Christophe Chambon

Differential proteome analysis of aging in rat skeletal muscle

To identify the mechanisms underlying muscle aging, we have undertaken a high‐resolution differential proteomic analysis of gastrocnemius muscle in young adults, mature adults, and old LOU/c/jall rats. Two‐dimensional gel electrophoresis and subsequent MALDI‐ToF mass spectrometry analyses led to the identification of 40 differentially expressed proteins. Strikingly, most differences characterized old (30‐month) animals, whereas young (7‐month) and mature (18‐month) adults exhibited similar patterns of expression. Important modifications in contractile (actin, myosin light‐chains, troponins‐T) and cytoskeletal (desmin, tubulin) proteins, and in essential regulatory proteins (gelsolin, myosin binding proteins, CapZ‐β, P23), likely account for dysfunctions in old muscle force generation and speed of contraction. Other features support decreases in cytosolic (triose‐phosphate isomerase, enolase, glycerol‐3‐P dehydrogenase, creatine kinase) and mitochondrial (isocitrate dehydrogenase, cytochrome‐c oxidase) energy metabolisms. Muscle aging is often associated with increased oxidative stress. Accordingly, we observed differential regulation of molecular chaperones (hsp20, hsp27, reticuloplasmin ER60) and of proteins implicated in reactive aldehyde detoxification (aldehyde dehydrogenase, glutathione transferase, glyoxalase). We further noticed up‐regulation of proteins involved in transcriptional elongation (RNA capping protein) and RNA‐editing (Apobec2). Most of these proteins were previously unrecognized as differentially expressed in old muscles, and they represent novel starting points for elucidating the mechanisms of muscle aging.

Proteome changes during pork meat ageing following use of two different pre-slaughter handling procedures.

The influence of postmortem storage time and pre-slaughter conditions (transport the day before slaughter or immediately before slaughter) on proteome changes of pork meat was investigated over a 72 h ageing period. Intensities of 37 spots varied significantly (p<0.05) with ageing time. Changes indicated proteolysis of troponin T, actin, α-crystallin, myokinase, creatine kinase and mitochondrial ATPase, but also of proteins constitutive of the Z-lines, namely cypher proteins and myozenin. Other modifications were the intensity increase of a full-length protein of the sarcoplasmic reticulum, which may be linked to its increased extractibility after membrane disruption, and a gradual shift in pHi towards alkaline values of some forms of myosin light chains (MLC) 2 and 3. The pre-slaughter conditions affected significantly (p<0.05) 8 spots. Mitochondrial ATPase was over-expressed in the group transported immediately before slaughter, also characterised by a faster pH fall, and the shift in pHi of MLC 2 was more pronounced. The pre-slaughter conditions had no significant effect on the above proteolytic events.

Small peptides (<5kDa) found in ready-to-eat beef meat.

Dietary proteins can have biological properties, many attributed to bioactive peptides (2-50 amino acids). Since little is known about peptides in meat, we investigated the postmortem occurrence of low molecular weight peptides (<5kDa) in bovine Pectoralis profundus muscle, after 14 days storage at 4°C and vacuum cooking for 90min at 75°C. The study combined quantitative (amino acid analysis) and qualitative approaches (mass spectrometry). Eighty-nine percent of peptidic amino acids in fresh muscle corresponded to carnosine, anserine and glutathione. Levels of these compounds were lower in cooked meat compared to fresh muscle. Concomitantly, numerous larger compounds, most probably peptides, were generated in a very reproducible manner during ageing and even more during cooking of meat. Seven peptides (fragments of troponin T, nebulin, procollagen and cypher proteins) were identified in cooked meat extracts.

The p300/CBP-associated factor (PCAF) is a cofactor of ATF4 for amino acid-regulated transcription of CHOP

When an essential amino acid is limited, a signaling cascade is triggered that leads to increased translation of the ‘master regulator’, activating transcription factor 4 (ATF4), and resulting in the induction of specific target genes. Binding of ATF4 to the amino acid response element (AARE) is an essential step in the transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation. We set out to identify proteins that interact with ATF4 and that play a role in the transcriptional activation of CHOP. Using a tandem affinity purification (TAP) tag approach, we identified p300/CBP-associated factor (PCAF) as a novel interaction partner of ATF4 in leucine-starved cells. We show that the N-terminal region of ATF4 is required for a direct interaction with PCAF and demonstrate that PCAF is involved in the full transcriptional response of CHOP by amino acid starvation. Chromatin immunoprecipitation analysis revealed that PCAF is engaged on the CHOP AARE in response to amino acid starvation and that ATF4 is essential for its recruitment. We also show that PCAF stimulates ATF4-driven transcription via its histone acetyltransferase domain. Thus PCAF acts as a coactivator of ATF4 and is involved in the enhancement of CHOP transcription following amino acid starvation.

Pig Longissimus lumborum proteome: Part II: Relationships between protein content and meat quality

Gender, rearing environment and breed of sire influenced 50.5% of the matched protein spots of the soluble fraction and some meat quality traits [Kwasiborski, A., Sayd, T., Chambon, C., Santé-Lhoutellier, V., Rocha, D., & Terlouw, C. (2008). Muscle proteome in pigs: Part I: Effects of genetic background, rearing environment and gender. Meat Science]. Multiple regression analyses determined that 1 or 2 proteins explained between 24% and 85% of variability in Longissimus meat quality. Regression models differed between treatment groups, but relationships between proteins and meat quality traits seemed to be related to common underlying mechanisms. Thus, proteins retained in models for ultimate pH, lightness, drip, thawing and cooking loss were related to the glycolytic pathway, phosphate transfer, or fibre type composition. Another model for thawing loss retained proteins related to denaturation of myofibrils or lipid content. The models for redness involved proteins related to post-mortem oxidative activity. Thus, proteins correlated with meat quality traits were related to biochemical mechanisms known to be involved in meat quality. Relative contributions of these mechanisms may vary according to gender, sire breed or rearing environment.

Proteomic and morphological analysis of early stages of wheat grain development

The identification of 249 proteins in the first 2 wks of wheat grain development enabled the chronological description of the early processes of grain formation. Cell division involved expression of the enzymes and proteins of the cytoskeleton and structure, DNA repair and replication enzymes and cellular metabolism enzymes (synthesis of amino acids, cell wall initiation, carbon fixation and energy production, cofactors and vitamins) with a peak expression at 125°Cday (degrees day after anthesis). After the first synthesis of amino acids, protein transport mechanisms, translation signals, sugar metabolism (polymerization of protein) and stress/defence proteins were activated with stable expression between 150 and 280°Cday. Proteins responsible for folding and degradation, including different subunits of proteasome, were highly expressed at 195°Cday. Proteins associated with starch granules (GBSS type 1) were present at the beginning of grain formation and increased regularly up to 280°Cday. Heat shock proteins (HSP70, 80, 90) were expressed throughout the early grain development stages.

In vivo proteome dynamics during early bovine myogenesis

Myogenesis is a complex process of which the underlying mechanisms are conserved between species, including birds and mammals. Despite a good understanding of the stages of myogenesis, many of the mechanisms involved in the regulation of proliferation of the successive myoblast generations, the cellular transitions cell proliferation/alignment of myoblasts/fusion of myoblasts into myotubes/differentiation of myofibres and the control of total myofibre number still remain unknown. An in vivo proteomic analysis of the semitendinosus muscle from Charolais foetuses, at three specific stages of myogenesis (60, 110 and 180 days postconception), was conducted using 2‐DE and MS. Expression profiles of more than 170 proteins were revealed and analysed using two way hierarchical clustering and statistical analysis. Our studies identify, for the first time, distinct proteins of varied biological functions and protein clusters with myogenic processes, such as the control of cell cycle activity and apoptosis, the establishment of cellular metabolism and muscle contractile properties and muscle cell reorganisation. These results are of fundamental interest to the field of myogenesis in general, and more specifically to the control of muscle development in meat producing animals.

Characterisation of PSE zones in semimembranosus pig muscle.

Pig semimembranosus muscles, sampled from normal hams or from PSE-zones of defective hams, were analysed by histochemistry and electrophoretic techniques. PSE zones were characterised by a disorganisation of fibre alignment and a significant increase of inter fibre spacing (26.2% vs. 16.9%, p<0.05). Protein solubility was significantly lower in defective muscle (55.4 vs. 91.5mg/g, p<0.001). SDS-PAGE evidenced in such samples a lower abundance of the 97, 40 and 26kDa bands in the sarcoplasmic fraction and a higher abundance of the 97, 58, 34, 31, 15 and 11kDa bands in the myofibrillar fraction. Intensity of the MHC band (200kDa) was lower in PSE zone samples. By 2-D electrophoresis, it was shown that troponin T, MLC 1 and alpha-crystallin were less proteolysed in defective muscles, while creatine kinase fragments were more represented. One form of HSP 27 was absent from PSE zone samples. Overall, meat from PSE-zones and fast pH fall-PSE meat show numerous histological and biochemical similarities, particularly in their protein characteristics.

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions

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.

Insight into the core and variant exoproteomes of Listeria monocytogenes species by comparative subproteomic analysis

While Listeria monocytogenes is responsible for listeriosis, it is also a saprophytic species with exceptional survival aptitudes. Secreted proteins are one of the main tools used by bacteria to interact with their environment. In order to take into account the biodiversity of L. monocytogenes species, exoproteomic analysis was carried out on 12 representative strains. Following 2‐DE and MALDI‐TOF MS, a total of 151 spots were identified and corresponded to 60 non‐orthologous proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach predicting final subcellular localization was carried out. Fifty‐two out of the 60 proteins identified (86.7%) were indeed predicted as localized in the extracellular milieu (gene ontology (GO): 0005576). Most of them (65.4%) were actually predicted as secreted via the Sec translocon. Comparative analysis allowed for proteins found in all or only in a subset of L. monocytogenes strains to be defined. While the core exoproteome included most proteins related to bacterial virulence, cell wall biogenesis, as well as proteins secreted by unknown pathways, a slight variation in the protein members of these categories were observed and constituted the variant exoproteome. This investigation resulted in the first definition of the core and variant exoproteomes of L. monocytogenes where corollaries on bacterial physiology are further discussed.