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1.
BMC Bioinformatics ; 18(1): 225, 2017 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-28454514

RESUMO

BACKGROUND: Whole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis. RESULTS: Here we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts. CONCLUSIONS: QueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/ .


Assuntos
Bases de Dados Genéticas , Variação Genética , Software , Doença/genética , Exoma , Genoma Humano , Humanos , Internet
2.
Cell Physiol Biochem ; 42(1): 169-184, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28531892

RESUMO

BACKGROUND: Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2) is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear. METHODS: Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H2O2 treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS) release and cell viability. RESULTS: Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart. CONCLUSIONS: For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.


Assuntos
Núcleo Celular/metabolismo , Lamina Tipo A/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Peróxido de Hidrogênio/toxicidade , Imunoprecipitação , Lamina Tipo A/química , Lamina Tipo A/genética , Microscopia de Fluorescência , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estresse Oxidativo/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Prenilação de Proteína/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética
3.
Histochem Cell Biol ; 146(5): 569-584, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27393496

RESUMO

Four human Ankrd2 transcripts, reported in the Ensembl database, code for distinct protein isoforms (360, 333, 327 and 300 aa), and so far, their existence, specific expression and localization patterns have not been studied in detail. Ankrd2 is preferentially expressed in the slow fibers of skeletal muscle. It is found in both the nuclei and the cytoplasm of skeletal muscle cells, and its localization is prone to change during differentiation and upon stress. Ankrd2 has also been detected in the heart, in ventricular cardiomyocytes and in the intercalated disks (ICDs). The main objective of this study was to distinguish between the Ankrd2 isoforms and to determine the contribution of each one to the general profile of Ankrd2 expression in striated muscles. We demonstrated that the known expression and localization pattern of Ankrd2 in striated muscle can be attributed to the isoform of 333 aa which is dominant in both tissues, while the designated cardiac and canonical isoform of 360 aa was less expressed in both tissues. The 360 aa isoform has a distinct nuclear localization in human skeletal muscle, as well as in primary myoblasts and myotubes. In contrast to the isoform of 333 aa, it was not preferentially expressed in slow fibers and not localized to the ICDs of human cardiomyocytes. Regulation of the expression of both isoforms is achieved at the transcriptional level. Our results set the stage for investigation of the specific functions and interactions of the Ankrd2 isoforms in healthy and diseased human striated muscles.


Assuntos
Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Humanos , Proteínas Musculares/análise , Proteínas Musculares/química , Músculo Esquelético/patologia , Miocárdio/patologia , Proteínas Nucleares/análise , Proteínas Nucleares/química , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/análise , Proteínas Repressoras/química , Alinhamento de Sequência
4.
Histochem Cell Biol ; 143(6): 583-97, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25585647

RESUMO

Muscle-specific mechanosensors Ankrd2/Arpp (ankyrin repeat protein 2) and Ankrd1/CARP (cardiac ankyrin repeat protein) have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. In skeletal muscle myofibrils, Ankrd2 has a structural role as a component of a titin associated stretch-sensing complex, while in the nucleus it exerts regulatory function as transcriptional co-factor. It is also involved in myogenic differentiation and coordination of myoblast proliferation. Although expressed in the heart, the role of Ankrd2 in the cardiac muscle is completely unknown. Recently, we have shown that hypertrophic and dilated cardiomyopathy pathways are altered upon Ankrd2 silencing suggesting the importance of this protein in cardiac tissue. Here we provide the underlying basis for the functional investigation of Ankrd2 in the heart. We confirmed reduced Ankrd2 expression levels in human heart in comparison with Ankrd1 using RNAseq and Western blot. For the first time we demonstrated that, apart from the sarcomere and nucleus, both proteins are localized to the intercalated disks of human cardiomyocytes. We further tested the expression and localization of endogenous Ankrd2 in rat neonatal cardiomyocytes, a well-established model for studying cardiac-specific proteins. Ankrd2 was found to be expressed in both the cytoplasm and nucleus, independently from maturation status of cardiomyocytes. In contrast to Ankrd1, it is not responsive to the cardiotoxic drug Doxorubicin, suggesting that different mechanisms govern their expression in cardiac cells.


Assuntos
Proteínas Musculares/análise , Músculo Esquelético/química , Miocárdio/química , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/análise , Proteínas Repressoras/análise , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Imuno-Histoquímica , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Proteínas Nucleares/metabolismo , Ratos , Ratos Wistar , Proteínas Repressoras/metabolismo
5.
Electrophoresis ; 36(24): 3097-100, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26383991

RESUMO

Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders morphologically characterized by foci of myofibril dissolution, disintegration of the Z-disk, and insoluble protein aggregates within the muscle fibers. The diagnosis is based on muscle biopsy. Light and electron microscopy has a central role in the diagnostic work up, and immunohistochemistry shows abnormal deposition of several proteins including αB-crystallin, desmin, and myotilin. In contrast, immunoblotting does not have any diagnostic value because it does not highlight differences in the amount of involved proteins. We investigated the pattern and level expression of desmin, αB-crystallin, myotilin, and ZASP (Z-band alternatively spliced PDZ motif-containing protein) in muscle of seven patients with MFMs by immunoblotting after SDS-PAGE and 2D-PAGE using two different solubilizing solutions, one radioimmunoprecipitation assay (RIPA) buffer, and the other urea-containing buffer. Our data demonstrated that urea-containing buffer improves the solubilization and recovery of desmin, αB-crystallin, myotilin, and ZASP as compared with RIPA buffer and that the total content of these proteins is increased in muscles of patients. The present results provide evidence that immunoblotting is an additional tool for confirming diagnosis of MFMs.


Assuntos
Biomarcadores/análise , Biomarcadores/química , Immunoblotting/métodos , Miopatias Congênitas Estruturais/diagnóstico , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/química , Conectina/análise , Conectina/química , Cristalinas/análise , Cristalinas/química , Eletroforese em Gel Bidimensional , Humanos , Proteínas com Domínio LIM/análise , Proteínas com Domínio LIM/química , Proteínas dos Microfilamentos
6.
Arch Biochem Biophys ; 569: 45-53, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25677450

RESUMO

Transcription factor Nkx2.5, essential for heart development, regulates cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted (GATA4 and dHAND) or ubiquitous (p300) transcription regulators. Here we demonstrate that Nkx2.5 and p53 synergistically activate the promoter of the striated muscle stress responsive transcriptional cofactor Ankrd2, involved in coordination of proliferation and apoptosis during myogenic differentiation. Moreover, the p53 protein is able to interact with both wild type Nkx2.5 and its mutant ΔNkx2.5 (aa 1-198) found in patients with diverse cardiac malformations. Nkx2.5 interaction site of p53 maps to the C terminal region, while p53 binding site on Nkx2.5 lies outside its C terminus. In addition, overexpression of Nkx2.5 has a modulatory, promoter dependent effect on p53 transactivation, while the mutant significantly abolished p53 activity on the Mdm2, p21(WAF1/CIP1) and Bax promoters. Their physical interaction contributes to the observed behavior in the case of the Mdm2 promoter. Our data provide a new evidence for the role of p53 in cardiac function through interaction with Nkx2.5.


Assuntos
Proteínas de Homeodomínio/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Sítios de Ligação/genética , Células COS , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação da Expressão Gênica , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
7.
Circ Res ; 109(7): 758-69, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21799151

RESUMO

RATIONALE: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique ß-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function. OBJECTIVE: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. METHODS AND RESULTS: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis ("mechanoptosis"). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. CONCLUSIONS: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.


Assuntos
Insuficiência Cardíaca/metabolismo , Coração/fisiopatologia , Mecanotransdução Celular , Proteínas Musculares/deficiência , Miocárdio/metabolismo , Adaptação Fisiológica , Animais , Animais Geneticamente Modificados , Apoptose , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Conectina , Modelos Animais de Doenças , Ecocardiografia , Fibrose , Genótipo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Miocárdio/patologia , Fenótipo , Interferência de RNA , Ratos , Sarcômeros/metabolismo , Estresse Mecânico , Transfecção , Proteína Supressora de Tumor p53/metabolismo
8.
Crit Rev Clin Lab Sci ; 48(5-6): 269-94, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22185618

RESUMO

Remodeling is a stringently controlled process that enables adequate response of muscle cells to constant physical stresses. In this process, different kinds of stimuli have to be sensed and converted into biochemical signals that ultimately lead to alterations of muscle phenotype. Several multiprotein complexes located in the sarcomere and organized on the titin molecular spring have been identified as stress sensors and signal transducers. In this review, we focus on Ankrd1/CARP and Ankrd2/Arpp proteins,which belong to the muscle ankyrin repeat protein family (MARP) involved in a mechano-signaling pathway that links myofibrillar stress response to muscle gene expression. Apart from the mechanosensory function, they have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. Their altered expression has been demonstrated in neuromuscular disorders, cardiovascular diseases, as well as in tumors, suggesting a role in pathological processes. Although analyzed in a limited number of patients, there is a considerable body of evidence that MARP proteins could be suitable candidates for prognostic and diagnostic biomarkers.


Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Adaptação Fisiológica/fisiologia , Biomarcadores/metabolismo , Doenças Cardiovasculares/metabolismo , Humanos , Desenvolvimento Muscular/fisiologia , Neoplasias/metabolismo , Doenças Neuromusculares/metabolismo
9.
Exp Cell Res ; 316(3): 297-313, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19932097

RESUMO

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.


Assuntos
Actinina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plectina/metabolismo , Animais , Proteínas de Transporte/química , Extratos Celulares , Células Cultivadas , Proteínas do Citoesqueleto/química , Distonina , Humanos , Soros Imunes , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Miocárdio/citologia , Miocárdio/ultraestrutura , Proteínas do Tecido Nervoso/química , Plectina/deficiência , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Sequências Repetitivas de Aminoácidos
10.
Sci Adv ; 7(22)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34049882

RESUMO

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.

11.
Arch Biochem Biophys ; 502(1): 60-7, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20599664

RESUMO

The muscle ankyrin repeat protein (MARP) family member Ankrd1/CARP is a part of the titin-mechanosensory signaling complex in the sarcomere and in response to stretch it translocates to the nucleus where it participates in the regulation of cardiac genes as a transcriptional co-repressor. Several studies have focused on its structural role in muscle, but its regulatory role is still poorly understood. To gain more insight into the regulatory function of Ankrd1/CARP we searched for transcription factors that could interact and modulate its activity. Using protein array methodology we identified the tumor suppressor protein p53 as an Ankrd1/CARP interacting partner and confirmed their interaction both in vivo and in vitro. We demonstrate a novel role for Ankrd1/CARP as a transcriptional co-activator, moderately up regulating p53 activity. Furthermore, we show that p53 operates as an upstream effector of Ankrd1/CARP, by up regulating the proximal ANKRD1 promoter. Our findings suggest that, besides acting as a transcriptional co-repressor, Ankrd1/CARP could have a stimulatory effect on gene expression in cultured skeletal muscle cells. It is probable that Ankrd1/CARP has a role in the propagation of signals initiated by myogenic regulatory factors (MRFs) during myogenesis.


Assuntos
Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Repetição de Anquirina , Sequência de Bases , Células COS , Linhagem Celular , Chlorocebus aethiops , Primers do DNA/genética , Humanos , Técnicas In Vitro , Camundongos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteína MyoD/genética , Proteína MyoD/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Análise Serial de Proteínas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Ativação Transcricional , Proteína Supressora de Tumor p53/genética
12.
Mol Cell Biol ; 27(1): 244-52, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17074808

RESUMO

Myotilin, palladin, and myopalladin form a novel small subfamily of cytoskeletal proteins that contain immunoglobulin-like domains. Myotilin is a thin filament-associated protein localized at the Z-disk of skeletal and cardiac muscle cells. The direct binding to F-actin, efficient cross-linking of actin filaments, and prevention of induced disassembly of filaments are key roles of myotilin that are thought to be involved in structural maintenance and function of the sarcomere. Missense mutations in the myotilin-encoding gene cause dominant limb girdle muscular dystrophy type 1A and spheroid body myopathy and are the molecular defect that can cause myofibrillar myopathy. Here we describe the generation and analysis of mice that lack myotilin, myo(-/-) mice. Surprisingly, myo(-/-) mice maintain normal muscle sarcomeric and sarcolemmal integrity. Also, loss of myotilin does not cause alterations in the heart or other organs of newborn or adult myo(-/-) mice. The mice develop normally and have a normal life span, and their muscle capacity does not significantly differ from wild-type mice even after prolonged physical stress. The results suggest that either myotilin does not participate in muscle development and basal function maintenance or other proteins serve as structural and functional compensatory molecules when myotilin is absent.


Assuntos
Deleção de Genes , Regulação da Expressão Gênica , Proteínas Musculares/fisiologia , Músculos/fisiologia , Actinas/metabolismo , Animais , Genótipo , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos , Músculo Esquelético/metabolismo , Músculos/metabolismo , Mutação de Sentido Incorreto , Miocárdio/metabolismo , Estrutura Terciária de Proteína , Sarcômeros/metabolismo , Fatores de Tempo
13.
Biochim Biophys Acta ; 1783(6): 1023-35, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18302940

RESUMO

Ankrd2 is a member of the Muscle Ankyrin Repeat Protein family (MARPs), consisting of sarcomere-associated proteins that can also localize in the nucleus. There are indications that MARPs might function as shuttle proteins between the cytoplasm and nucleus, likely sending information to the nucleus concerning the changes in the structure or function of the contractile machinery. Even though recent findings suggest that the MARP gene family is not essential for the basal functioning of skeletal muscle, its influence on the gene expression program of skeletal muscle cells was highlighted. To investigate this regulatory role we produced and examined both morphological and functional features of myocytes stable overexpressing or silencing the Ankrd2 protein. The transcriptional profiles of the myocytes revealed that the molecular pathways perturbed by changes in Ankrd2 protein level are congruent with the morpho-physiological and biochemical data obtained in Ankrd2-modified myoblasts induced to differentiate. Our results suggest that Ankrd2 gives an important contribution to the coordination of proliferation and apoptosis during myogenic differentiation in vitro, mainly through the p53 network.


Assuntos
Ciclo Celular/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/citologia , Mioblastos/metabolismo , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Expressão Gênica/fisiologia , Perfilação da Expressão Gênica , Camundongos , Células Musculares/citologia , Células Musculares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Transfecção
14.
Cell Tissue Res ; 337(3): 463-76, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19603185

RESUMO

In humans, mutations in ZASP (the gene for Z-band alternatively spliced PDZ-motif protein) are associated with dilated cardiomyopathy and left ventricular non-compaction. In particular, mutations in or around the Zasp motif seem to be related to myofibrillar myopathy. Thus, "zaspopathies" include symptoms such as Z-line disgregation, proximal and distal muscle weakness, cardiomyopathies, and peripheral neuropathies. In order to understand the role of ZASP in muscle structure and function, we have performed a molecular characterization of the Drosophila ortholog of human ZASP and a functional analysis following the post-transcriptional silencing of the Drosophila gene. Transcriptional analysis of dzasp has revealed six additional exons, with respect to the known 16, and multiple splice variants. We have produced transgenic lines harboring constructs that, through the use of the UAS/Gal4 binary system, have enabled us to drive dsRNA interference of dzasp in a tissue-specific manner. Knockdown individuals show locomotor defects associated with alterations of muscle structure and ultrastructure, consistent with a role of dzasp specifically in the maintenance of muscular integrity.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inativação Gênica , Interferência de RNA , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Hipertrofia/metabolismo , Proteínas com Domínio LIM , Larva , Locomoção/genética , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/diagnóstico por imagem , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ultrassonografia
15.
Acta Neuropathol ; 117(3): 293-307, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19151983

RESUMO

Myofibrillar myopathies (MFMs) are rare inherited or sporadic progressive neuromuscular disorders with considerable clinical and genetic heterogeneity. In the current study, we have analyzed histopathological and immunohistochemical characteristics in genetically identified MFMs. We performed a morphological and morphometrical study in a cohort of 24 genetically identified MFM patients (12 desmin, 6 alphaB-crystallin, 4 ZASP, 2 myotilin), and an extensive immunohistochemical study in 15 of these patients, using both well-known and novel antibodies directed against distinct compartments of the muscle fibers, including Z-disc and M-band proteins. Our morphological data revealed some significant differences between the distinct MFM subgroups: the consistent presence of 'rubbed-out' fibers in desminopathies and alphaB-crystallinopathies, an elevated frequency of vacuoles in ZASPopathies and myotilinopathies, and the presence of a few necrotic fibers in the two myotilinopathy patients. Immunohistochemistry showed that in MFM only a subset of Z-disc proteins, such as filamin C and its ligands myotilin and Xin, exhibited significant alterations in their localization, whereas other Z-disc proteins like alpha-actinin, myopodin and tritopodin, did not. In contrast, M-band proteins revealed no abnormalities in MFM. We conclude that the presence of 'rubbed-out' fibers are a suggestive feature for desminopathy or alphaB-crystallinopathy, and that MFM is not a general disease of the myofibril, but primarily affects a subgroup of stress-responsive Z-disc proteins.


Assuntos
Proteínas Contráteis/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/patologia , Miofibrilas/patologia , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Biópsia , Estudos de Coortes , Conectina , Desmina/genética , Feminino , Filaminas , Humanos , Imuno-Histoquímica , Proteínas com Domínio LIM , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Miofibrilas/metabolismo , Necrose/patologia , Vacúolos/patologia , Cadeia B de alfa-Cristalina/genética
16.
Oxid Med Cell Longev ; 2019: 7318796, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428229

RESUMO

Ankrd2 (ankyrin repeats containing domain 2) or Arpp (ankyrin repeat, PEST sequence, and proline-rich region) is a member of the muscle ankyrin repeat protein family. Ankrd2 is mostly expressed in skeletal muscle, where it plays an intriguing role in the transcriptional response to stress induced by mechanical stimulation as well as by cellular reactive oxygen species. Our studies in myoblasts from Emery-Dreifuss muscular dystrophy 2, a LMNA-linked disease affecting skeletal and cardiac muscles, demonstrated that Ankrd2 is a lamin A-binding protein and that mutated lamins found in Emery-Dreifuss muscular dystrophy change the dynamics of Ankrd2 nuclear import, thus affecting oxidative stress response. In this review, besides describing the latest advances related to Ankrd2 studies, including novel discoveries on Ankrd2 isoform-specific functions, we report the main findings on the relationship of Ankrd2 with A-type lamins and discuss known and potential mechanisms involving defective Ankrd2-lamin A interplay in the pathogenesis of muscular laminopathies.


Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Humanos , Lamina Tipo A/metabolismo , Mecanotransdução Celular , Proteínas Musculares/química , Distrofia Muscular de Emery-Dreifuss/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/química
17.
PLoS One ; 13(9): e0204312, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30252882

RESUMO

Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.


Assuntos
Repetição de Anquirina , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Condicionamento Físico Animal , Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Humanos , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Filogenia , Alinhamento de Sequência , Estresse Fisiológico , Sintenia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
18.
Structure ; 12(4): 611-22, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15062084

RESUMO

Z band alternately spliced PDZ-containing protein (ZASP) is a sarcomere Z disk protein expressed in human cardiac and skeletal muscle that is thought to be involved in a dominant familial dilated cardiomyopathy. The N-terminal PDZ domain of ZASP interacts with the C terminus of alpha-actinin-2, the major component of the Z disk, probably by forming a ternary complex with titin Z repeats. We have determined the structure of ZASP PDZ by NMR and showed that it is a classical class 1 PDZ domain that recognizes the carboxy-terminal sequence of an alpha-actinin-2 calmodulin-like domain with micromolar affinity. We also characterized the role of each component in the ternary complex ZASP/alpha-actinin-2/titin, showing that the alpha-actinin-2/ZASP PDZ interaction involves a binding surface distinct from that recognized by the titin Z repeats. ZASP PDZ structure was used to model other members of the enigma family by homology and to predict their abilities to bind alpha-actinin-2.


Assuntos
Proteínas de Transporte/química , Proteínas de Homeodomínio/química , Sarcômeros/química , Actinina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Conectina , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas com Domínio LIM , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Musculares/metabolismo , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Sarcômeros/metabolismo
19.
Biochim Biophys Acta ; 1588(1): 33-40, 2002 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-12379311

RESUMO

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G). We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. The possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/biossíntese , Doenças Neuromusculares/genética , Actinina/análise , Biópsia , Conectina , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/genética , Histocitoquímica , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/análise , Proteínas Musculares/genética , Atrofia Muscular Espinal/genética , Distrofias Musculares/genética , Distrofia Muscular de Duchenne/genética , Mutação , Miopatias da Nemalina/genética , Doenças Neuromusculares/metabolismo , Sarcômeros/metabolismo
20.
J Am Coll Cardiol ; 42(11): 2014-27, 2003 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-14662268

RESUMO

OBJECTIVES: We evaluated the role of Cypher/ZASP in the pathogenesis of dilated cardiomyopathy (DCM) with or without isolated non-compaction of the left ventricular myocardium (INLVM). BACKGROUND: Dilated cardiomyopathy, characterized by left ventricular dilation and systolic dysfunction with signs of heart failure, is genetically transmitted in 30% to 40% of cases. Genetic heterogeneity has been identified with mutations in multiple cytoskeletal and sarcomeric genes causing the phenotype. In addition, INLVM with a hypertrophic dilated left ventricle, ventricular dysfunction, and deep trabeculations, is also inherited, and the genes identified to date differ from those causing DCM. Cypher/ZASP is a newly identified gene encoding a protein that is a component of the Z-line in both skeletal and cardiac muscle. METHODS: Diagnosis of DCM was performed by echocardiogram, electrocardiogram, and physical examination. In addition, levels of the muscular isoform of creatine kinase were measured to evaluate for skeletal muscle involvement. Cypher/ZASP was screened by denaturing high performance liquid chromatography (DHPLC) and direct deoxyribonucleic acid sequencing. RESULTS: We identified and screened 100 probands with left ventricular dysfunction. Five mutations in six probands (6% of cases) were identified in patients with familial or sporadic DCM or INLVM. In vitro studies showed cytoskeleton disarray in cells transfected with mutated Cypher/ZASP. CONCLUSIONS: These data suggest that mutated Cypher/ZASP can cause DCM and INLVM and identify a mechanistic basis.


Assuntos
Cardiomiopatia Dilatada/genética , Proteínas de Transporte/genética , Ventrículos do Coração/patologia , Proteínas de Homeodomínio/genética , Proteínas Musculares/genética , Mutação , Disfunção Ventricular Esquerda/genética , Proteínas Adaptadoras de Transdução de Sinal , Northern Blotting , Western Blotting , Cardiomiopatia Dilatada/diagnóstico , Cromatografia Líquida de Alta Pressão , Ecocardiografia , Humanos , Imuno-Histoquímica , Proteínas com Domínio LIM , Mutagênese , Transfecção
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