Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.007
Filtrar
1.
Food Chem ; 340: 127914, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32889207

RESUMO

The objective of this study was to investigate the influence of oxidation on heat shock protein 27 (HSP27) and cytochrome c translocation, myofibrils degradation and endogenous enzymes activities, perfecting tenderization mechanism after slaughter. Bovine muscle (longissimus thoracis) was obtained at 30 min postmortem. Bovine muscle was cut and exposed to saline solution with or without H2O2 at 4 °C for 0.25, 1, 3 and 5 days, followed by detection of proteins degradation, location and enzymes activities. Results showed that oxidation promoted the translocation of HSP27 and cytochrome c from the cytoplasm to the cell membrane, which reduced µ-calpain activity, but increased caspase-3 activity through mediating the interaction with the two enzymes. Oxidation retarded troponin-T degradation, but accelerated desmin degradation, which is probably because oxidative modification of myofibrils induced different susceptibility to proteolysis. Therefore, oxidation leads to different regulatory mechanism on µ-calpain and caspase-3, as well as the degree of degradation of myofibrillar proteins, possibly through mediating HSP27 and cytochrome c.


Assuntos
Calpaína/metabolismo , Caspase 3/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Animais , Bovinos , Citocromos c/metabolismo , Peróxido de Hidrogênio/química , Masculino , Músculo Esquelético/química , Oxirredução , Mudanças Depois da Morte , Proteólise , Carne Vermelha , Troponina T/metabolismo
2.
Mem Inst Oswaldo Cruz ; 115: e200142, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33053076

RESUMO

BACKGROUND: Calpains are present in almost all organisms and comprise a family of calcium-dependent cysteine peptidases implicated in crucial cellular functions. Trypanosoma cruzi, the causative agent of Chagas disease, presents an expansion on this gene family with unexplored biological properties. OBJECTIVES: Here, we searched for calpains in the T. cruzi genome, evaluated the mRNA levels, calpain activity and the protein expression and determined the cellular localisation in all three parasite life cycle forms. METHODS/FINDINGS: Sixty-three calpain sequences were identified in T. cruzi CL Brener genome, with fourteen domain arrangements. The comparison of calpain mRNA abundance by quantitative polymerase chain reaction (qPCR) revealed seven up-regulated sequences in amastigotes and/or bloodstream trypomastigotes and five in epimastigotes. Western Blotting analysis revealed seven different molecules in the three parasite forms, and one amastigote-specific, while no proteolytic activity could be detected. Flow cytometry assays revealed a higher amount of intracellular calpains in amastigotes and/or trypomastigotes in comparison to epimastigotes. Finally, ultrastructural analysis revealed the presence of calpains in the cytoplasm, vesicular and plasma membranes of the three parasite forms, and in the paraflagellar rod in trypomastigotes. CONCLUSION: Calpains are differentially expressed and localised in the T. cruzi life cycle forms. This study adds data on the calpain occurrence and expression pattern in T. cruzi.


Assuntos
Calpaína/genética , Trypanosoma cruzi/genética , Animais , Western Blotting , Calpaína/metabolismo , Doença de Chagas , Estágios do Ciclo de Vida , RNA Mensageiro/genética
3.
Neuron ; 108(1): 180-192.e5, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32827455

RESUMO

During development, endothelial tip cells (ETCs) located at the leading edge of growing vascular plexus guide angiogenic sprouts to target vessels, and thus, ETC pathfinding is fundamental for vascular pattern formation in organs, including the brain. However, mechanisms of ETC pathfinding remain largely unknown. Here, we report that Piezo1-mediated Ca2+ activities at primary branches of ETCs regulate branch dynamics to accomplish ETC pathfinding during zebrafish brain vascular development. ETC branches display spontaneous local Ca2+ transients, and high- and low-frequency Ca2+ transients cause branch retraction through calpain and branch extension through nitric oxide synthase, respectively. These Ca2+ transients are mainly mediated by Ca2+-permeable Piezo1 channels, which can be activated by mechanical force, and mutating piezo1 largely impairs ETC pathfinding and brain vascular patterning. These findings reveal that Piezo1 and downstream Ca2+ signaling act as molecular bases for ETC pathfinding and highlight a novel function of Piezo1 and Ca2+ in vascular development.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Encéfalo/irrigação sanguínea , Cálcio/metabolismo , Células Endoteliais/metabolismo , Canais Iônicos/genética , Neovascularização Fisiológica/genética , Proteínas de Peixe-Zebra/genética , Animais , Encéfalo/crescimento & desenvolvimento , Sinalização do Cálcio , Calpaína/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Mutação , Óxido Nítrico Sintase/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
4.
Biochim Biophys Acta Mol Cell Res ; 1867(10): 118788, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32603758

RESUMO

Muscle atrophy is an inevitable sequel of fasting, denervation, aging, exposure to microgravity, and many human diseases including, cancer, type-2 diabetes, and renal failure. During atrophy the destruction of the muscle's fundamental contractile machinery, the myofibrils, is accelerated leading to a reduction in muscle mass, weakness, frailty, and physical disability. Recent findings indicate that atrophy can be a major cause of death in affected individuals, and inhibition of muscle wasting is likely to prolong survival. Major advances in our understanding of the mechanisms for myofibril breakdown in atrophy include the discovery of biological pathways and key components that play prominent roles. On fasting or denervation, degradation of myofibrillar proteins requires an initial dissociation of the desmin cytoskeleton, whose integrity is critical for myofibril stability. This loss of desmin filaments involves phosphorylation, ubiquitination, and subsequent depolymerization by calpain-1, and appears to reduce myofibrils integrity and facilitate their destruction. Consequently, depolymerization of desmin filament in atrophy seems to be an early key event for overall proteolysis. A focus of this review is to discuss these new insights and the specific role of calpain-1 in promoting desmin filaments loss, and to highlight important key questions that merit further study.


Assuntos
Calpaína/metabolismo , Desmina/metabolismo , Atrofia Muscular/metabolismo , Polimerização , Animais , Humanos , Miofibrilas/metabolismo , Ubiquitinação
5.
Am J Chin Med ; 48(5): 1179-1202, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32668972

RESUMO

Over-expression of calpains in tumor tissues can be associated with cancer progression. Thus, inhibition of calpain activity using specific inhibitors has become a novel approach to control tumor growth. In this study, the anticancer potential of cryptotanshinone in combination with calpain inhibitor had been investigated in colon cancer cells and tumor xenograft. Cryptotanshinone elicited an initial endoplasmic reticular (ER) stress response, whereas prolonged stress would result in the promotion of apoptosis. It was then discovered that cryptotanshinone could cause rapid and sustained increase in cytosolic calcium in colon cancer cells accompanied by early GRP78 overexpression, which could be attenuated by pre-treatment of the calcium chelator BAPTA-AM. Cryptotanshinone also facilitated an early increase in calpain activity, which could be blocked by BAPTA-AM or the calpain inhibitor PD150606. A dynamic interaction between GRP78 and calpain during the action of cryptotanshinone was unveiled. This together with the altered NF-[Formula: see text]B signaling could be abolished by calpain inhibitor. GRP78 knockdown increased the sensitivity of cancer cells to cryptotanshinone-evoked apoptosis and reduction of cancer cell colony formation. Such sensitization of drug action had been confirmed to be p53-dependent by using p53-mutated (HT-29) and p53-deficient (HCT116 p53-∕-) cells. The synergistic antitumor effect of cryptotanshinone and calpain inhibitor was further exhibited in vivo. Taken together, findings in this study exemplify a new chemotherapeutic regimen comprising cryptotanshinone and calpain inhibitor by regulation of calpain and calcium homeostasis. This has provided us with new insights in the search of a potential target-specific neoadjuvant therapy against colon cancer.


Assuntos
Antineoplásicos Fitogênicos , Apoptose/efeitos dos fármacos , Apoptose/genética , Cálcio/metabolismo , Calpaína/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Homeostase/efeitos dos fármacos , Fenantrenos/farmacologia , Fenantrenos/uso terapêutico , Fitoterapia , Proteína Supressora de Tumor p53/metabolismo , Animais , Calpaína/genética , Neoplasias do Colo/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos Nus , Células Tumorais Cultivadas
6.
Nat Chem Biol ; 16(9): 988-996, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661379

RESUMO

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors-molecules that 'light up' in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce nonlinear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract.


Assuntos
Acústica/instrumentação , Técnicas Biossensoriais/instrumentação , Enzimas/metabolismo , Trato Gastrointestinal/enzimologia , Ultrassonografia/métodos , Animais , Bactérias/enzimologia , Bactérias/genética , Técnicas Biossensoriais/métodos , Calpaína/análise , Calpaína/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Endopeptidases/análise , Endopeptidases/metabolismo , Enzimas/análise , Desenho de Equipamento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Potyvirus/enzimologia , Probióticos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Razão Sinal-Ruído , Ultrassonografia/instrumentação
7.
Anim Sci J ; 91(1): e13423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32648277

RESUMO

Meat tenderness is considered as the most important criterion for meat quality by consumers and can be improved by the actions of endogenous proteases, mainly calpains, during postmortem storage at 0-5°C. The purpose of this study, therefore, was to examine the postmortem calpain activation and proteolysis in breast (BM) and leg and thigh (LM) muscles of White Roman goose. BM and LM were taken from goose carcasses (n = 15) at 0 (10-15 min postmortem), 1, 3, and 7 days of storage at 5°C. The decrease in postmortem pH, calpain-1 and -11 activities, and contents of the calpain-1 80 kDa subunit and desmin was more rapid (p < .05) in BM than in LM. Our results show that postmortem proteolysis was more extensive in BM than in LM of White Roman goose, not only because the difference in fiber type composition between two muscles, but because the rate and extent of calpain activation were greater in BM as well. These results may provide useful information to optimize meat processing for different muscles in goose industry.


Assuntos
Calpaína/metabolismo , Qualidade dos Alimentos , Gansos , Carne , Músculo Esquelético/metabolismo , Proteólise , Animais , Calpaína/análise , Temperatura Baixa , Manipulação de Alimentos , Armazenamento de Alimentos/métodos , Concentração de Íons de Hidrogênio , Carne/análise , Mudanças Depois da Morte , Fatores de Tempo
8.
Adv Exp Med Biol ; 1246: 129-151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32399829

RESUMO

Although the cytosolic Ca2+ signalling event in phagocytosis is well established, and the mechanism for generating such signals also understood, the target for the Ca2+ signal and how this relates to the phagocytic outcome is less clear. In this chapter, we present the evidence for a role of the Ca2+ activated protease, calpain, in phagocytosis. The abundant evidence for Ca2+ changes and calpain activation during cell shape changes is extended to include the specific cell shape change which accompanies phagocytosis. The discussion therefore includes a brief description of the domain structure of calpain and their functions. Also the mechanism by which calpain activation is limited at the cell periphery subdomains, and how this would allow phagocytic pseudopodia to form locally.


Assuntos
Cálcio/metabolismo , Calpaína/metabolismo , Fagocitose , Animais , Forma Celular , Citosol/metabolismo , Ativação Enzimática , Humanos , Pseudópodes/metabolismo
9.
Nat Commun ; 11(1): 2019, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332747

RESUMO

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.


Assuntos
Calpaína/metabolismo , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , Proteínas E7 de Papillomavirus/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/genética , Acrilatos/farmacologia , Calpaína/antagonistas & inibidores , Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ciclopentanos/farmacologia , Fatores de Transcrição E2F/metabolismo , Feminino , Células HEK293 , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Humanos , Proteína NEDD8/antagonistas & inibidores , Proteína NEDD8/metabolismo , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
10.
Food Chem ; 324: 126892, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32339789

RESUMO

To investigate calpain's effect on protein degradation, myowater properties, and the water-holding capacity (WHC), porcine longissimus muscles were incubated with control buffer, PD150,606 (calpain-specific inhibitor) and MG-262 (multiple-protease inhibitor) and assigned to an ageing period of 1, 4 or 7 d. Over 7 d of storage, no significant differences (P > 0.05) were observed in desmin or integrin expression between the MG-262 and PD150,606 groups, which indicated that calpain played a major role in protein proteolysis. Compared to those in the control group, muscle samples subjected to PD150,606 and MG-262 exhibited higher water mobility and a poorer WHC. Additionally, there were no significant differences in myowater properties or the WHC between the two groups at 1 d postmortem (P > 0.05). Calpain regulated the distribution and mobility of myowater, which contributed to a higher WHC in the early postmortem period (before 4 d), but other proteases tended to take over at a later stage.


Assuntos
Calpaína/metabolismo , Músculo Esquelético/química , Água/metabolismo , Acrilatos/química , Acrilatos/metabolismo , Animais , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Calpaína/antagonistas & inibidores , Desmina/metabolismo , Armazenamento de Alimentos , Integrinas/metabolismo , Carne/análise , Músculo Esquelético/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Proteólise , Suínos , Água/análise
11.
Oncol Rep ; 43(6): 2093-2104, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32236617

RESUMO

Human epidermal growth factor receptor 2 (HER2) is composed of an extracellular domain (ECD), a lipophilic transmembrane region and an intracellular domain (ICD). The most commonly used method to determine the status of HER2 is immunohistochemistry. However, false­negative results are sometimes given, which causes some patients to lose the opportunity for anti­HER2 therapy. We found that calpain­10 may prohibit HER2­ECD into peripheral blood resulting in a HER2­negative result by the immunohistochemical method. We enrolled 289 patients into our experiment to assess the relationship between sHER2­ECD and calpain­10. The results showed that there was a positive correlation between sHER2­ECD and calpain­10. Moreover, we also investigated the prognostic values of sHER2­ECD and calpain­10 in breast cancer patients. According to the follow­up results, positive sHER2­ECD and tissue calpain­10 were indicative of a poor prognosis in breast cancer patients. Subsequently, we further validated the relationship between the two molecules in in vitro experiments. In the in vitro experiments, the level of HER2­ECD in the culture medium was increased or decreased with a decrease or increase in calpain­10 by transfection technology, showing an inverse association. The results indicated that sHER2­ECD and tissue calpain­10 levels were powerful factors to assess the status of HER2. In combination with tissue HER2 detection, the occurrence of false­negative HER2 was reduced, providing patients with additional treatment opportunities. In conclusion, sHER2­ECD and tissue calpain­10 may be used as new prognostic indices for breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Calpaína/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Calpaína/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Domínios Proteicos , Regulação para Cima
12.
Food Chem ; 321: 126677, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32247180

RESUMO

Myosin heavy chain (MHC) isoforms in goat muscles and their possible relationships with meat quality have not been fully elucidated. This study characterized the MHC isoforms in different caprine muscles using sodium dodecyl sulphate glycerol gel electrophoresis (SDS-GGE). The relationships between MHC isoforms, calpain systems and meat quality characteristics of different muscles in goats were examined. Four muscles, namely infraspinatus (IF), longissimus dorsi (LD), psoas major (PM) and supraspinatus (SS) were obtained from ten Boer crossbred bucks (7-10 months old; 26.5 ± 3.5 kg, BW). The percentages of MHC I, MHC IIa and MHC IIx in SS, IF, PM and LD were 47.2, 38.3, 32.1, 11.9; 28.0, 42.1, 33.0, 36.4; and 24.8, 19.6, 34.9 and 51.7, respectively. IF and SS had higher levels of calpastatin, total collagen and insoluble collagen contents than did PM and LD. PM had longer sarcomere length than did other muscles. LD had higher collagen solubility, troponin-T degradation products and glycogen content than did other muscles. These results infer that variable fiber-type composition could account partially for the differences in the physicochemical properties of goat muscles.


Assuntos
Calpaína/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Eletroforese , Eletroforese em Gel de Poliacrilamida , Cabras , Carne/análise , Músculo Esquelético/química , Isoformas de Proteínas/metabolismo , Troponina T/metabolismo
13.
Ann Clin Lab Sci ; 50(1): 24-30, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32161009

RESUMO

OBJECTIVE: Calpain 6 (CAPN6) is one of the calcium-dependent intracellular nonlysosomal proteases that are dysregulated in uterine leiomyomas (UtLMs). However, its function and mechanism in UtLMs is still unknown. METHODS: The correlation between CAPN6 expression and UtLMs was analyzed by the Gene Expression Omnibus (GEO). The expression of CAPN6 and Rac1 was detected by quantitative real-time PCR (qPCR) and western blot analysis. Cell proliferation ability was analyzed by a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry. RESULTS: CAPN6 was overexpressed in UtLMs compared with uterine smooth muscle cells (UtSMCs). The downregulation of CAPN6 resulted in decreased cell proliferation and increased apoptosis of UtLMs. Furthermore, mechanical investigations revealed that these inhibitory effects were correlated with Rac1/PAK1 signaling pathways. Silencing the expression of CAPN6 resulted in decreased Rac1 and phospho-PAK1. On the other hand, upregulated Rac1 expression could reverse the reduced phosphorylation of PAK1 induced by CAPN6 silencing. CONCLUSIONS: This data suggests that CAPN6 regulates UtLMs proliferation and apoptosis while being mediated through the Rac1/PAK1 signaling pathway.


Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Calpaína/metabolismo , Proliferação de Células , Leiomioma/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Biomarcadores Tumorais/genética , Calpaína/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leiomioma/metabolismo , Leiomioma/cirurgia , Proteínas Associadas aos Microtúbulos/genética , Miócitos de Músculo Liso/metabolismo , Prognóstico , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genética
14.
Food Chem ; 319: 126571, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32169769

RESUMO

This study aims to investigate the changes in mitochondrial apoptotic factors and proteolysis of two porcine muscles (psoas major - PM and longissimus dorsi - LD) during aging. Results found that during 2-168 h postmortem mitochondrial membrane permeability, mitochondrial lipid peroxidation, Ca2+ levels were increased, while the reduction level and abundance of cytochrome c were decreased (P < 0.05) in both muscle types. Furthermore, the activation of caspase-3 along with increases in troponin-T and desmin degradation, and µ-calpain autolysis were found (P < 0.05), regardless of muscle type. PM maintained higher mitochondrial apoptotic factors, but had more intact desmin, less troponin-T degradation and less extent of autolyzed products of µ-calpain compared to LD (P < 0.05). These results indicate that the rapid onset of mitochondrial apoptosis of PM would not lead to a subsequent impact on myofibrillar protein degradation, suggesting that the mitochondrial apoptosis mediated tenderization process could be muscle-specific.


Assuntos
Apoptose , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Carne Vermelha , Animais , Autólise/metabolismo , Calpaína/metabolismo , Desmina/metabolismo , Miofibrilas/metabolismo , Proteólise , Suínos , Fatores de Tempo , Troponina T/metabolismo
15.
Food Chem ; 318: 126516, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32146313

RESUMO

This work investigated the effects of L-arginine (Arg) and L-lysine (Lys) on the tenderness of chicken breast and explored the possible mechanisms underlying this effect for the first time. The results showed that both Arg and Lys decreased the shear force and increased the pH value, sarcomere length and myofibrillar fragmentation index as well as degraded the troponin-T by keeping calpain activity in chicken breast. In addition, Arg effectively reduced Ca2+/Mg2+-ATPase activities and promoted actomyosin dissociation. These results indicated that both Arg and Lys could enhance the tenderness of chicken breast, and it could also explain why Arg was more effective than Lys in improving the tenderness of chicken breast. These results will help facilitate the development of industrial-scale methods for improving the tenderness of meat products.


Assuntos
Actomiosina/química , Arginina/farmacologia , Galinhas , Lisina/farmacologia , Produtos Avícolas , Troponina T/química , Animais , Arginina/química , Calpaína/química , Calpaína/metabolismo , Qualidade dos Alimentos , Concentração de Íons de Hidrogênio , Lisina/química
16.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140411, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32200007

RESUMO

Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta-EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.


Assuntos
Calpaína/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Animais , Calpaína/química , Calpaína/genética , Linhagem Celular , Motivos EF Hand , Feminino , Predisposição Genética para Doença/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Mutagênese Insercional , Mutação , Domínios Proteicos
17.
Int J Mol Sci ; 21(6)2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32204400

RESUMO

Synovial sarcoma is a rare but highly malignant and metastatic disease. Despite its relative sensitivity to chemotherapies, the high recurrence and low 5-year survival rate for this disease suggest that new effective therapeutic agents are urgently needed. Marine antimicrobial peptide epinecidin-1 (epi-1), which was identified from orange-spotted grouper (Epinephelus coioides), exhibits multiple biological effects, including bactericidal, immunomodulatory, and anticancer activities. However, the cytotoxic effects and mechanisms of epi-1 on human synovial sarcoma cells are still unclear. In this study, we report that epi-1 exhibits prominent antisynovial sarcoma activity in vitro and in a human SW982 synovial sarcoma xenograft model. Furthermore, we determined that calcium overload-induced calpain activation and subsequent oxidative stress and mitochondrial dysfunction are required for epi-1-mediated cytotoxicity. Interestingly, reactive oxygen species (ROS)-mediated activation of extracellular signal-regulated kinase (ERK) plays a protective role against epi-1-induced cytotoxicity. Our results provide insight into the molecular mechanisms underlying epi-1-induced cell death in human SW982 cells.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Cálcio/metabolismo , Calpaína/metabolismo , Proteínas de Peixes/farmacologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sarcoma Sinovial/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Masculino , Camundongos Nus , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
18.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 2): 81-85, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32039889

RESUMO

Calpain is a Ca2+-activated, heterodimeric cysteine protease consisting of a large catalytic subunit and a small regulatory subunit. Dysregulation of this enzyme is involved in a range of pathological conditions such as cancer, Alzheimer's disease and rheumatoid arthritis, and thus calpain I is a drug target with potential therapeutic applications. Difficulty in the production of this enzyme has hindered structural and functional investigations in the past, although heterodimeric calpain I can be generated by Escherichia coli expression in low yield. Here, an unexpected structure discovered during crystallization trials of heterodimeric calpain I (CAPN1C115S + CAPNS1ΔGR) is reported. A novel co-crystal structure of the PEF(S) domain from the dissociated regulatory small subunit of calpain I and the RNA-binding chaperone Hfq, which was likely to be overproduced as a stress response to the recombinant expression conditions, was obtained, providing unexpected insight in the chaperone function of Hfq.


Assuntos
Calpaína/química , Fator Proteico 1 do Hospedeiro/química , Chaperonas Moleculares/química , Conformação Proteica , Multimerização Proteica , Calpaína/metabolismo , Cristalografia por Raios X , Fator Proteico 1 do Hospedeiro/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Domínios Proteicos
19.
Arch Biochem Biophys ; 683: 108299, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32061585

RESUMO

BACKGROUND: ER (endoplasmic reticulum) stress leads to decreased complex I activity in cardiac mitochondria. The aim of the current study is to explore the potential mechanisms by which ER stress leads to the complex I defect. ER stress contributes to intracellular calcium overload and oxidative stress that are two key factors to induce mitochondrial dysfunction. Since oxidative stress is often accompanied by intracellular calcium overload during ER stress in vivo, the role of oxidative stress and calcium overload in mitochondrial dysfunction was studied using in vitro models. ER stress results in intracellular calcium overload that favors activation of calcium-dependent calpains. The contribution of mitochondrial calpain activation in ER stress-mediated complex I damage was studied. METHODS: Thapsigargin (THAP) was used to induce acute ER stress in H9c2 cells and C57BL/6 mice. Exogenous calcium (25 µM) and H2O2 (100 µM) were used to induce modest calcium overload and oxidative stress in isolated mitochondria. Calpain small subunit 1 (CAPNS1) is essential to maintain calpain 1 and calpain 2 (CPN1/2) activities. Deletion of CAPNS1 eliminates the activities of CPN1/2. Wild type and cardiac-specific CAPNS1 deletion mice were used to explore the role of CPN1/2 activation in calcium-induced mitochondrial damage. RESULTS: In isolated mitochondria, exogenous calcium but not H2O2 treatment led to decreased oxidative phosphorylation, supporting that calcium overload contributes a key role in the mitochondrial damage. THAP treatment of H9c2 cells decreased respiration selectively with complex I substrates. THAP treatment activated cytosolic and mitochondrial CPN1/2 in C57BL/6 mice and led to degradation of complex I subunits including NDUFS7. Calcium treatment decreased NDUFS7 content in wild type but not in CAPNS1 knockout mice. CONCLUSION: ER stress-mediated activation of mitochondria-localized CPN1/2 contributes to complex I damage by cleaving component subunits.


Assuntos
Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Cardiopatias/metabolismo , Mitocôndrias/metabolismo , Animais , Calpaína/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Deleção de Genes , Peróxido de Hidrogênio/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo , Fosforilação , Ratos , Tapsigargina/farmacologia
20.
Nat Commun ; 11(1): 922, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066742

RESUMO

Ryanodine receptors (RyR) are ion channels responsible for the release of Ca2+ from the sarco/endoplasmic reticulum and play a crucial role in the precise control of Ca2+ concentration in the cytosol. The detailed permeation mechanism of Ca2+ through RyR is still elusive. By using molecular dynamics simulations with a specially designed Ca2+ model, we show that multiple Ca2+ ions accumulate in the upper selectivity filter of RyR1, but only one Ca2+ can occupy and translocate in the narrow pore at a time, assisted by electrostatic repulsion from the Ca2+ within the upper selectivity filter. The Ca2+ is nearly fully hydrated with the first solvation shell intact during the whole permeation process. These results suggest a remote knock-on permeation mechanism and one-at-a-time occupation pattern for the hydrated Ca2+ within the narrow pore, uncovering the basis underlying the high permeability and low selectivity of the RyR channels.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Simulação de Dinâmica Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/química , Calpaína/metabolismo , Cátions Bivalentes/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Eletricidade Estática , Relação Estrutura-Atividade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA