[Show abstract][Hide abstract] ABSTRACT: Muscle ageing contributes to both loss of functional autonomy and increased morbidity. Muscle atrophy accelerates after 50 years of age, but the mechanisms involved are complex and likely result from the alteration of a variety of interrelated functions. In order to better understand the molecular mechanisms underlying muscle chronological ageing in human, we have undertaken a top-down differential proteomic approach to identify novel biomarkers after the fifth decade of age.
Muscle samples were compared between adult (56 years) and old (78 years) post-menopausal women. In addition to total muscle extracts, low-ionic strength extracts were investigated to remove high abundance myofibrillar proteins and improve the detection of low abundance proteins. Two-dimensional gel electrophoreses with overlapping IPGs were used to improve the separation of muscle proteins. Overall, 1919 protein spots were matched between all individuals, 95 were differentially expressed and identified by mass spectrometry, and they corresponded to 67 different proteins. Our results suggested important modifications in cytosolic, mitochondrial and lipid energy metabolism, which may relate to dysfunctions in old muscle force generation. A fraction of the differentially expressed proteins were linked to the sarcomere and cytoskeleton (myosin light-chains, troponin T, ankyrin repeat domain-containing protein-2, vinculin, four and a half LIM domain protein-3), which may account for alterations in contractile properties. In line with muscle contraction, we also identified proteins related to calcium signal transduction (calsequestrin-1, sarcalumenin, myozenin-1, annexins). Muscle ageing was further characterized by the differential regulation of several proteins implicated in cytoprotection (catalase, peroxiredoxins), ion homeostasis (carbonic anhydrases, selenium-binding protein 1) and detoxification (aldo-keto reductases, aldehyde dehydrogenases). Notably, many of the differentially expressed proteins were central for proteostasis, including heat shock proteins and proteins involved in proteolysis (valosin-containing protein, proteasome subunit beta type-4, mitochondrial elongation factor-Tu).
This study describes the most extensive proteomic analysis of muscle ageing in humans, and identified 34 new potential biomarkers. None of them were previously recognized as differentially expressed in old muscles, and each may represent a novel starting point to elucidate the mechanisms of muscle chronological ageing in humans.
[Show abstract][Hide abstract] ABSTRACT: Annexin A1 deregulation is often associated with cancer. Indeed we have shown that annexin A1 is overexpressed in melanoma and promotes metastases by formyl peptide receptor stimulation and MMP2 expression. Here, we demonstrated in different melanoma cell lines that annexin A1-MMP2 induction is mediated by MAPK and STAT3 pathways. To decipher endogenous annexin A1 action mode, we showed that annexin A1 is externalized in A375 cells and cleaved by a membrane-associated serine protease, allowing the release of a pro-invasive annexin A1 peptide in the extra cellular environment. Finally, a biochemical and proteomic approach allowed to enrich eight out of 12 members of the annexin family and to identify an original annexin A1 cleavage site localized between Ser(28) and Lys(29). Altogether, these data identify signaling pathways involved in annexin A1 pro-invasive role and suggest that externalized full-length annexin A1 interacts with formyl peptide receptors in a juxtacrine manner while ANXA 2-28 release allows autocrine and paracrine interaction.
Archives for Dermatological Research 11/2014; · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aleurone layer (AL) is an inner tissue of the wheat grain. It contains micronutrients, vitamins, antioxidants and fibre, and can greatly increase the nutritional quality of flour if it is not removed from the kernel with the bran. The AL of mature kernels of three varieties of the two major cultivated wheat species T. aestivum (genome A, B and D) and T. monococcum (genome A) were manually dissected and analyzed using two-dimensional gel-based proteomics. In T. monococcum although composed of only genome A, the maximum number of Coomassie stained AL spots was close to the number found in the bread wheat varieties (1320 and 1258, respectively). Inter-variety variation in spots was higher in the three T. monococcum varieties (103 spots) than in the three T. aestivum varieties (79 spots). Comparison of the two species revealed that only 88 spots differed significantly either in abundance or presence/absence. The B and D genome did not drastically modify the AL proteome, as demonstrated by the fact that 93% of the spots present in T. Monococcum AL spots were also present in T. aestivum. Proteins which differed within and between species were identified using MALDI-TOF and LC-MS/MS Mass Spectrometry. Among the 182 spots that differed, 115 were identified, 53 differed between the two species and 44 (83%) were globulin (Glo) storage proteins. The remarkable environmental stability of the AL proteome previously observed in T. durum and T. aestivum species was confirmed in the variety T. monococcum DV92, grown for two consecutive years in field conditions. Only 15 proteins (out of 1320 AL spots) exhibited significant quantitative variations.
Current Proteomics 07/2014; 11(1). · 0.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Long chain omega 3 polyunsaturated fatty acids (LC-n-3PUFAs) exert potent anti-atherosclerotic action but the mechanisms of action at the vascular level remain unclear. The present study used a non-targeted nutrigenomic approach to investigate the modulations of the aortic proteome in atherosclerotic prone mice following DHA supplementation. LDLR−/− male mice received an atherogenic diet (20 weeks) in parallel with daily oral gavages with either oleic acid-rich oil (Control) or a mixture of oils providing 2% of energy as DHA. The overall protein expression was determined by a proteomic approach followed by bioinformatics analysis. In addition, protein oxidative modifications, namely 4-hydroxynonenal (4-HNE) protein adducts, were detected with a specific antibody followed by MS analysis of the identified spots. Nineteen differentially expressed proteins have been identified in the aorta of the DHA group. The top 5 canonical pathways analysis associated all proteins to metabolic pathways and showed that most of them were related to glucose or lipid metabolism. Up-regulation of superoxide dismutase also suggests an impact of DHA supplementation on vascular antioxidant defenses. The analysis of 4-HNE-protein adducts indicated that DHA supplementation did not enhance oxidative modification of proteins with 4-HNE. This study also identified some new proteins with 4-HNE adducts at the aortic level. In summary, DHA supplementation induces significant modifications of aorta proteome which point out the importance of metabolic changes occurring in the vascular tissue in the presence of LC-n-3PUFAs.
European Journal of Lipid Science and Technology 12/2013; 115(12). · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.
Applied Microbiology and Biotechnology 11/2013; 98(2). · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared to mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main biological functions altered during human muscle aging were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.
[Show abstract][Hide abstract] ABSTRACT: Meat is an appropriate source of proteins and minerals for human nutrition. Technological treatments modify the physical-chemical properties of proteins, making them liable to decrease the nutritional potential of meat. To counteract this damage, antioxidants and chaperone proteins in muscle cells can prevent oxidation, restore the function of denatured proteins, and thus prevent aggregation. This study aimed to explore the impact of indoor vs outdoor-reared meat protein composition on digestion and to associate protein markers to in vitro digestion parameters. Indoor-reared meat tended to show less oxidation and denaturation than outdoor-reared meat and was characterised by an overexpression of contractile and chaperone proteins. Outdoor-reared meat showed amplification of antioxidant and detoxification metabolism defending against oxidised compounds. Impacts on digestion remained minor. Several protein markers of in vitro digestion parameters were found for aged and cooked meat, linked to the detoxification process and to muscle contraction.
[Show abstract][Hide abstract] ABSTRACT: As part of the Sec translocase, the accessory ATPase SecA2 is present in some pathogenic Gram-positive bacteria. In Listeria monocytogenes, deletion of secA2 results in filamentous cells that forms rough colonies and have lower virulence. However, only a few proteins have been identified that are secreted by this pathway. This investigation aims to provide the first exoproteomic analysis of the SecA2-dependent secretion in L. monocytogenes EGD-e. By using media and temperatures relevant to bacterial physiology, we demonstrated that the rough colony and elongated bacterial cell morphotypes are highly dependent on growth conditions. Subsequently, comparative exoproteomic analyses of the ΔsecA2 versus wt strains were performed in chemically defined medium at 20°C and 37°C. Analyzing the proteomic data following the secretomics-based method, part of the proteins appeared routed towards the Sec pathway and exhibited an N-terminal signal peptide. For another significant part, they were primarily cytoplasmic proteins, thus lacking signal peptide and with no predictable secretion pathway. In total, 13 proteins were newly identified as secreted via SecA2, which were essentially associated with cell-wall metabolism, adhesion and/or biofilm formation. From this comparative exoproteomic analysis, new insights into the L. monocytogenes physiology are discussed in relation to its saprophytic and pathogenic lifestyle.
Journal of proteomics 01/2013; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to identify specific markers of lipid oxidation generated in meat during refrigerated storage and cooking an analysis was conducted to investigate the relationships between the early post-mortem sarcoplasmic proteome, which contains the majority of enzymes involved in the oxidative process, and the level of lipid oxidation. This study was performed in Longissimus lumborum pig muscle. Proteome was analysed by 2-D electrophoresis in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and lipid oxidation was estimated by the TBA reactive substances (TBA-RS) measurement. Many markers of lipid oxidation were identified, but no single marker covered the oxidative process in its entirety. The role of five protein groups (albumin, redoxins, annexins, lipid transporters and enzymes of aerobic respiration), from which a link with lipid oxidation can be established, is discussed. This study, which completes a precedent work focused on protein oxidation, clearly demonstrates that a combination of several markers is needed to assess the sensitivity of meat to oxidation during both ageing and cooking.
[Show abstract][Hide abstract] ABSTRACT: Fat loss during cooking of duck 'foie gras' is the main problem for both manufacturers and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yields remains high and uncontrolled. To understand the biochemical effects of post-slaughter processing on fat loss during cooking, we characterize for the first time the protein expression of fatty liver during chilling using a proteomic approach. For this purpose we separated the proteins according to their solubility: the protein fraction soluble in a buffer of low ionic strength (S) and the protein fraction insoluble in the same buffer (IS). To analyze the S fraction, we used two-dimensional electrophoresis and mass spectrometry for the identification of spots of interest. This analysis revealed 36 (21 identified proteins) and 34 (26 identified proteins) spots of interests in the low-fat-loss and high-fat-loss groups respectively. The expression of proteins was lower after chilling, which revealed a suppressive effect of chilling on biological processes. To analyze the IS fraction, we used the shot-gun strategy with the identification of all the proteins by mass spectrometry. It allowed us to identify 554 and 562 proteins in the low-fat-loss and high-fat-loss groups respectively. Among these proteins, only the proteins that were up-regulated in the high-fat-loss group were significant (p value = 3.17 10-3) and corresponded to protein from the cytoskeleton and its associated proteins. Taken together, our results suggest that the variability of technological yield observed in processing plants could be explained by different ageing states of fatty livers during chilling, most likely associated with different proteolytic patterns.
Journal of Agricultural and Food Chemistry 12/2012; · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND & AIMS: Postprandial lipemia has been associated with inflammation, oxidative stress and vascular dysfunction. This metabolic disturbance represents a complex process only partly understood. The purpose of this study was to identify variations in plasma proteome after a high-fat challenge in healthy middle-aged men. METHODS: Two-dimensional electrophoresis was used to compare plasma from seven subjects, drawn before and 4 h after a high-fat challenge. RESULTS: Among the 231 spots detected and analyzed, 22 were present at different levels in postprandial hyperlipemic plasma compared to preprandial plasma. For 10 of them, corresponding proteins were identified by mass spectrometry. Some of them are related to the hemostatic system (tetranectin and fibrinogen) or the complement system (complement component 3 and 4 and ficollin-3) and have been previously associated to atherothrombosis. CONCLUSION: These results provide new perspectives and broaden our understanding of the biological processes of postprandial metabolic stress, as well as its links with the development of atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: The aleurone layer (AL) is one of inner tissues removed from the grain with the wheat bran. It is the main source of vitamins, minerals and antioxidants of potential nutritional value in the wheat kernel. The AL of three varieties of each of the two main species of wheat, Triticum aestivum (ABD) and Triticum durum (AB), were manually dissected and analysed using two-dimensional gel-based proteomics. A total of 1258 and 1109 Coomassie-stained spots were detected in the AL of representatives of the ABD and AB genomes. In two varieties (T. aestivum Chinese Spring and T. durum Bidi17), grown in two different years with full fungicide protection, no quantitative or qualitative (presence/absence) differences in spots were detected, suggesting that AL proteome is strongly genetically controlled. Comparison within and between species revealed a total of 339 AL significant protein spots. Among these spots, 30.8% differed within T. aestivum and 56.5% within T. durum varieties, whereas only 12.7% differed between the two species. Among the 142 AL proteins identified using MALDI-TOF and LC-MS/MS, 57% were globulin type storage proteins (Glo-3, Glo-3B, Glo-3C, Glo-2), 16.2% were involved in carbohydrate metabolism and 17.6% in defence/stress pathways. These variations in AL proteome are discussed.
Journal of Cereal Science 05/2012; 55(3):323–330. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to describe developmental changes in human salivary peptidome, whole saliva was obtained from 98 infants followed longitudinally at 3 and 6months of age. Data on teeth eruption and diet at the age of 6months were also recorded. Salivary peptide extracts were characterised by label-free MALDI-MS. Peptides differentially expressed between the two ages, and those significantly affected by teeth eruption or introduction of solid foods were identified by MALDI TOF-TOF and LC ESI MS-MS. Out of 81 peaks retained for statistical analysis, 26 were overexpressed at the age of 6months. Exposure to solid foods had a more pronounced effect on profiles (overexpression of nine peaks) than teeth eruption (overexpression of one peak). Differential peaks corresponded to fragments of acidic and basic PRPs, statherin and histatin. Comparison with existing knowledge on adult saliva peptidome revealed that proteolytic processing of salivary proteins is qualitatively quite comparable in infants and in adults. However, age and diet are modulators of salivary peptidome in human infants.
Journal of proteomics 04/2012; 75(12):3665-73. · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteomic analysis of albumins and globulins (alg) present in starchy endosperm of wheat (Triticum aestivum cv Récital), at 21 stages of grain development, led to the identification of 487 proteins. Four main developmental phases of these metabolic proteins, with three subphases in phase three and two in phase four, were shown. Hierarchical cluster analysis revealed nine major expression profiles throughout grain development. Classification of identified proteins in 17 different biochemical functions provided a uniform picture of temporal coordination among cellular processes. Proteins involved in cell division, transcription/translation, ATP interconversion, protein synthesis, protein transport, along with amino acid, lipid, carbohydrate and nucleotide metabolisms were highly expressed in early and early mid stages of development. Protein folding, cytoskeleton, and storage proteins peaked during the middle of grain development, while in later stages stress/defense, folic acid metabolism, and protein turn over were the abundant functional categories. Detailed analysis of stress/defense enzymes revealed three different evolutionary profiles. A global map with their predicted subcellular localizations and placement in grain developmental scale was constructed. The present study of complete grain development enriched our knowledge on proteome expression of alg, successively from endosperm cell division and differentiation to programmed cell death.
Journal of Proteome Research 03/2012; 11(5):2754-73. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated a protein profile evolution during steatosis in ducks using 2-dimensional electrophoresis gels to better understand the mechanisms underlying liver steatosis at the level of hepatic proteins in waterfowl. Two-dimensional electrophoresis gels were performed in the liver at different stages of steatosis in the duck. Mule ducks were slaughtered after 0, 14, or 23 meals of overfeeding, according to commercial conditions. Thirty-one proteic spots were differentially expressed between 3 or 2 durations of overfeeding: 3 spots were differentially expressed between the 3 times and 28 spots were differentially expressed between 2 times. The identified proteins (14) could be regrouped into 5 categories: enzymes, translation factors, proteins involved in cell structure, proteins with antioxidant properties, and proteins that can link calcium. This study opens new research areas in the understanding of steatosis in waterfowl, such as cell structure and oxidative stress.
[Show abstract][Hide abstract] ABSTRACT: Fat loss during cooking of duck "foie gras" is the main quality issue for both processors and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yield remains high and uncontrolled. To better understand the biological basis of this phenomenon, a proteomic study was conducted. To analyze the protein fraction soluble at low ionic strength (LIS), we used bidimensional electrophoresis and mass spectrometry for the identification of spots of interest. To analyze the protein fraction not soluble at low ionic strength (NS), we used the shotgun strategy. The analysis of data acquired from both protein fractions suggested that at the time of slaughter, livers with low fat loss during cooking were still in anabolic processes with regard to energy metabolism and protein synthesis, whereas livers with high fat loss during cooking developed cell protection mechanisms. The variability in the technological yield observed in processing plants could be explained by a different physiological stage of liver steatosis.
Journal of Agricultural and Food Chemistry 12/2011; 59(23):12617-28. · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.
Journal of Experimental Botany 11/2011; 63(2):1001-11. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Defined as proteins actively transported via secretion systems, secreted proteins can have radically different subcellular destinations in monoderm (Gram-positive) bacteria. From degradative enzymes in saprophytes to virulence factors in pathogens, secreted proteins are the main tools used by bacteria to interact with their surroundings. The etiological agent of listeriosis, Listeria monocytogenes, is a Gram-positive facultative intracellular foodborne pathogen, whose ecological niche is the soil and as such should be primarily considered as a ubiquitous saprophyte. Recent advances on protein secretion systems in this species prompted us to investigate the exoproteome. First, an original and rational bioinformatic strategy was developed to mimic the protein exportation steps leading to the extracellular localization of secreted proteins; 79 exoproteins were predicted as secreted via Sec, 1 exoprotein via Tat, 4 bacteriocins via ABC exporters, 3 exoproteins via holins, and 3 exoproteins via the WXG100 system. This bioinformatic analysis allowed for defining a databank of the mature protein set in L. monocytogenes, which was used for generating the theoretical exoproteome and for subsequent protein identification by proteomics. 2-DE proteomic analyses were performed over a wide pI range to experimentally cover the largest protein spectrum possible. A total of 120 spots could be resolved and identified, which corresponded to 50 distinct proteins. These exoproteins were essentially virulence factors, degradative enzymes, and proteins of unknown functions, which exportation would essentially rely on the Sec pathway or nonclassical secretion. This investigation resulted in the first comprehensive appraisal of the exoproteome of L. monocytogenes EGD-e based on theoretical and experimental secretomic analyses, which further provided indications on listerial physiology in relation with its habitat and lifestyle. The novel and rational strategy described here is generic and has been purposely designed for the prediction of proteins localized extracellularly in monoderm bacteria.
Journal of Proteome Research 08/2011; · 5.06 Impact Factor