Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50-70 kDa), F2 (Mr:40-60 kDa) and F3 (Mr:10-15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.

On the behaviour of nanoparticles in oil-in-water emulsions with different surfactants.

The distribution of narrowly dispersed gold nanoparticles in hexane-in-water emulsions was studied for different surfactants. Good surfactants such as SDS and Triton X-100 block the oil-water interfaces and confine particles in the droplet. Other surfactants (Tween 85 and Span 20) form synergistic mixtures with the nanoparticles at the interfaces that lower the surface tension more than any component. Supraparticles with fully defined particle distribution form in the droplets only for surfactants that block the interface. Other surfactants promote the formation of fcc agglomerates. Nanoparticles in emulsions behave markedly different from microparticles-their structure formation is governed by free energy minimization, while microparticles are dominated by kinetics.

Early postmortem degradation of actin muscle protein in Algerian Sahraoui dromedaries.

The present study aimed to evaluate actin degradation during the early postmortem time in Longissimus Lumborum muscle according to Sahraoui dromedary's age. A sample of eight males, young (2 years old) and adult (8 years old) dromedaries, was used to investigate meat quality traits and actin proteolysis during the conversion of muscle to meat. Results demonstrated higher pH values in young compared to adult with a polyphasic pH drop profile. While, age did not affect drip loss (DL) and the values at 72 h postmortem varied from 5 to 9%. Western blot revealed that actin proteolysis occurred since 1 h postmortem and that it was affected by age and postmortem time. In particular, the 32 and 25 kDa actin fragments could be potential markers of ongoing meat tenderization.

Calpain 1 binding capacities of the N1-line region of titin are significantly enhanced by physiological concentrations of calcium.

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.

An original SERPINA3 gene cluster: elucidation of genomic organization and gene expression in the Bos taurus 21q24 region.

BACKGROUND: The superfamily of serine proteinase inhibitors (serpins) is involved in numerous fundamental biological processes as inflammation, blood coagulation and apoptosis. Our interest is focused on the SERPINA3 sub-family. The major human plasma protease inhibitor, alpha1-antichymotrypsin, encoded by the SERPINA3 gene, is homologous to genes organized in clusters in several mammalian species. However, although there is a similar genic organization with a high degree of sequence conservation, the reactive-centre-loop domains, which are responsible for the protease specificity, show significant divergences. RESULTS: We provide additional information by analyzing the situation of SERPINA3 in the bovine genome. A cluster of eight genes and one pseudogene sharing a high degree of identity and the same structural organization was characterized. Bovine SERPINA3 genes were localized by radiation hybrid mapping on 21q24 and only spanned over 235 Kilobases. For all these genes, we propose a new nomenclature from SERPINA3-1 to SERPINA3-8. They share approximately 70% of identity with the human SERPINA3 homologue. In the cluster, we described an original sub-group of six members with an unexpected high degree of conservation for the reactive-centre-loop domain, suggesting a similar peptidase inhibitory pattern. Preliminary expression analyses of these bovSERPINA3s showed different tissue-specific patterns and diverse states of glycosylation and phosphorylation. Finally, in the context of phylogenetic analyses, we improved our knowledge on mammalian SERPINAs evolution. CONCLUSION: Our experimental results update data of the bovine genome sequencing, substantially increase the bovSERPINA3 sub-family and enrich the phylogenetic tree of serpins. We provide new opportunities for future investigations to approach the biological functions of this unusual subset of serine proteinase inhibitors.

Proteomic identification of actin-derived oligopeptides in dry-cured ham.

An intense proteolysis of muscle proteins, mainly due to the action of endogenous proteolytic enzymes, has been reported to occur during the processing of dry-cured ham. This gives rise to an important generation of free amino acids and peptides of small size that contribute directly or indirectly to flavor characteristics of the final product. The nature and properties of the free amino acids generated during postmortem proteolysis have been well established. However, little is known about the identity of peptides generated during the processing of dry-cured ham. In the present paper, we describe the isolation (by ethanol precipitation followed by size exclusion and reverse phase chromatographies) and identification (by matrix-assisted laser desorption/ionization time-of-flight MS and collision-induced dissociation MS/MS) in a Spanish dry-cured ham of the following four oligopeptides: (A) TKQEYDEAGPSIVHR, (B) ITKQEYDEAGPSIVHRK, (C) DSGDGVTHNVPIYE, and (D) DSGDGVTHNVPIYEG. This is the first time that these peptide fragments have been reported in dry-cured ham at the end of processing. Sequence homology analysis revealed that the four peptides originated from muscle actin, indicating an extensive hydrolysis of this protein during dry-curing. Some of the cleavage sites corresponding to these fragments in actin were reproduced by other authors through the incubation of this myofibrillar protein in the presence of cathepsin D (EC 3.4.23.5), thus supporting a relevant action of this enzyme during the processing of dry-cured ham.

Purification of the skeletal muscle protein Endopin 1B and characterization of the genes encoding Endopin 1A and 1B isoforms.

In the present work, a new endopin-like serpin designed mEndopin 1B was purified from bovine muscle. Biochemical characterizations (amino acid sequencing and Maldi-Tof mass spectrometry peptide mapping) demonstrated that the purified protein is different from the previously described Endopin 1, renamed mEndopin 1A. The genes and cDNA of both endopins were characterized. The cDNA sequence of mEndopin 1B encodes a predicted protein of 411 amino-acids with a molecular mass of 43808Da. The mEndopin 1B gene comprised four coding exons and an additional 5' untranslated exon. The reactive site sequence of mEndopin 1B is somewhat different from that of mEndopin 1A. Nevertheless, both serpins have a similar peptidase inhibitory pattern against examined proteases (elastase, trypsin, plasmin and chymotrypsin). The high expression of both mEndopin 1A and 1B in bovine serum and tissues and their high efficiency to inhibit elastase (k(ass) approximately 10(6)-10(7) M(-1) s(-1)) suggested that these serpins might play a major role in inflammatory processes.

Muscle endopin 1, a muscle intracellular serpin which strongly inhibits elastase: purification, characterization, cellular localization and tissue distribution.

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 microg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41x10(7) M(-1).s(-1)) and trypsin (kass=3.92x10(6) M(-1).s(-1)), whereas plasmin (kass=1.78x10(3) M(-1).s(-1)) and chymotrypsin (kass=1.0x10(2) M(-1).s(-1)) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.

Revisiting the conversion of muscle into meat and the underlying mechanisms.

The conversion of muscle into meat is a complex process in which all mechanisms responsible for the development of meat qualities are very likely interdependent. Colour and flavour are thus both dependent on oxidative mechanisms. Oxidation and proteolysis are probably two processes involved in the development of meat tenderness. This paper reviewed the consequences of programmed cell death or apoptosis on muscle cells structure and biochemistry and on meat qualities as well. We therefore look at different new hypothesis susceptible to highlight the meat science field and provide new supports for a more dynamic meat research. One of them which would have appeared evident for our purpose since a decade, deals with the fact that, after animal bleeding, muscle cells have no other alternative to only enter the programmed cell death procedure or apoptosis. If we introduce an early phase corresponding to apoptosis, taking place before the rigor onset and overlapping it, we will see that the known consequences of that process bring forward possible answers to still unexplained observations. After an overview of the actual state-of-the-art in meat science, we will introduce the programmed cell death and its underlying mechanisms. We then described the strong analogies between the known consequences of apoptosis and the postmortem changes affecting a set of different muscle characteristics.

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril.

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.

Caspases and Thrombin Activity Regulation by Specific Serpin Inhibitors in Bovine Skeletal Muscle.

In living cells, after activation, protein inhibitors constitute the last step of proteases activity regulation. This review intends to provide original information about a group of bovine muscle serine proteases inhibitors belonging to the Serpin superfamily and characterized at the gene and protein level. This report is the only one and the first to provide much information on this group of proteases inhibitors of the serpin type and their potential biological functions. Amongst the eight genes identified in bovine, three serpins were purified from the muscle tissue and characterized. These are two members of the bovSERPINA3 family, i.e., bovSERPINA3-1 and A3-3, and the last one is antithrombin III (AT-III or BovSERPINC1). BovSERPINA3 family comprises at least eight protein members encoded by different genes mapped on chromosome 7q23-q26 cluster. BovSERPINA3-1 and A3-3 were shown to locate within muscle cells and are cross-class inhibitors strongly active against trypsin as well as against human initiator and effector caspases 8 and 3. They constitute a key apoptosis control in mammals. They were thus expressed in proliferating and confluent myoblasts phases where cells must be alive but not in myotubes. Antithrombin III inhibits trypsin and, in a heparin dependent manner, thrombin. AT-III and its mRNA were expressed in muscle cells and in differentiating primary myoblasts in culture.

Myofibrillar tightly bound calcium in skeletal muscle fibers: a possible role of this cation in titin strands aggregation.

In muscle cells, part of the calcium is tightly bound to the N1- and N2-line of the sarcomere but its physiological significance was unknown. In the present work we reported the ability of a recombinant titin fragment spanning titin domains Z9 to I1 to tightly bind calcium ions with a K(d) of 0.049+/-0.004 nM. We further showed that calcium induced a spontaneous aggregation of the titin fragment and that the major aggregate is a tetramer. The implication of these findings on the organization of the six titin strands that emanate from the end of the thick filament within the I-band is discussed.

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma.

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr approximately 30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8-3.3 and 3.0-3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose-response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r(2) of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319+/-237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.

Calpain 3 is expressed in astrocytes of rat and Microcebus brain.

The calcium-dependent protease calpain is involved in numerous functions, including the control of cell survival, plasticity and motility. Whereas the isoforms calpain 1 and 2 have been described as ubiquitously expressed enzymes, calpain 3 has been called "muscle-specific", although trace amounts of calpain 3 mRNA have been detected by Northern blot in brain homogenates. In this study, we validated antibodies raised either against the peptides that were specific for a given isoform or the peptides present in all the three isoforms. We then used the anti-calpain 3 antibodies together with antibodies directed against cell-type-specific proteins to determine by double- and triple-labelling immunocytochemistry if the protease is expressed in specific cell populations of rat as well as lesser mouse lemur (Microcebus murinus) brain. Calpain 3 was almost exclusively found in cells displaying astrocyte morphology. These cells, most of which co-expressed glial fibrillary acidic protein, were particularly numerous close to the striatal subventricular zone (where numerous neurones forming the rostral migratory stream (RMS) towards the olfactory bulbs are generated) and the RMS itself. Other immunoreactive cells were found close to the pial surface of the forebrain, in the corpus callosum and in the dentate gyrus. Calpain 3 may be involved in astrocyte plasticity and/or motility.

Biomarkers of meat tenderness: present knowledge and perspectives in regards to our current understanding of the mechanisms involved.

Biomarkers of the meat quality are of prime importance for meat industry, which has to satisfy consumers' expectations and, for them, meat tenderness is and will remain the primary and most important quality attribute. The tenderization of meat starts immediately after animal death with the onset of apoptosis followed by a cooperative action of endogenous proteolytic systems. Before consideration of the biomarkers identified so far, we present here some new features on the apoptotic process. Among them, the most important is the recent discovery of a complex family of serpins capable to inhibit, in a pseudo-irreversible manner, caspases, the major enzymes responsible of cell dismantling during apoptosis. The biomarkers so far identified have been then sorted and grouped according to their common biological functions. All of them refer to a series of biological pathways including glycolytic and oxidative energy production, cell detoxification, protease inhibition and production of Heat Shock Proteins. Some unusual biomarkers are also presented: annexins, galectins and peroxiredoxins. On this basis, a detailed analysis of these metabolic pathways allowed us to identify some domains of interest for future investigations. It was thus emphasized that mitochondria, an important organelle in the production of energy from carbohydrates, lipids and proteins are a central element in the initiation and development of apoptosis. It was therefore stressed forward that, in fact, very little is known about the postmortem fate of these organelles and their multiple associated activities. Other topics discussed here would provide avenues for the future in the context of identifying reliable predictors of the ultimate meat tenderness.

Cardiac ankyrin repeat protein is a marker of skeletal muscle pathological remodelling.

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, alpha-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin-proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21(WAF1/CIP1), is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21(WAF1/CIP1) is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

Inhibition of human initiator caspase 8 and effector caspase 3 by cross-class inhibitory bovSERPINA3-1 and A3-3.

Serpins are a superfamily of structurally conserved proteins. Inhibitory serpins use a suicide substrate-like mechanism. Some are able to inhibit cysteine proteases in cross-class inhibition. Here, we demonstrate for the first time the strong inhibition of initiator and effector caspases 3 and 8 by two purified bovine SERPINA3s. SERPINA 3-1 (uniprotkb:Q9TTE1) binds tighly to human CASP3 (uniprotkb:P42574) and CASP8 (uniprotkb:Q14790) with k(ass) of 4.2x10(5) and 1.4x10(6) M(-1)s(-1), respectively. A wholly similar inhibition of human CASP3 and CASP8 by SERPINA3-3 (uniprotkb:Q3ZEJ6) was also observed with k(ass) of 1.5x10(5) and 2.7x10(6) M(-1)s(-1), respectively and form SDS-stable complexes with both caspases. By site-directed mutagenesis of bovSERPINA3-3, we identified Asp(371) as the potential P1 residue for caspases. The ability of other members of this family to inhibit trypsin and caspases was analysed and discussed.

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts.

The bovine PRKAG3 gene encodes the AMPK gamma3 subunit, one isoform of the regulatory gamma subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the gamma subunits have been shown to influence AMPK activity. The gamma3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly influences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3' UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.

The calpain 1-alpha-actinin interaction. Resting complex between the calcium-dependent protease and its target in cytoskeleton.

Calpain 1 behaviour toward cytoskeletal targets was investigated using two alpha-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA [Kd(-Ca2+) = 0.5 +/- 0.1 microM] was improved in the presence of 1 mm calcium ions [Kd(+Ca2+) = 0.05 +/- 0.01 microM]. Location of the binding structures shows that the C-terminal domain of alpha-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III-IV). The in vivo colocalization of calpain 1 and alpha-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and alpha-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations.