In Vitro Proteolysis of Myofibrillar Proteins from Beef Skeletal Muscle by Caspase-3 and Caspase-6

2011 ◽  
Vol 59 (17) ◽  
pp. 9658-9663 ◽  
Author(s):  
Feng Huang ◽  
Ming Huang ◽  
Guanghong Zhou ◽  
Xinglian Xu ◽  
Mei Xue
Keyword(s):  
Skeletal Muscle ◽  
Caspase 3 ◽  
Myofibrillar Proteins ◽  
Caspase 6

The effect of recombinant caspase 3 on porcine myofibrillar proteins

2007 ◽  
Vol 2007 ◽  
pp. 112-112
Author(s):  
CM. Kemp ◽  
T. Parr
Keyword(s):  
Skeletal Muscle ◽  
Cell Death ◽  
Preliminary Data ◽  
Shear Force ◽  
Caspase 3 ◽  
Proteolytic Enzymes ◽  
Myofibrillar Proteins ◽  
Caspase Activity ◽  
Conditioning Period

Meat tenderisation results from the weakening of the myofibrillar structures and has been attributed to endogenous proteolytic enzymes. It has been proposed that tenderization is a mulitenzymatic system and the process of slaughter and exsanguination would engage muscle cells in a form of cell death (Ouali et al., 2006). Caspases are primarily associated with apoptosis and once activated they target and cleave a number of substrates including components of the Z-disk and costameres. Recent studies have shown that caspases are active in skeletal muscle during the postmortem conditioning period and our preliminary data indicates that there is a relationship between caspase activity and shear force (Kemp et al., 2006). The aim of this study was to investigate whether recombinant caspase 3 was capable of porcine degrading myofibrillar proteins in vitro.

Effect of Oxidation on In Vitro Digestibility of Skeletal Muscle Myofibrillar Proteins

2007 ◽  
Vol 55 (13) ◽  
pp. 5343-5348 ◽  
Author(s):  
Veronique Sante-Lhoutellier ◽  
Laurent Aubry ◽  
Philippe Gatellier
Keyword(s):  
Skeletal Muscle ◽  
In Vitro Digestibility ◽  
Myofibrillar Proteins

Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro: implications for microfilament reorganization during apoptosis

1999 ◽  
Vol 112 (23) ◽  
pp. 4475-4482 ◽  
Author(s):  
A. Sgorbissa ◽  
R. Benetti ◽  
S. Marzinotto ◽  
C. Schneider ◽  
C. Brancolini
Keyword(s):  
Caspase 3 ◽  
Early Stage ◽  
Proteolytic Processing ◽  
Caspase 7 ◽  
Microfilament System ◽  
Caspase 6 ◽  
Shape Changes ◽  
Mcf 7

Apoptosis is characterized by proteolysis of specific cellular proteins by a family of cystein proteases known as caspases. Gas2, a component of the microfilament system, is cleaved during apoptosis and the cleaved form specifically regulates microfilaments and cell shape changes. We now demonstrate that Gas2 is a substrate of caspase-3 but not of caspase-6. Proteolytic processing both in vitro and in vivo is dependent on aspartic residue 279. Gas2 cleavage was only partially impaired in apoptotic MCF-7 cells which lack caspase-3, thus indicating that different caspases can process Gas2 in vivo. In vitro Gas2 was processed, albeit with low affinity, by caspase-7 thus suggesting that this caspase could be responsible for the incomplete Gas2 processing observed in UV treated MCF-7 cells. In vivo proteolysis of Gas2 was detected at an early stage of the apoptotic process when the cells are still adherent on the substrate and it was coupled to the specific rearrangement of the microfilament characterizing cell death. Finally we also demonstrated that Gas2 in vitro binds to F-actin, but this interaction was unaffected by the caspase-3 dependent proteolytic processing.

scholarly journals Selective Cleavage of BLM, the Bloom Syndrome Protein, during Apoptotic Cell Death

2001 ◽  
Vol 276 (15) ◽  
pp. 12068-12075 ◽  
Author(s):  
Oliver Bischof ◽  
Sanjeev Galande ◽  
Farzin Farzaneh ◽  
Terumi Kohwi-Shigematsu ◽  
Judith Campisi
Keyword(s):  
Caspase 3 ◽  
Apoptotic Cell ◽  
Autosomal Recessive Disorder ◽  
Bloom Syndrome ◽  
Recognition Sequence ◽  
Blm Helicase ◽  
Caspase 6 ◽  
Syndrome Protein ◽  
Condensed Dna

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6,in vitro. The caspase 3 recognition sequence412TEVD415was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.

Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure

2007 ◽  
Vol 292 (2) ◽  
pp. H1009-H1017 ◽  
Author(s):  
Catherine Coirault ◽  
Aziz Guellich ◽  
Thomas Barbry ◽  
Jane Lise Samuel ◽  
Bruno Riou ◽  
...  
Keyword(s):  
Heart Failure ◽  
Skeletal Muscle ◽  
Chronic Heart Failure ◽  
Oxidative Damage ◽  
Caspase 3 ◽  
Mechanical Performance ◽  
Molecular Motor ◽  
Fast Myosin ◽  
Skeletal Muscle Dysfunction

Intrinsic muscle abnormalities affecting skeletal muscle are often reported during chronic heart failure (CHF). Because myosin is the molecular motor of force generation, we sought to determine whether its dysfunction contributes to skeletal muscle weakness in CHF and, if so, to identify the underlying causative factors. Severe CHF was induced in rats by aortic stenosis. In diaphragm and soleus muscles, we investigated in vitro mechanical performance, myosin-based actin filament motility, myosin heavy (MHC) and light (MLC) chain isoform compositions, MLC integrity, caspase-3 activation, and oxidative damage. Diaphragm and soleus muscles from CHF exhibited depressed mechanical performance. Myosin sliding velocities were 16 and 20% slower in CHF than in sham in diaphragm (1.9 ± 0.1 vs. 1.6 ± 0.1 μm/s) and soleus (0.6 ± 0.1 vs. 0.5 ± 0.1 μm/s), respectively (each P < 0.05). The ratio of slow-to-fast myosin isoform did not differ between sham and CHF. Immunoblots with anti-MLC antibodies did not detect the presence of protein fragments, and no activation of caspase-3 was evidenced. Immunolabeling revealed oxidative damage in CHF muscles, and MHC was the main oxidized protein. Lipid peroxidation and expression of oxidized MHC were significantly higher in CHF than in shams. In vitro myosin exposure to increasing ONOO− concentrations was associated with an increasing amount of oxidized MHC and a reduced myosin velocity. These data provide experimental evidence that intrinsic myosin dysfunction occurs in CHF and may be related to oxidative damage to myosin.

Abstract 197: Emerging Role of Toll-like Receptor 4 and Heat Shock Protein 60&70 in Ischaemia-Induced Skeletal Muscle Damage In Vitro

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Ali Navi ◽  
Rebekah Yu ◽  
Xu Shi-Wen ◽  
Sidney Shaw ◽  
Daryll Baker ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Inflammatory Cytokine ◽  
Caspase 3 ◽  
Neutralizing Antibody ◽  
Cytokine Release ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Skeletal Muscle Damage ◽  
Tnf Α

OBJECTIVES The innate immune response contributes to the skeletal muscle damage in patients with critical limb ischaemia (CLI); however, the detailed signaling mechanisms are not fully understood. We hypothesized that simulated ischaemia induces inflammatory cytokine release from skeletal myotubes, via a mechanism that involves heat shock protein (HSP) 60&70, known endogenous ligands of Toll-like receptor 4 (TLR4), in vitro. METHODS Human gastrocnemius muscle biopsies were taken from patients with CLI undergoing major lower limb amputation and from patients with no peripheral arterial disease (PAD). Human myoblasts were isolated, cultured to myotubes and then pre-treated with TLR4 neutralizing antibody prior to exposure to simulated ischaemia. Fluorescent immunostaining was carried out to confirm cell differentiation; ELISA analysis were carried out to quantify IL6 and TNF-α release; and Western blot was used to assess expression of HSP60&70, TLR4 and cleaved caspase-3 as a marker of apoptosis. RESULTS Myotubes from patients with CLI expressed greater levels of cleaved caspase-3 and TLR4 as compared to those from patients with no PAD. When exposed to ischaemic conditions, increased IL6 and TNF-α release and upregulation of HSP60&70, cleaved caspase-3 and TLR4 were observed in myotubes from both groups of patients compared to culturing in normoxic conditions (P<0.05). Pre-treatment of myotubes from patients with CLI with TLR4 neutralizing antibody prior to simulated ischaemia was associated with reduced expression of HSP60&70, IL6, TNF-α and cleaved caspase-3 (P<0.05). CONCLUSIONS Increased cytokine release, apoptosis and expression of HSP60&70 and TLR4 occur in ischaemic skeletal muscle in vitro. TLR4 antagonism was associated with reduced apoptosis and inflammatory cytokine release and down-regulation of HSP60&70 expression. This suggests a potential pathway where TLR4 and its endogenous ligands contribute to a positive feedback loop to maintain a proinflammatory environment during ischaemia.

scholarly journals Apoptosis in skeletal muscle with aging

2002 ◽  
Vol 282 (2) ◽  
pp. R519-R527 ◽  
Author(s):  
Amie Dirks ◽  
Christiaan Leeuwenburgh
Keyword(s):  
Skeletal Muscle ◽  
Cytochrome C ◽  
Gastrocnemius Muscle ◽  
Caspase 3 ◽  
Age Related ◽  
Fiber Number ◽  
Old Rats ◽  
Cytosolic Cytochrome

Sarcopenia may be partly due to a loss in total fiber number by apoptosis. We have investigated age-related alterations in the mitochondria-mediated pathway leading to apoptosis in the gastrocnemius muscle from 6-mo-old and 24-mo-old male Fisher 344 rats. Apoptosis (mono- and oligonucleosome fragmentation) in the gastrocnemius muscle was increased by 50% in the old rats compared with the adult animals. Furthermore, there was a significant correlation between cytosolic cytochrome c and caspase-3 activity, although neither cytochrome c nor caspase-3 activity increased significantly with age. Furthermore, there was a significant correlation between caspase-3 activity and mono- and oligonucleosome fragmentation in the old rats only. Mitochondrial Bcl-2 and Bax were not altered with age. In vitro experiments demonstrated that activation of the caspase cascade in skeletal muscle might be limited by procaspase-9 activation. This is the first study to explore the role of apoptosis in sarcopenia and suggests that subtle changes in apoptosis are involved.

scholarly journals alphaII-Spectrin is an in vitro target for caspase-2, and its cleavage is regulated by calmodulin binding

2004 ◽  
Vol 378 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Björn ROTTER ◽  
Yolande KROVIARSKI ◽  
Gaël NICOLAS ◽  
Didier DHERMY ◽  
Marie-Christine LECOMTE
Keyword(s):  
Cell Shape ◽  
Caspase 3 ◽  
Membrane Skeleton ◽  
Calmodulin Binding ◽  
Caspase 7 ◽  
Caspase 6 ◽  
Caspase 2 ◽  
The Stability ◽  
Main Components

The spectrin–actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains. These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape. Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold. It is also one of the major processes involved in membrane remodelling. Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and µ-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis). In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases. Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to αII-spectrin, while the short isoform does not. Like caspase-3, caspase-2 cleaves αII-spectrin in vitro at residue Asp-1185. This study emphasizes a role of executioner caspase for caspase-2. We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro. This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin. In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176. These results indicate that αII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton.

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