Myosin Heavy Chain Fragments Research Articles

Skeletal muscle myosin heavy chain protein fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We sought to examine how resistance exercise (RE), cycling exercise, and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation in humans. In the first study (1boutRE), younger adult men (n=8; 5±2 years of RE experience) performed a lower body RE bout with vastus lateralis (VL) biopsies obtained immediately before, 3-, and 6-hours post-exercise. In the second study (10weekRT), VL biopsies were obtained in untrained younger adults (n=36, 18 men and 18 women) before and 24 hours (24h) after their first/naïve RE bout. These participants also engaged in 10 weeks (24 sessions) of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined from a third acute cycling study (n=7) and a fourth study involving two weeks of leg immobilization (n=20, 15 men and 5 women) to determine how MyHC fragmentation was affected. In the 1boutRE study, the fragmentation of all MyHC isoforms (MyHCTotal) increased 3 hours post-RE (~ +200%, p=0.018) and returned to pre-exercise levels by 6 hours post-RE. Immunoprecipitation of MyHCTotal revealed ubiquitination levels remained unaffected at the 3- and 6-hour post-RE time points. Interestingly, a greater increase in magnitude for MyHC type IIa versus I isoform fragmentation occurred 3-hours post-RE (8.6±6.3-fold versus 2.1±0.7-fold, p=0.018). In all 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, respectively; p<0.001); however, the last RE bout response was attenuated compared to the first bout (p=0.045). The first/naïve bout response was significantly elevated in females only (p<0.001), albeit females also demonstrated a last bout attenuation response (p=0.002). Although an acute cycling bout did not alter MyHCTotal fragmentation, ~8% VL atrophy with two weeks of leg immobilization led to robust MyHCTotal fragmentation (+108%, p<0.001), and no sex-based differences were observed. In summary, RE and disuse atrophy increase MyHC protein fragmentation. A dampened response with 10 weeks of resistance training, and more refined responses in well-trained men, suggest this is an adaptive process. Given the null polyubiquitination IP findings, more research is needed to determine how MyHC fragments are processed. Moreover, further research is needed to determine how aging and disease-associated muscle atrophy affect these outcomes, and whether MyHC fragmentation is a viable surrogate for muscle protein turnover rates.

Exacerbation of autoimmune myocarditis by an immune checkpoint inhibitor is dependent on its time of administration in mice

BackgroundAlthough immune checkpoint inhibitors (ICIs) have made an immense breakthrough in cancer therapeutics, they can exert unique, immune-related adverse events. Among them, myocarditis is less frequent, but it is serious and often follows a lethal course. MethodsTo examine the changes in cardiac autoimmunity after ICI administration, we developed a mouse experimental autoimmune myocarditis (EAM) model via intraperitoneal administration of murine α-cardiac myosin heavy chain (MyHC-α) fragment. Thereafter, the mouse anti-PD-1 antibody (mPD1ab) was administered at two time points, subsequent to and concurrent with MyHC-α fragment administration. ResultsSevere EAM developed in 3 weeks; wide inflammatory lesions were observed in the cardiac sections. Furthermore, inflammatory/fibrotic genes, such as interleukin 1β, interleukin 6, and collagen 1, were upregulated, although the cardiac function was not significantly affected. The subsequent administration of mPD1ab at 2 weeks post administration of the first MyHC-α fragment exacerbated EAM, whereas the administration of mPD1ab concurrent with MyHC-α fragment administration did not exacerbate EAM. The subsequent administration of mPD1ab significantly increased the infiltration of cluster of differentiation (CD)4- and F4/80-positive cells, whereas the concurrent administration of mPD1ab significantly decreased the infiltration of CD4-positive cells, indicating that the concurrent and subsequent administration of mPD1ab had opposite effects on immune/inflammatory cell infiltration. ConclusionsThese data suggest that the appearance of ICI-induced autoimmune myocarditis might be related to autoimmune system activity before ICI administration. Although ICIs do not adversely affect patients with normal immune systems, we propose that ICI administration should be avoided in patients with autoimmune disorders.

Contribution and Value of Biomarkers in Acute Aortic Syndromes.

Acute aortic syndromes, being mostly underdiagnosed due to unspecific symptoms, are associated with high morbidity and mortality. Diagnosis carried out by transesophageal echocardiography, computed tomography and magnetic resonance imaging. However, there are lots of biochemical assays being investigated, but none of them used reliably to identify acute aortic syndromes. Biomarkers could accelerate the diagnostic time with cost effective way and could get place in definitive diagnostic algorithm of acute aortic syndrome. This review aims to identify contribution and value of biomarkers in acute aortic syndromes. We searched the contribution and value of biomarkers such as D - Dimer, Smooth muscle myosin heavy chain (sm - MHC), Calponin, and Soluble elastin fragments (sELAF) in acute aortic syndromes at the literature. Twenty two actual papers were included in this review for searching the contribution and value of biomarkers in the acute aortic syndromes. Biomarkers accelerate the diagnosis and direct patients to imaging modalities with a risk classification. Plenty of biomarkers have been investigated so far but none of them were used in clinical routine. Currently none of the biomarkers can reliably identify acute aortic syndromes. Each of them has some limitation in term of sensitivity or specificity. Although, there is no single biomarker that can be safely used but a combination of the assays may increase the sensitivities and specificities.

Proteolytic resistance of actin but not of myosin heavy chain during processing of Italian PDO (protected designation of origin) dry-cured hams

Proteomics is the approach of choice to study the fate of specific proteins, and it is very useful in identifying quality molecular markers. A combination of immunochemical and mass spectrometry analysis was used to assess the occurrence of proteolytic changes of actin and myosin heavy chain (MHC) proteins in pig biceps femoris skeletal muscle during processing of three Italian PDO dry-cured hams. Early post-mortem muscle displayed low levels of actin and myosin fragments. In spite of a high proteolysis index and the presence of active cathepsin D until the final ripening phase, during dry-cured ham processing, very low actin proteolysis and no generation of fragments from α-skeletal muscle isoform were found, while the identified fragments derived mainly from the cardiac actin isoform. On the other hand, MHC showed a remarkable degradation of its catalytic head, generating a C-terminal 135-kDa fragment. Based on its ability to interact with actin in vitro, this MHC fragment might have a role in stabilisation of actin. In conclusion, these results suggest that maintenance of skeletal muscle α-actin could reflect limited dismantling of the sarcomeric structure and be a useful marker to monitor the events that result in the typical texture of dry-cured ham.

Phosphorylation Modulates the Dynamics of the N-Terminal Tail in Cardiac RLC

Background: Phosphorylation of the Regulatory Light Chain (RLC) in cardiac myosin has been shown to be critical for the normal heart performance. In particular, different cardiomyopathy-related mutations have been found to be associated with decreased levels of RLC phosphorylation. RLC is part of the myosin lever arm, whose power stroke is the result of the amplification of conformational changes occurring in the motor domain during the contractile cycle. Several findings indicate that RLC phosphorylation can have significant effects on myosin mechanics. However, the associated molecular events are still unknown.Methods and Results: Here, we use computational methods to model the dynamics of human cardiac RLC bound to a fragment of myosin heavy chain (MyBS). Molecular Dynamics simulations are used to explore the space of conformations accessible to the N-terminal tail, which contains the RLC phosphorylation site. The conformational free energy landscape is reconstructed and the dynamic behaviour is analysed in terms of probability distributions of key residues of the tail around RLC and MyBS. Remarkably, simulations of phosphorylated RLC show significantly modified tail distributions, associated with a shifting in the population of the preferred tail conformations.Results from simulations are compared with experimental NMR data and the effects of mutations implicated in the pathogenesis of hypertrophic cardiomyopathy (HCM) are discussed.Conclusions: Molecular Dynamics simulations of cardiac RLC reveal phosphorylation-induced changes in the dynamics of the N-terminal tail and in its interaction with the rest of the molecule. These modifications can potentially regulate the interactions between RLC and other myosin components and interactors.This research is supported by the British Heart Foundation.

Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion

Ischemia/reperfusion (I/R) injury is a major consequence of a cardiovascular intervention. The study of changes of the left and right ventricle proteomes from hearts subjected to I/R may be a key to revealing the pathological mechanisms underlying I/R-induced heart contractile dysfunction. Isolated rat hearts were perfused under aerobic conditions or subjected to 25 min global ischemia and 30 min reperfusion. At the end of perfusion, right and left ventricular homogenates were analyzed by 2DE. Contractile function and coronary flow were significantly reduced by I/R. 2DE followed by mass spectrometry identified ten protein spots whose levels were significantly different between aerobic left and right ventricles, eight protein spots whose levels were different between aerobic and I/R left ventricle, ten protein spots whose levels were different between aerobic and I/R right ventricle ten protein spots whose levels were different between the I/R groups. Among these protein spots were ATP synthase beta subunit, myosin light chain 2, myosin heavy chain fragments, peroxiredoxin-2, and heat shock proteins, previously associated with cardiovascular disease. These results reveal differences between proteomes of left and right ventricle both under aerobic conditions and in response to I/R that contribute to a better understanding of I/R injury.

Asymmetric Mode of Ca2+-S100A4 Interaction with Nonmuscle Myosin IIA Generates Nanomolar Affinity Required for Filament Remodeling

SummaryFilament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca2+-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca2+-dependent binding sites of each subunit in the dimer. Synergy between these sites leads to a very tight association (KD ∼1 nM) that is unique in the S100 family. Single-residue mutations that remove this synergy weaken binding and ameliorate the effects of S100A4 on NMIIA filament assembly and cell spreading in A431 human epithelial carcinoma cells. We propose a model for NMIIA filament disassembly by S100A4 in which initial binding to the unstructured NMIIA tail initiates unzipping of the coiled coil and disruption of filament packing.

Crystal structure of the S100A4–nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction.

Protein nitration is associated with increased proteolysis in skeletal muscle of bile duct ligation–induced cirrhotic rats

Cirrhosis is characterized by skeletal muscle wasting. In this study, the effects of nitric oxide production on skeletal muscle protein nitration and degradation in cirrhosis were investigated. Cirrhosis was induced by bile duct ligation (BDL) in Sprague-Dawley rats for 4 weeks. The BDL-induced cirrhotic rats and sham-operated rats were then injected daily with either saline or N G - l-nitro-arginine methyl ester ( l-NAME) for 7 days from week 4 to week 5, after which nitrite/nitrate, glutathione reduction, as well as protein nitration, ubiquitination, and degradation were assessed in skeletal muscle. Elevated muscular nitrite/nitrate concentrations, protein nitration, total ubiquitin conjugates, and degradation fragments of myosin heavy chain as well as diminished glutathione reduction levels were observed in BDL-induced cirrhotic rats as compared with controls. Administration of l-NAME for 1 week led to reduction of nitrite/nitrate levels; protein nitration was also decreased in the skeletal muscle. In addition, ubiquitination of muscular proteins and degradation of myosin heavy chain were significantly diminished after treatment of l-NAME. In conclusion, nitrosative stress occurred in the skeletal muscle of BDL-induced cirrhotic rats and may lead to increased proteolysis of muscle-specific structural proteins.

Counterpoint: Estrogen and Sex do not Significantly Influence Post-Exercise Indexes of Muscle Damage, Inflammation, and Repair

Exercise-induced muscle damage (EIMD) occurs following strenuous, novel exercise and is assessed directly via tissue analysis and characterized indirectly by muscle dysfunction, inflammation, delayed onset muscle soreness, and increased levels of muscle proteins [e.g., creatine kinase (CK),

Pathophysiology, diagnosis, and management of aortic dissection

Aortic dissection is an uncommon but potentially fatal disease with catastrophic complications. A high level of suspicion is required for successful diagnosis as presenting symptoms are so variable that dissection may be overlooked in up to 39% of cases. It most commonly presents in the elderly population with a history of chronic hypertension. Rapid intervention is necessary as delay leads to higher mortality. Despite advances in diagnostic and therapeutic techniques, morbidity and mortality remains high. Advances in diagnostic imaging have raised the awareness of variants of aortic dissection, including intramural hemorrhage and penetrating aortic ulcer. This distinction is important as the clinical course of these variants differs from that of classical aortic dissection, and thus treatment may also differ. Understanding of these variants has also led to the recognition of markers that may help predict progression to classical aortic dissection and thus warrant closer vigilance in selected patient populations. The recognition that rapid diagnosis is required for management of aortic dissection has led to the investigation of serum tests as diagnostic aids. Serum smooth muscle myosin heavy chain, d-dimer, and serum soluble elastin fragments are promising tests that may help raise suspicion for the diagnosis of acute aortic dissection. The high mortality associated with surgical therapy has led to investigation of alternative approaches. Endovascular therapy has emerged as a viable option in patients with type B dissection who are too unstable for surgery. However, long-term follow up is required to validate this procedure.

Abnormal cytoskeletal protein expression in cultured skin fibroblasts from type 1 diabetes mellitus patients with nephropathy: A proteomic approach

Diabetic nephropathy (DN) develops in about 40% of insulin-dependent type 1 diabetes mellitus (T1DM) patients, and is associated not only with diabetes duration and metabolic control, but also with a genetic predisposition. Constitutive alterations of cytoskeletal proteins may play a role in the development of DN. We investigated the expression of these proteins in cultured skin fibroblasts, obtained from long-term T1DM patients with and without DN but comparable metabolic control, and from matched healthy subjects, by means of 2-DE electrophoresis and MS-MALDI analyses. In T1DM with DN, compared to the other two groups, quantitative analyses revealed an altered expression of 17 spots (p<0.05-p<0.01), corresponding to 12 unique proteins. In T1DM with DN, beta-actin and three isoforms of tubulin beta-2 chain, tropomodulin-3, and LASP-1 were decreased, whereas two tubulin beta-4 chain isoforms, one alpha actinin-4 isoform, membrane-organizing extension spike protein (MOESIN), FLJ00279 (corresponding to a fragment of myosin heavy chain, non-muscle type A), vinculin, a tropomyosin isoform, and the macrophage capping protein were increased. A shift in caldesmon isoforms was also detected. These results demonstrate an association between DN and the constitutive expression of cytoskeleton proteins in cultured skin fibroblasts from T1DM with DN, which may retain pathophysiologycal implications.

Parasites, proteomics and performance: effects of gregarine gut parasites on dragonfly flight muscle composition and function

In previous work, we found that dragonflies infected with gregarine gut parasites have reduced muscle power output, loss of lipid oxidation in their flight muscles, and a suite of symptoms similar to mammalian metabolic syndrome. Here, we test the hypothesis that changes in muscle protein composition underlie the observed changes in contractile performance. We found that gregarine infection was associated with a 10-fold average reduction in abundance of a approximately 155 kDa fragment of muscle myosin heavy chain (MHC; approximately 206 kDa intact size). Insect MHC gene sequences contain evolutionarily conserved amino acid motifs predicted for calpain cleavage, and we found that calpain digestion of purified dragonfly MHC produced a peptide of approximately 155 kDa. Thus, gut parasites in dragonflies are associated with what appears to be a reduction in proteolytic degradation of MHC. MHC155 abundance showed a strong negative relationship to muscle power output in healthy dragonflies but either no relationship or a weakly positive relationship in infected dragonflies. Troponin T (TnT) protein isoform profiles were not significantly different between healthy and infected dragonflies but whereas TnT isoform profile was correlated with power output in healthy dragonflies, there was no such correlation in infected dragonflies. Multivariate analyses of power output based on MHC155 abundance and a principal component of TnT protein isoform abundances explained 98% of the variation in muscle power output in healthy dragonflies but only 29% when data from healthy and infected dragonflies were pooled. These results indicate that important, yet largely unexplored, functional relationships exist between (pathways regulating) myofibrillar protein expression and (post-translational) protein processing. Moreover, infection by protozoan parasites of the midgut is associated with changes in muscle protein composition (i.e. across body compartments) that, either alone or in combination with other unmeasured changes, alter muscle contractile performance.

Targeted Rearrangement of Floxed Alleles in Smooth Muscle Cells in Vivo

To the Editor: In their biologically and technically important article “Development of a Smooth Muscle-targeted Cre Recombinase Mouse Reveals Novel Insights Regarding Smooth Muscle Myosin Heavy Chain Promoter Regulation”,1 Regan et al reported generation of mice that express cre recombinase from a fragment of the smooth muscle myosin heavy chain (SMMHC) promoter. The biological importance of this article was that it provided strong evidence of either temporally or spatially restricted expression of the SMMHC promoter in smooth muscle cells in vivo. The paper also had two important technical aspects. First, it demonstrated that expression of a SMMHC promoter-cre construct in cre-indicator mice was a more sensitive means of detecting promoter activity than a construct in which the SMMHC promoter expressed lacZ. Second, the SMMHC-cre mice themselves were said to “provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth-muscle-specific gene activation or inactivation.” Although the SMMHC-cre mice reported by Regan et al have been used by one group for gene activation,2,3 we—as well as other groups that shared their results with us4 …

Modifications of Trout (Oncorhynchus mykiss) Muscle Proteins by Preslaughter Activity

The effect of two different preslaughter procedures (limited or 15-min intense muscular activity) on muscle trout proteins was investigated. Muscle was sampled 45 min and 24 h post-mortem, proteins were separated using two-dimensional electrophoresis, and spots of interest were tentatively identified by MALDI-TOF spectrometry. Twenty-nine and 4 spots were differentially represented between the two groups of fish at 45 min and 24 h post-mortem, respectively. Spots that could be identified corresponded mainly to proteins involved in energy-producing pathways (triosephosphate isomerase, enolase, pyruvate dehydrogenase) or to structural proteins (desmin, cap-Z, myosin heavy chain fragment). Persistent under-representation of desmin, a key cytoskeletal protein, in fish submitted to intense muscular activity suggests that such a preslaughter treatment can have an effect on post-mortem muscle integrity.

Bispecific Enzyme-Linked Signal-Enhanced Immunoassay with Subattomole Sensitivity

A bispecific enzyme-linked signal-enhanced immunoassay (BiELSIA) was developed with markedly increased sensitivity. Antimyosin, the detection antibody, was linked to the signal probespecific antibody. Probes consisted of diethylenetriamine pentaacetic acids attached to polylysine modified with up to seven or eight horseradish peroxidase (HRP) units. Each bispecific antibody bound two polymer probes, providing twice the signal. Using BiELSIA in a competitive inhibition immunoassay format with an average of 1.5, 3, 4.5, 6, and 7.5 HRP units per polymer-probe, the sensitivity of standard enzyme-linked immunosorbent assay (10(13) mole) was increased to 10(15), 10(18), 10(19), 10(20), and 10(-21) mol (< or = 1,000 molecules), respectively. BiELSIA detected cardiac myosin heavy chain fragments in sera of patients obtained at the time of emergency department admission for acute myocardial infarction, but not in normal sera. This technology should be applicable for detection of cancer, human immunodeficiency virus, prion, and other antigens that are present in concentrations too low for detection by current immunoassays.

Proteome Changes In Rat Heart Induced By Exercise Training

PURPOSE Chronic exercise training elicits adaptations in the heart that improve pump function and confer cardioprotection. To identify molecular mechanisms by which exercise training stimulates this favorable phenotype, a proteomic approach was employed to test the hypothesis that rat cardiac proteins would be differentially expressed or modified after exercise training. METHODS Exercise-trained rats (n=8) underwent six weeks of progressive treadmill training, five days per week, with high intensity sprints interspersed periodically with running at a moderate speed. Sedentary control rats (n=8) were age-matched and weight-matched to exercise-trained rats. Hearts were harvested and quick-frozen in liquid nitrogen at various times (0–72 hr) after the last bout of exercise and were subsequently used to generate 2-dimensional electrophoretic (2-DE) proteome maps. PDQuest software was used to analyze 2-DE gels, quantify spot densities, and compare expression levels between groups. Protein spots were identified with mass spectrometry of trypsin digests. RESULTS Exercise-trained rats exhibited a 14% greater left ventricular weight-to-body weight ratio than sedentary rats (p < 0.05). Compared with hearts of sedentary rats, 26 protein spot intensities were significantly altered in hearts of exercise-trained rats (p < 0.05), and 15 additional spots appeared exclusively on gels from the exercise-trained rats. Among candidate proteins with increased levels of expression in hearts of exercise-trained rats were a heat shock protein, a mitochondrial elongation factor, a proteasome subunit, desmin, and two steroid modifying enzymes. Proteins that exhibited decreased expression levels in the hearts of exercise-trained rats included a mitochondrial ATP synthase and a myosin heavy chain fragment. CONCLUSIONS Exercise training alters the cardiac proteome of the rat. The proteome changes include several candidate proteins that require further study to determine their role in the adaptation of the heart to exercise training. Supported by American Heart Association Midwest Affiliate and NIH R03 AG022625.

Proteome Changes In Rat Heart Induced By Exercise Training

PURPOSE Chronic exercise training elicits adaptations in the heart that improve pump function and confer cardioprotection. To identify molecular mechanisms by which exercise training stimulates this favorable phenotype, a proteomic approach was employed to test the hypothesis that rat cardiac proteins would be differentially expressed or modified after exercise training. METHODS Exercise-trained rats (n=8) underwent six weeks of progressive treadmill training, five days per week, with high intensity sprints interspersed periodically with running at a moderate speed. Sedentary control rats (n=8) were age-matched and weight-matched to exercise-trained rats. Hearts were harvested and quick-frozen in liquid nitrogen at various times (0–72 hr) after the last bout of exercise and were subsequently used to generate 2-dimensional electrophoretic (2-DE) proteome maps. PDQuest software was used to analyze 2-DE gels, quantify spot densities, and compare expression levels between groups. Protein spots were identified with mass spectrometry of trypsin digests. RESULTS Exercise-trained rats exhibited a 14% greater left ventricular weight-to-body weight ratio than sedentary rats (p < 0.05). Compared with hearts of sedentary rats, 26 protein spot intensities were significantly altered in hearts of exercise-trained rats (p < 0.05), and 15 additional spots appeared exclusively on gels from the exercise-trained rats. Among candidate proteins with increased levels of expression in hearts of exercise-trained rats were a heat shock protein, a mitochondrial elongation factor, a proteasome subunit, desmin, and two steroid modifying enzymes. Proteins that exhibited decreased expression levels in the hearts of exercise-trained rats included a mitochondrial ATP synthase and a myosin heavy chain fragment. CONCLUSIONS Exercise training alters the cardiac proteome of the rat. The proteome changes include several candidate proteins that require further study to determine their role in the adaptation of the heart to exercise training. Supported by American Heart Association Midwest Affiliate and NIH R03 AG022625.

Proteomic analysis of water soluble and myofibrillar protein changes occurring in dry-cured hams

The myofibrillar fraction of raw ham muscles and dry-cured hams with different ripening times was extracted in denaturing and reducing conditions and subjected to two-dimensional gel electrophoresis. The two-dimensional maps gave overall pictures of the already noted progressive disappearance of actin, tropomyosin and myosin light chains during ripening. In addition, two fragments from Myosin Heavy Chain proteolysis, marked as myosin chain fragments MCF1 and MCF2, were identified by immunodetection and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Furthermore, a new form of actin on two-dimensional gel was identified by MALDI-TOF peptide mapping. In 12-month-old dry-cured ham, most myofibrillar proteins were completely hydrolyzed. At this stage of ripening, in fact, in some Parma and S. Daniele dry-cured ham samples, myosin heavy chain fragments and other unidentified neo-formed spots were found. Some of the sarcoplasmic proteins in water extracts from pork meat markedly decreased in amount or disappeared totally, during ripening. Surprisingly, two-dimensional gel electrophoresis maps of the water soluble protein fraction from dry-cured ham showed the presence of two spots identified as tropomyosin α- and β-chain. This result suggests that some of the saline soluble myofibrillar proteins can disappear from this fraction because of salt solubilization and not due to complete enzyme action. Two-dimensional gel electrophoresis (2-DGE) has proved a powerful tool to evaluate the enzymatic susceptibility of meat proteins and the evolution of protein map fragmentation throughout ripening process as well as a means of obtaining a standard fingerprinting map characterizing the final product.

Interaction of metastasis-inducing S100A4 protein in vivo by fluorescence lifetime imaging microscopy.

Elevated levels of the calcium-binding regulatory protein, S100A4, have been shown to be causative of a metastatic phenotype in models of cancer metastasis and to be associated with reduced patient survival in breast cancer patients. Recombinant S100A4 protein interacts in vitro in a calcium-dependent manner with the heavy chain of non-muscle myosin isoform A at a protein kinase C phosphorylation site. At present, the mechanism of metastasis induction by S100A4 in vivo is almost completely unknown. The binding of S100A4 to a C-terminal recombinant fragment of non-muscle myosin heavy chain in living HeLa cells has now been shown using confocal microscopy, fluorescence lifetime imaging microscopy and time-correlated single-photon counting. The association between S100A4 and non-muscle myosin heavy chain was studied by determining fluorescence resonance energy transfer-derived changes in the fluorescence lifetime of enhanced cyan fluorescent protein fused to S100A4 in the presence of a recombinant fragment of the C-terminal region of non-muscle myosin heavy chain (rNMMHCIIA) fused to enhanced yellow fluorescent protein. There was no interaction between the non-muscle myosin heavy chain fragment and a calcium-binding-deficient mutant of S100A4 protein which has been shown to be defective in the induction of metastasis in model systems in vivo. The results demonstrate, for the first time, not only direct interaction between S100A4 and a target rNMMHCIIA in live mammalian cells, but also that the interaction between S100A4 and the non-muscle myosin heavy chain in vivo could contribute to the mechanism of metastasis induction by a high level of S100A4 protein.