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1.
Food Chem ; 308: 125576, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31648092

RESUMO

This study investigated the effects of cold storage at different temperatures (4, -0.5, -3, and -20 °C) on protein degradation and its relationship to structural changes of black carp muscle. At -0.5 and 4 °C, major structural changes occurred, including the formation of gaps between myofibers and myofibrils, breakage of myofibrils and myofibers, and degradation of sarcoplasmic reticulum. Gel-based proteomic analysis showed that these structural changes were accompanied by degradation of a series of myofibrillar proteins, including titin, nebulin, troponin, myosin, myomesin, myosin-binding protein, and α-actinin. Loss of extractable gelatinolytic and caseinolytic protease activities was also observed. At -3 and -20 °C, formation of ice crystals was the most noticeable change. The major proteins were degraded at different locations in the black carp muscle, and gelatinolytic and caseinolytic proteases appear to contribute to the degradation of those proteins.


Assuntos
Carpas/metabolismo , Actinina/metabolismo , Animais , Proteínas de Transporte , Temperatura Baixa , Conectina/metabolismo , Cyprinidae/metabolismo , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Proteólise , Proteômica
2.
Food Chem ; 301: 125278, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387033

RESUMO

Softening is always a problem in fish preservation. This study was aimed to investigate the role of myofibrillar structural proteins degradation in fish softening. The changes of myofibrillar structural proteins, muscle ultrastructure, myofibril fragmentation, and shear force were studied. The results indicated that during the superchilled preservation of grass carp (Ctenopharyngodon idella), small (low-molecular-weight) myofibrillar structural proteins like desmin and troponin-T initiated textural deterioration, leading to Z-disk weakening and actin loosening. In contrast, giant (high-molecular-weight) myofibrillar structural proteins like titin and nebulin were degraded in more amount in the later storage, contributing to Z-disk and M-band disassembly and vague of light and dark regions (I and A bands). Compared to each other, desmin and titin played more important part in softening. All these changes were involved in the increase of muscle fibril segments and the sharp decrease of shear force.


Assuntos
Carpas/metabolismo , Produtos Pesqueiros , Proteínas de Peixes/química , Miofibrilas/química , Animais , Conectina/química , Conectina/metabolismo , Desmina/química , Desmina/metabolismo , Proteínas de Peixes/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Proteólise , Troponina T/química , Troponina T/metabolismo
3.
Adv Clin Chem ; 90: 1-23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31122607

RESUMO

Titin/connectin, encoded by the TTN gene, is the largest protein in humans. It acts as a molecular spring in the sarcomere of striated muscles. Although titin is degraded in the skeletal muscles of patients with muscular dystrophies, studies of titin have been limited by its mammoth size. Mutations in the TTN gene have been detected not only in skeletal muscle diseases but in cardiac muscle diseases. TTN mutations result in a wide variety of phenotypes. Recent proteome analysis has found that titin fragments are excreted into the urine of patents with Duchenne muscular dystrophy (DMD). Enzyme-linked immunosorbent assays (ELISAs) have shown that urinary titin is a useful noninvasive biomarker for the diagnosis and screening of not only DMD, but also of neuromuscular diseases, for predicting the outcome of cardiomyopathy and for evaluating physical activities. The development of ELISA systems to measure urinary titin has opened a door to studying muscle degradation directly and noninvasively. This review provides current understanding of urinary titin and future prospects for measuring this protein.


Assuntos
Conectina/urina , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismo , Biomarcadores/urina , Conectina/metabolismo , Humanos
4.
Am J Physiol Endocrinol Metab ; 316(5): E967-E976, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30912963

RESUMO

Alcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1) both fast and slow muscle fibers are subjected to atrophy; 2) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content.


Assuntos
Transtornos Induzidos por Álcool/metabolismo , Alcoolismo/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Doenças Musculares/metabolismo , Músculo Quadríceps/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenilato Quinase/metabolismo , Adulto , Transtornos Induzidos por Álcool/patologia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/metabolismo , Conectina/metabolismo , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Pessoa de Meia-Idade , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/patologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/patologia , Tamanho do Órgão , Fosfoproteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Músculo Quadríceps/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
5.
J Anim Physiol Anim Nutr (Berl) ; 103(1): 283-294, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30284316

RESUMO

The objective of this study was to examine markers of whole-body and muscle protein metabolism in aged horses fed a diet typical for North American aged horses, supplemented with amino acids. In a replicated Latin square design, six aged horses (20 ± 1.1 years) were studied while receiving each of three isocaloric, isonitrogenous diets, a control treatment concentrate (CON; 100 mg/kg-1 BW day-1 lysine, 84 mg kg-1  day-1 threonine, 51 mg kg-1  day-1 methionine), LYS/THR (134 mg kg-1 BW day-1 lysine, 110 mg kg-1 BW day-1 threonine, 52 mg kg-1 BW day-1 methionine) and LYS/THR/MET (132 mg kg-1 BW day-1 lysine, 112 mg kg-1 BW day-1 threonine, 62 mg kg-1 BW day-1 methionine). In each 15-days period, urine and faeces were collected for assessment of nitrogen balance. Blood samples were collected before and after feeding for analysis of plasma urea nitrogen (PUN), glucose, insulin and plasma amino acid concentrations. Skeletal muscle samples were collected for measurement of proteins associated with muscle protein synthesis and degradation, and horses underwent stable isotope infusion procedures for comparison of differences in whole-body rates of protein synthesis and degradation. There was no effect of treatment on relative abundance of proteins involved in protein synthesis, nitrogen retention or phenylalanine kinetics. PUN concentrations tended to be higher for LYS/THR (p = 0.054) and were higher for LYS/THR/MET (p = 0.0056) than for CON. Atrogin-1 abundance tended to be higher in the post-absorptive state for the CON treatment (p = 0.07), indicating that amino acid supplementation resulted in less muscle protein degradation when horses were in the post-absorptive state. However, lack of differences in nitrogen retention and phenylalanine kinetics indicated that whole-body protein metabolism was not improved, and higher PUN concentrations in the supplemented diets suggest that the supplemented amino acids may have been catabolized. Amino acid availability was not limiting protein synthesis in the sedentary aged horses in this study when fed the CON diet.


Assuntos
Envelhecimento , Aminoácidos/administração & dosagem , Cavalos/fisiologia , Proteínas Musculares/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Glicemia , Nitrogênio da Ureia Sanguínea , Conectina/efeitos dos fármacos , Conectina/metabolismo , Estudos Cross-Over , Dieta/veterinária , Suplementos Nutricionais , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/sangue , Proteínas Musculares/genética , Distribuição Aleatória
6.
Redox Biol ; 21: 101074, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30584979

RESUMO

The nanomechanics of sarcomeric proteins is a key contributor to the mechanical output of muscle. Among them, titin emerges as a main target for the regulation of the stiffness of striated muscle. In the last years, single-molecule experiments by Atomic Force Microscopy (AFM) have demonstrated that redox posttranslational modifications are strong modulators of the mechanical function of titin. Here, we provide an overview of the recent development of the redox mechanobiology of titin, and suggest avenues of research to better understand how the stiffness of molecules, cells and tissues are modulated by redox signaling in health and disease.


Assuntos
Suscetibilidade a Doenças , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Oxirredução , Sarcômeros/metabolismo , Animais , Fenômenos Biomecânicos , Conectina/química , Conectina/genética , Conectina/metabolismo , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Especificidade de Órgãos , Relação Estrutura-Atividade
7.
Biomed Res Int ; 2018: 5816875, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30581860

RESUMO

Several studies have compared molecular components between red and white skeletal muscles in mammals. However, mammalian skeletal muscles are composed of mixed types of muscle fibers. In the current study, we analyzed and compared the distributions of titin, lipid, phosphate ions, and fatty acid levels in red and white muscles using a fish model (Tilapia), which is rich in red and white muscles, and these are well separated. Oil-red O staining showed that red muscle had more-abundant lipids than did white muscle. A time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmitic acid and oleic acid, but white muscle contained more phosphate ions. Moreover, elastica-van Gieson (EVG) and Mito-Tracker green FM staining showed that collagen and elastic fibers were highly, respectively, distributed in connective tissues and mitochondria in red muscle. An electron micrographic analysis indicated that red muscle had a relatively higher number of mitochondria and longer sarcomere lengths and Z-line widths, while myofibril diameters were thicker in white muscle. Myofibrillar proteins separated by SDS-PAGE showed that the major giant protein, titin, was highly expressed in white muscle than in red muscle. Furthermore, ratios of titin to myosin heavy chain (MHC) (titin/MHC) were about 1.3 times higher in white muscle than red muscle. We postulated that white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres, whereas red muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres.


Assuntos
Conectina/metabolismo , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/metabolismo , Animais , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Elasticidade/fisiologia , Peixes , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Modelos Animais , Miofibrilas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Sarcômeros/metabolismo , Alimentos Marinhos
8.
Proc Natl Acad Sci U S A ; 115(48): E11284-E11293, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30413621

RESUMO

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-ß Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.


Assuntos
Conectina/química , Ribossomos/metabolismo , Conectina/genética , Conectina/metabolismo , Humanos , Cinética , Modelos Moleculares , Biossíntese de Proteínas , Dobramento de Proteína , Ribossomos/química , Ribossomos/genética
9.
Can J Cardiol ; 34(10): 1369.e5-1369.e7, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30269836

RESUMO

Because of the rare co-occurrence, it remains a question whether cardiomyopathy is a true association of neurofibromatosis type 1. A boy with café-au-lait spots manifested restrictive cardiomyopathy. Whole exome sequencing confirmed the genetic diagnosis of neurofibromatosis and further identified a novel titin (TTN) missense variant. The significance of the variant is supported by its de novo origin, in silico predictions, and evolutionary conservation. Modern genetics raises an intriguing explanation for the unexpected phenotype and adds to the evolving role of TTN variants in cardiomyopathy.


Assuntos
Cardiomiopatias/etiologia , Conectina/genética , DNA/genética , Mutação , Neurofibromatose 1/complicações , Cateterismo Cardíaco , Cardiomiopatias/diagnóstico , Cardiomiopatias/genética , Pré-Escolar , Conectina/metabolismo , Análise Mutacional de DNA , Humanos , Masculino , Miocárdio/patologia , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/genética
10.
Protein Pept Lett ; 25(11): 973-979, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30289063

RESUMO

INTRODUCTION: The giant muscular proteins titin and obscurin bind to each other at the Zdisk during muscle development. This binding event is mediated through two domains from each protein: ZIg9/10 from titin and Ig58/59 from obscurin. This interaction helps stabilize and organize the sarcomere; ablation of this binding leads to muscular dystrophy. OBJECTIVE: Here we solve the high-resolution solution structure of titin ZIg10 and further delineate which sections of titin bind to obscurin. MATERIALS AND METHODS: Solution NMR, Circular Dichroism, and SEC-MALS were used to biophysically characterize the titin domains involved in this titin-obscurin interaction. RESULTS AND CONCLUSION: We present the high-resolution solution structure of titin ZIg10. Additionally, we show that titin ZIg9 drives the titin-obscurin interaction, while ZIg10 does not actively participate in the titin-obscurin interaction but instead acts to stabilize ZIg9.


Assuntos
Conectina/química , Conectina/metabolismo , Proteínas Musculares/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
11.
Sci Rep ; 8(1): 14575, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30275509

RESUMO

Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A - F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A - actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A - actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin - actin binding occurs in skeletal muscle sarcomeres in vivo.


Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Conectina/metabolismo , Ligação Proteica
12.
Nat Commun ; 9(1): 4316, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333491

RESUMO

A family history of atrial fibrillation constitutes a substantial risk of developing the disease, however, the pathogenesis of this complex disease is poorly understood. We perform whole-exome sequencing on 24 families with at least three family members diagnosed with atrial fibrillation (AF) and find that titin-truncating variants (TTNtv) are significantly enriched in these patients (P = 1.76 × 10-6). This finding is replicated in an independent cohort of early-onset lone AF patients (n = 399; odds ratio = 36.8; P = 4.13 × 10-6). A CRISPR/Cas9 modified zebrafish carrying a truncating variant of titin is used to investigate TTNtv effect in atrial development. We observe compromised assembly of the sarcomere in both atria and ventricle, longer PR interval, and heterozygous adult zebrafish have a higher degree of fibrosis in the atria, indicating that TTNtv are important risk factors for AF. This aligns with the early onset of the disease and adds an important dimension to the understanding of the molecular predisposition for AF.


Assuntos
Fibrilação Atrial/genética , Conectina/genética , Adulto , Idoso , Animais , Fibrilação Atrial/patologia , Estudos de Casos e Controles , Estudos de Coortes , Conectina/metabolismo , Feminino , Fibrose , Variação Genética , Humanos , Masculino , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Adulto Jovem , Peixe-Zebra
13.
Proc Natl Acad Sci U S A ; 115(45): 11525-11530, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30341218

RESUMO

An immense repertoire of protein chemical modifications catalyzed by enzymes is available as proteomics data. Quantifying the impact of the conformational dynamics of the modified peptide remains challenging to understand the decisive kinetics and amino acid sequence specificity of these enzymatic reactions in vivo, because the target peptide must be disordered to accommodate the specific enzyme-binding site. Here, we were able to control the conformation of a single-molecule peptide chain by applying mechanical force to activate and monitor its specific cleavage by a model protease. We found that the conformational entropy impacts the reaction in two distinct ways. First, the flexibility and accessibility of the substrate peptide greatly increase upon mechanical unfolding. Second, the conformational sampling of the disordered peptide drives the specific recognition, revealing force-dependent reaction kinetics. These results support a mechanism of peptide recognition based on conformational selection from an ensemble that we were able to quantify with a torsional free-energy model. Our approach can be used to predict how entropy affects site-specific modifications of proteins and prompts conformational and mechanical selectivity.


Assuntos
Conectina/química , Endopeptidases/química , Peptídeos/química , Poliproteínas/química , Biocatálise , Fenômenos Biomecânicos , Conectina/genética , Conectina/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Entropia , Expressão Gênica , Cinética , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Poliproteínas/genética , Poliproteínas/metabolismo , Conformação Proteica , Engenharia de Proteínas , Desdobramento de Proteína , Proteólise , Especificidade por Substrato
14.
J Cell Biochem ; 119(12): 9986-9996, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30133019

RESUMO

Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction, and muscle stiffness. TTN splicing generates variable protein sizes with different functions. Therefore, understanding TTN splicing is important to develop a novel treatment for TTN-based diseases. The I-band TTN splicing regulated by RNA binding motif 20 (RBM20) has been extensively studied. However, the Z- and M-band splicing and regulation remain poorly understood. Herein, we aimed to define the Z- and M-band splicing in striated muscles and determined whether RBM20 regulates the Z- and M-band splicing. We discovered four new Z-band TTN splicing variants, and one of them dominates in mouse, rat, sheep, and human hearts. But only one form can be detected in frog and chicken hearts. In skeletal muscles, three new Z repeats (Zr) were detected, and Zr4 to 6 exclusion dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z-band. In the M-band, two new variants were discovered with alternative 3' splice site in exon363 (Mex5) and alternative 5' splice site in intron 362. However, only the sheep heart expresses two new variants rather than other species. Skeletal muscles express three M-band variants with altered ratios of Mex5 inclusion to Mex5 exclusion. Finally, we revealed that RBM20 does not regulate the Z- and M-band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z- and M-band splicing and provided the first evidence of the role of RBM20 in the Z- and M-band TTN splicing.


Assuntos
Conectina/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Conectina/genética , Humanos , Camundongos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sarcômeros/metabolismo , Ovinos/genética , Ovinos/metabolismo
15.
Sci Rep ; 8(1): 12337, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120340

RESUMO

Loss of cardiomyocyte proliferative capacity after birth is a major obstacle for therapeutic heart regeneration in adult mammals. We and others have recently shown the importance of hypoxic in utero environments for active foetal cardiomyocyte proliferation. Here, we report the unexpected expression of novex-3, the short splice variant of the giant sarcomeric protein connectin (titin), in the cardiomyocyte nucleus specifically during the hypoxic foetal stage in mice. This nuclear localisation appeared to be regulated by the N-terminal region of novex-3, which contains the nuclear localisation signal. Importantly, the nuclear expression of novex-3 in hypoxic foetal cardiomyocytes was repressed at the postnatal stage following the onset of breathing and the resulting elevation of oxygen tension, whereas the sarcomeric expression remained unchanged. Novex-3 knockdown in foetal cardiomyocytes repressed cell cycle-promoting genes and proliferation, whereas novex-3 overexpression enhanced proliferation. Mechanical analysis by atomic force microscopy and microneedle-based tensile tests demonstrated that novex-3 expression in hypoxic foetal cardiomyocytes contributes to the elasticity/compliance of the nucleus at interphase and facilitates proliferation, by promoting phosphorylation-induced disassembly of multimer structures of nuclear lamins. We propose that novex-3 has a previously unrecognised role in promoting cardiomyocyte proliferation specifically at the hypoxic foetal stage.


Assuntos
Conectina/metabolismo , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Biomarcadores , Ciclo Celular/genética , Núcleo Celular/metabolismo , Conectina/química , Conectina/genética , Imunofluorescência , Expressão Gênica , Hipóxia/genética , Interfase/genética , Laminas/química , Laminas/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Sinais Direcionadores de Proteínas , Transporte Proteico
16.
Meat Sci ; 145: 308-319, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30015160

RESUMO

Reverse Phase Protein Arrays (RPPA) were applied for the quantification and validation of protein biomarkers of beef qualities on M. longissimus thoracis sampled early post-mortem from young Charolais bulls. pHu was related to six proteins, three of which are glycolytic enzymes (ENO1, ENO3 and TPI1), while others belong to structural (TTN and α-actin) and proteolytic (µ-calpain) pathways. For color traits, several correlations were found, interestingly with structural proteins. The relationships were in some cases trait-dependent. To understand the mechanisms and explore animal variability, color data were categorized into three classes. α-actin and TTN allowed efficient separation of the classes and were strongly related with all color traits. Biomarkers belonging to heat stress and metabolism pathways were also involved. Two identified proteins, namely Four and a half LIM domains 1 (FHL1) and Tripartite motif-containing 72 (TRIM72), were for the first time related to beef color. Overall, these relationships could be used to develop muscle-specific processing strategies to improve beef color stability.


Assuntos
Cor , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Carne Vermelha/análise , Actinas/metabolismo , Animais , Biomarcadores , Cruzamento , Calpaína/metabolismo , Bovinos , Conectina/metabolismo , Glicólise , Proteínas de Choque Térmico/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas com Domínio LIM/metabolismo , Masculino , Fosfopiruvato Hidratase/metabolismo , Análise Serial de Proteínas/métodos , Proteólise , Triose-Fosfato Isomerase , Proteínas com Motivo Tripartido/metabolismo
17.
PLoS One ; 13(6): e0198492, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29889873

RESUMO

Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20. Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.


Assuntos
Cardenolídeos/farmacologia , Conectina/genética , Descoberta de Drogas , Genes Reporter , Processamento de RNA/efeitos dos fármacos , Cardenolídeos/química , Cardenolídeos/metabolismo , Conectina/antagonistas & inibidores , Conectina/metabolismo , Digitoxina/química , Digitoxina/metabolismo , Digitoxina/farmacologia , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transcrição Genética/efeitos dos fármacos
18.
Int J Biol Sci ; 14(4): 369-380, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29725258

RESUMO

RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.


Assuntos
Conectina/metabolismo , Redes Reguladoras de Genes , Miocárdio/metabolismo , Proteínas de Ligação a RNA/fisiologia , Animais , Conectina/genética , Conectina/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos Sprague-Dawley
19.
Circ Res ; 123(3): 342-355, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29760016

RESUMO

RATIONALE: Increased titin-dependent cardiomyocyte tension is a hallmark of heart failure with preserved ejection fraction associated with type-2 diabetes mellitus. However, the insulin-related signaling pathways that modify titin-based cardiomyocyte tension, thereby contributing to modulation of diastolic function, are largely unknown. OBJECTIVE: We aimed to determine how impaired insulin signaling affects titin expression and phosphorylation and thus increases passive cardiomyocyte tension, and whether metformin or neuregulin-1 (NRG-1) can correct disturbed titin modifications and increased titin-based stiffness. METHODS AND RESULTS: We used cardiac biopsies from human diabetic (n=23) and nondiabetic patients (n=19), cultured rat cardiomyocytes, left ventricular tissue from apolipoprotein E-deficient mice with streptozotocin-induced diabetes mellitus (n=12-22), and ZSF1 (obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid) rats (n=5-6) and analyzed insulin-dependent signaling pathways that modulate titin phosphorylation. Titin-based passive tension was measured using permeabilized cardiomyocytes. In human diabetic hearts, we detected titin hypophosphorylation at S4099 and hyperphosphorylation at S11878, suggesting altered activity of protein kinases; cardiomyocyte passive tension was significantly increased. When applied to cultured cardiomyocytes, insulin and metformin increased titin phosphorylation at S4010, S4099, and S11878 via enhanced ERK1/2 (extracellular signal regulated kinase 1/2) and PKCα (protein kinase Cα) activity; NRG-1 application enhanced ERK1/2 activity but reduced PKCα activity. In apolipoprotein E-deficient mice, chronic treatment of streptozotocin-induced diabetes mellitus with NRG-1 corrected titin phosphorylation via increased PKG (protein kinase G) and ERK1/2 activity and reduced PKCα activity, which reversed the diabetes mellitus-associated changes in titin-based passive tension. Acute application of NRG-1 to obese ZSF1 rats with type-2 diabetes mellitus reduced end-diastolic pressure. CONCLUSIONS: Mechanistically, we found that impaired cGMP-PKG signaling and elevated PKCα activity are key modulators of titin-based cardiomyocyte stiffening in diabetic hearts. We conclude that by restoring normal kinase activities of PKG, ERK1/2, and PKCα, and by reducing cardiomyocyte passive tension, chronic NRG-1 application is a promising approach to modulate titin properties in heart failure with preserved ejection fraction associated with type-2 diabetes mellitus.


Assuntos
Conectina/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Insulina/farmacologia , Miócitos Cardíacos/metabolismo , Neuregulina-1/farmacologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Animais , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação , Proteína Quinase C-alfa/metabolismo , Ratos , Ratos Zucker
20.
Hellenic J Cardiol ; 59(5): 254-261, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29807197

RESUMO

Left ventricular enlargement and dysfunction are fundamental components of dilated cardiomyopathy (DCM). DCM is a major cause of heart failure and cardiac transplantation. A wide variety of etiologies underlie acquired and familial DCM. Familial disease is reported in 20% to 35% of cases. A genetic substrate is recognized in at least 30% of familial cases. A recently proposed scheme defines DCM as a continuum of subclinical and clinical phenotypes. The evolution of classification systems permitted use of effective treatment strategies in disorders sharing the same structural and functional characteristics and common clinical expression. The major causes of death are progressive heart failure and sudden cardiac death secondary to ventricular arrhythmias or less commonly bradyarrhythmias. Remarkable progress has been made in survival owing to well-defined evidence-based therapies and appropriate guidelines for risk stratification and sudden cardiac death prevention measures. Neurohormonal antagonists and device therapy decreased all-cause mortality in adult patients with DCM. However, additional red flags in diagnosis have to be addressed in everyday practice, and cardiologists have to be aware of the subsequent effect on risk stratification and treatment plan. Genetic substrate cannot be modified, but the presence of a peculiar type of gene mutation modifies thresholds for implantable cardioverter defibrillator (ICD) implantation. DCM is part of the spectrum of heart failure which is a syndrome with certain morphological and functional characteristics. Although significant progress has been achieved in the management of patients with DCM, it seems that the future treatments of this entity will be related to the specific pathological substrate.


Assuntos
Cardiomiopatia Dilatada/diagnóstico , Morte Súbita Cardíaca/prevenção & controle , Testes Genéticos/métodos , Insuficiência Cardíaca/complicações , Arritmias Cardíacas/epidemiologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Cardiomiopatia Dilatada/tratamento farmacológico , Cardiomiopatia Dilatada/epidemiologia , Cardiomiopatia Dilatada/genética , Conectina/metabolismo , Citoesqueleto/metabolismo , Morte Súbita Cardíaca/epidemiologia , Desfibriladores Implantáveis/normas , Feminino , Humanos , Masculino , Mutação/genética , Prevalência , Medição de Risco , Sarcômeros/metabolismo , Troponina T/metabolismo
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