)

The Saccharomyces cerevisiae homolog of the ATP-dependent Lon protease, Pim1p, is essential for mitochondrial protein quality control, DNA maintenance, and respiration. Here, we demonstrate that Pim1p activity declines in aging cells and that Pim1p deficiency shortens the replicative life span of yeast mother cells. This accelerated aging of pim1Δ cells is accompanied by elevated cytosolic levels of oxidized and aggregated proteins, as well as reduced proteasome activity. Overproduction of Hsp104p greatly diminishes aggregation of oxidized cytosolic proteins, rescues proteasome activity, and restores life span of pim1Δ cells to near wild-type levels. Our results show that defects in mitochondrial protein quality control have global intracellular effects leading to the increased generation of misfolded proteins and cytosolic protein aggregates, which are linked to a decline in replicative potential., (Copyright © 2012 Elsevier Inc. All rights reserved.)

ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Pim1, a Lon-like serine protease in Saccharomyces cerevisiae, is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Pim1, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to lose integrity of mitochondrial genome, and to be respiration-deficient. Because of the severity of phenotypes associated with the depletion of Pim1, this protease appears to be an essential component of the protein quality control machinery in mitochondria and to exert crucial functions during the biogenesis of this organelle. Nevertheless, its physiological substrates and partners are not fully characterized. Therefore, we used the combination of different proteomic techniques to assess the nature of oxidized protein substrates and physiological partners of Pim1 protease under non-repressing growth conditions. The results presented here supply evidence that Pim1-mediated proteolysis is required for elimination of oxidatively damaged proteins in mitochondria.

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.

Friedreich ataxia has frequently been associated with an increased susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich ataxia to study the physiological consequences of a shift from anaerobiosis to aerobiosis. Cells lacking frataxin (Deltayfh1) showed no growth defect when cultured anaerobically. Under these conditions, a significant amount of aconitase was functional, with an intact 4 Fe/4 S cluster. When shifted to aerobic conditions, aconitase was rapidly degraded, and oxidatively modified proteins (carbonylated and HNE-modified proteins) accumulated in both the cytosol and the mitochondria. The ATP-dependent mitochondrial protease Pim1 (Lon) was strongly activated, although its expression level remained unchanged, and the cytosolic activity of the 20S proteasome was greatly decreased, compared to that in wild-type cells. Analysis of the purified proteasome revealed that the decrease in proteasome activity was likely due to both direct inactivation of the enzyme and inhibition by cytosolic oxidized proteins. These features indicate that the cells were subjected to major oxidative stress triggered by oxygen. Accumulation of oxidatively modified proteins, activation of Pim1, and proteasome inhibition did not directly depend on the amount of mitochondrial iron, because these phenotypes remained unchanged when the cells were grown under iron-limiting conditions, and these phenotypes were not observed in another mutant (Deltaggc1) which overaccumulates iron in its mitochondrial compartment. We conclude that oxygen is primarily involved in generating the deleterious phenotypes that are observed in frataxin-deficient yeast cells.

Cystatins are protein inhibitors of cystein proteinases belonging to the papain family. In cowpea, cystatin-like polypeptides and a cDNA have been identified from seeds and metabolic functions have been attributed to them. This paper describes VuC1, a new cystatin cDNA isolated from cowpea leaves (Vigna unguiculata (L.) Walp.). Sequence analysis revealed a multicystatin structure with two cystatin-like domains. The recombinant VUC1 protein (rVUC1) was expressed in an heterologous expression system and purified to apparent homogeneity. It appeared to be an efficient inhibitor of papain activity on a chromogenic substrate. Polyclonal antibodies against rVUC1 were obtained. Involvement of the VuC1 cDNA in the cellular response to various abiotic stresses (progressive drought-stress, dessication and application of exogenous abscissic acid) was studied, using Northern blot and Western blot analysis, in the leaf tissues of cowpea plants corresponding to two cultivars with different capacity to tolerate drought-stress. Surprisingly, these abiotic stresses induced accumulation of two VuC1-like messages both translated into VUC1-like polypeptides. Difference in the transcript accumulation patterns was observed between the two cultivars and related to their respective tolerance level. Presence of multiple cystatin-like polypeptides and their possible involvement in the control of leaf protein degradation by cysteine proteinases is discussed.

A cowpea (Vigna unguiculata cv. EPACE-1) aspartic proteinase (AP) gene was isolated by genomic Library screening. Sequence analysis shows that this AP gene follows the same pattern of intron/exon number and organization as the other isolated plant AP genes, which are distinct from other solved AP genes. Northern blot analysis revealed that cowpea AP accumulates in leaves and stems but not in roots, indicating tissue-specific expression. An increased accumulation of transcripts during senescence suggests enzyme involvement in this process.

FOXL2 is a putative transcription factor involved in ovarian development and function. Its mutations in humans are responsible for the blepharophimosis syndrome, characterized by eyelid malformations and premature ovarian failure (POF). Here we have performed a comparative sequence analysis of FOXL2 sequences of ten vertebrate species. We demonstrate that the entire open reading frame (ORF) is under purifying selection leading to strong protein conservation. We also review recent data on FOXL2 transcript and protein expression. FOXL2 has been shown 1) to be the earliest known sex dimorphic marker of ovarian determination/differentiation in vertebrates, 2) to have, at least in mammals, an ovarian expression persisting until adulthood. The conservation of its sequence and pattern of expression suggests that FOXL2 might be a key factor in the early development of the vertebrate female gonad and involved later in adult ovarian function. Finally, we provide arguments for the existence of an alternative transcript in rodents, that may arise from a differential polyadenylation. Although it has only been demonstrated in rodents, its presence/absence in other species deserves further investigation., (Copyright 2003 S. Karger AG, Basel)

Four cultivars of related species, common bean and cowpea, which exhibit different degrees of drought resistance, were submitted to water stress by withholding irrigation. Drought induced an increase in endoproteolytic activity, being higher in susceptible cultivars (bean) than in tolerant ones (cowpea). An aspartic protease activity was found to be strongly induced especially in bean. From a cowpea leaf cDNA library, a full length aspartic protease precursor cDNA was obtained. Transcript accumulation in response to water stress indicated that the expression of the gene was constitutive in cowpea and transcriptionally up-regulated in bean. The results showed that drought-tolerant and drought-susceptible bean plants differ regarding aspartic protease precursor gene expression.

Phospholipase D, a major lipid-degrading enzyme in plants, was studied in two cultivars of Vigna unguiculata L.Walp, differing in their tolerance to drought (cv. EPACE-1, drought-tolerant, and cv. 1183, drought-susceptible). Enzymatic activities, measured with 14C-PC as substrate, increased when plants were submitted to water stress, the increase being much higher in the drought-sensitive cultivar. A 2911 bp cDNA encoding a putative phospholipase D (VuPLD1) was isolated from a cDNA library prepared from V. unguiculata leaves. The deduced amino acid sequence (809 residues) shows 85.5% identity and 91.3% similarity to that of PLD from Ricinus communis. The expression of the VuPLD1 gene in the leaves is differently modulated by water deficit, depending on the intensity of stress and the tolerance or sensitivity of the plants. In the drought-susceptible V. unguiculata cv. 1183, it readily increased under water stress, reaching maximum values at mild water deficit (-1.5 MPa). In the drought-tolerant cv. EPACE-1, VuPLD1 mRNA remained low throughout the whole drought treatment. Dehydration of leaves led to a dramatic increase in transcript level in both cultivars. Changes in protein amounts semi-quantified by immunoblotting correlated well with variations in transcript steady-state level. Taken together, these results showed that phospholipase D in cowpea plants is essentially regulated at the transcriptional level, and that gene expression is strongly stimulated even by moderate water deficit in the drought-sensitive plant. On the contrary, the drought-tolerant plant presents a remarkable stability of PLD gene expression in conditions of water stress.