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1.
Mol Carcinog ; 58(10): 1897-1907, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31313392

RESUMO

The mechanism of hepatocellular carcinoma (HCC) metastasis remains poorly understood. Tropomodulin 3 (TMOD3) is a member of the pointed end capping protein family that contributes to invasion and metastasis in several types of malignancies. It has been found to be crucial for the membranous skeleton and embryonic development, although, its role in HCC progression remains largely unclear. We observed increased levels of Tmod3 in HCCs, especially in extrahepatic metastasis. High Tmod3 expression correlated with aggressive carcinoma and poor patient with HCC survival. Loss-of-function studies conducted by us determined Tmod3 as an oncogene that promoted HCC growth and metastasis. Mechanistically, Tmod3 increases transcription of matrix metalloproteinase-2, -7, and -9 which required PI3K-AKT. Interaction between Tmod3 and epidermal growth factor receptor (EGFR) that supports the activation of EGFR phosphorylation, is essential for signaling activation of PI3K-AKT viral oncogene homolog. These findings reveal that Tmod3 enhances aggressive behavior of HCC both in vitro and in vivo by interacting with EFGR and by activating the PI3K-AKT signaling pathway.


Assuntos
Carcinoma Hepatocelular/genética , Proliferação de Células/genética , Neoplasias Hepáticas/genética , Tropomodulina/genética , Animais , Carcinoma Hepatocelular/patologia , Progressão da Doença , Intervalo Livre de Doença , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/genética
2.
PLoS One ; 14(7): e0219932, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339916

RESUMO

The distal tubule and collecting duct in kidney regulate water homeostasis. TMOD1 is an actin capping protein that plays an important role in controlling the organization of actin filaments. In this study, we found TMOD1 was specifically expressed in distal tubules and collecting ducts. To investigate the role of TMOD1, we created Tmod1flox/flox mice and bred them with Ksp-Cre mice to generate tubule-specific Tmod1 knockout mice, Tmod1flox/flox/Ksp-Cre+ (designated as TFK). As compared with control mice, TFK mice showed oliguria, hyperosmolality urine, and high blood pressure. To determine the mechanisms underlying this phenotype, we performed label-free quantitative proteomics on kidneys of TFK and control mice. Total of 83 proteins were found differentially expressed. Bioinformatic analysis indicated that biological processes, including protein phosphorylation and metabolic process, were involved in TMOD1 regulatory network. Gene set enrichment analysis showed that multiple pathways, such as phosphatidylinositol signaling system and GnRH signaling pathway, were strongly associated with Tmod1 knockout. Western blot validated the down-regulation of three proteins, TGFBR2, SLC25A11, and MTFP1, in kidneys of TFK mice. Our study provides valuable information on the molecular functions and the regulatory network of Tmod1 gene in kidney, as well as the new mechanisms for the regulation of water balance.


Assuntos
Redes Reguladoras de Genes , Proteoma/genética , Tropomodulina/genética , Equilíbrio Hidroeletrolítico , Animais , Rim/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteoma/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Tropomodulina/deficiência
3.
Oncol Rep ; 41(5): 3060-3068, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864730

RESUMO

Tropomodulin 3 (TMOD3) is a member of the pointed­end capping protein family that contributes to invasion and metastasis in several types of malignancies. TMOD3 has been found to be crucial for membranous skeleton and embryonic development; however, little is known regarding the role of TMOD3 in liver cancer progression. In addition, to the best of our knowledge, no previous studies have investigated the mechanism underlying the TMOD3­regulated promotion of liver cancer. The aim of the present study was to determine whether TMOD3 is associated with liver cancer progression. TMOD3 expression was found to be elevated in liver cancer cells and tissues. In the in vitro experiments, liver cancer cell proliferation, invasion and migration were inhibited by TMOD3 knockdown and promoted by ectopic expression of TMOD3. Furthermore, mechanistic analysis indicated that TMOD3 overexpression activated mitogen­activated protein kinase (MAPK)/extracellular signal­regulated kinase (ERK) signaling and increased the levels of other targets of this pathway, including matrix metalloproteinase (MMP)2, MMP9 and cyclin D1. TMOD3 overexpression was associated with changes in liver cancer cell morphology and altered expression of epithelial and mesenchymal markers. High TMOD3 expression was hypothesized to promote epithelial­to­mesenchymal transition in liver cancer cells. In conclusion, TMOD3 was shown to promote liver cancer cell growth, invasion and migration through the MAPK/ERK signaling pathway, and it may serve as a candidate biomarker and therapeutic target in liver cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases , Tropomodulina/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Tropomodulina/genética
4.
J Cell Sci ; 132(4)2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30659118

RESUMO

The actin cytoskeleton is subjected to dynamic mechanical forces over time and the history of force loading may serve as mechanical preconditioning. While the actin cytoskeleton is known to be mechanosensitive, the mechanisms underlying force regulation of actin dynamics still need to be elucidated. Here, we investigated actin depolymerization under a range of dynamic tensile forces using atomic force microscopy. Mechanical loading by cyclic tensile forces induced significantly enhanced bond lifetimes and different force-loading histories resulted in different dissociation kinetics in G-actin-G-actin and G-actin-F-actin interactions. Actin subunits at the two ends of filaments formed bonds with distinct kinetics under dynamic force, with cyclic mechanical reinforcement more effective at the pointed end compared to that at the barbed end. Our data demonstrate force-history dependent reinforcement in actin-actin bonds and polarity of the actin depolymerization kinetics under cyclic tensile forces. These properties of actin may be important clues to understanding regulatory mechanisms underlying actin-dependent mechanotransduction and mechanosensitive cytoskeletal dynamics.This article has an associated First Person interview with the first author of the paper.


Assuntos
Actinas/química , Proteínas Aviárias/química , Proteína de Capeamento de Actina CapZ/química , Mecanotransdução Celular , Imagem Individual de Molécula/métodos , Tropomodulina/química , Citoesqueleto de Actina , Actinas/genética , Actinas/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Galinhas , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Microscopia de Força Atômica , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/instrumentação , Estresse Mecânico , Tropomodulina/genética , Tropomodulina/metabolismo
5.
Eur J Hum Genet ; 27(1): 150-159, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30254217

RESUMO

The Central Asian Kyrgyz highland population provides a unique opportunity to address genetic diversity and understand the genetic mechanisms underlying high-altitude pulmonary hypertension (HAPH). Although a significant fraction of the population is unaffected, there are susceptible individuals who display HAPH in the absence of any lung, cardiac or hematologic disease. We report herein the analysis of the whole-genome sequencing of healthy individuals compared with HAPH patients and other controls (total n = 33). Genome scans reveal selection signals in various regions, encompassing multiple genes from the first whole-genome sequences focusing on HAPH. We show here evidence of three candidate genes MTMR4, TMOD3 and VCAM1 that are functionally associated with well-known molecular and pathophysiological processes and which likely lead to HAPH in this population. These processes are (a) dysfunctional BMP signaling, (b) disrupted tissue repair processes and (c) abnormal endothelial cell function. Whole-genome sequence of well-characterized patients and controls and using multiple statistical tools uncovered novel candidate genes that belong to pathways central to the pathogenesis of HAPH. These studies on high-altitude human populations are pertinent to the understanding of sea level diseases involving hypoxia as a main element of their pathophysiology.


Assuntos
Hipertensão Pulmonar/genética , Polimorfismo Genético , Altitude , Estudo de Associação Genômica Ampla , Humanos , Quirguistão , Proteínas Tirosina Fosfatases não Receptoras/genética , Tropomodulina/genética , Molécula 1 de Adesão de Célula Vascular/genética
6.
Arch Biochem Biophys ; 630: 18-26, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28732641

RESUMO

Correct assembly of thin filaments composed of actin and actin-binding proteins is of crucial importance for properly functioning muscle cells. Tropomyosin (Tpm) mediates the binding of tropomodulin (Tmod) and leiomodin (Lmod) at the slow-growing, or pointed, ends of the thin filaments. Together these proteins regulate thin filament lengths and actin dynamics in cardiac muscle. The K15N mutation in the TPM1 gene is associated with familial dilated cardiomyopathy (DCM) but the effect of this mutation on Tpm's function is unknown. In this study, we introduced the K15N mutation in striated muscle α-Tpm (Tpm1.1) and investigated its interaction with actin, Tmod and Lmod. The mutation caused a ∼3-fold decrease in the affinity of Tpm1.1 for actin. The binding of Lmod and Tmod to Tpm1.1-covered actin filaments also decreased in the presence of the K15N mutation. Furthermore, the K15N mutation in Tpm1.1 disrupted the inhibition of actin polymerization and affected the competition between Tmod1 and Lmod2 for binding at the pointed ends. Our data demonstrate that the K15N mutation alters pointed end dynamics by affecting molecular interactions between Tpm1.1, Lmod2 and Tmod1.


Assuntos
Cardiomiopatia Dilatada/genética , Mutação de Sentido Incorreto , Tropomiosina/química , Tropomiosina/genética , Substituição de Aminoácidos , Cardiomiopatia Dilatada/metabolismo , Tropomodulina/química , Tropomodulina/genética , Tropomodulina/metabolismo , Tropomiosina/metabolismo
7.
Biophys J ; 112(9): 1742-1760, 2017 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-28494946

RESUMO

Cytoskeletal structures characterized by actin filaments with uniform lengths, including the thin filaments of striated muscles and the spectrin-based membrane skeleton, use barbed and pointed-end capping proteins to control subunit addition/dissociation at filament ends. While several proteins cap the barbed end, tropomodulins (Tmods), a family of four closely related isoforms in vertebrates, are the only proteins known to specifically cap the pointed end. Tmods are ∼350 amino acids in length, and comprise alternating tropomyosin- and actin-binding sites (TMBS1, ABS1, TMBS2, and ABS2). Leiomodins (Lmods) are related in sequence to Tmods, but display important differences, including most notably the lack of TMBS2 and the presence of a C-terminal extension featuring a proline-rich domain and an actin-binding WASP-Homology 2 domain. The Lmod subfamily comprises three somewhat divergent isoforms expressed predominantly in muscle cells. Biochemically, Lmods differ from Tmods, acting as powerful nucleators of actin polymerization, not capping proteins. Structurally, Lmods and Tmods display crucial differences that correlate well with their different biochemical activities. Physiologically, loss of Lmods in striated muscle results in cardiomyopathy or nemaline myopathy, whereas complete loss of Tmods leads to failure of myofibril assembly and developmental defects. Yet, interpretation of some of the in vivo data has led to the idea that Tmods and Lmods are interchangeable or, at best, different variants of two subfamilies of pointed-end capping proteins. Here, we review and contrast the existing literature on Tmods and Lmods, and propose a model of Lmod function that attempts to reconcile the in vitro and in vivo data, whereby Lmods nucleate actin filaments that are subsequently capped by Tmods during sarcomere assembly, turnover, and repair.


Assuntos
Proteínas Musculares/metabolismo , Tropomodulina/metabolismo , Animais , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Músculos/metabolismo , Tropomodulina/química , Tropomodulina/genética
8.
Invest Ophthalmol Vis Sci ; 57(10): 4084-99, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27537257

RESUMO

PURPOSE: To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein. METHODS: We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers. RESULTS: F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, ß2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, ß2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers. CONCLUSIONS: These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a ß2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.


Assuntos
Actinas/genética , DNA/genética , Cristalino/ultraestrutura , Mutação , Tropomodulina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Diferenciação Celular , Células Cultivadas , Análise Mutacional de DNA , Modelos Animais de Doenças , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Tropomodulina/metabolismo
9.
Mol Biol Cell ; 27(16): 2565-75, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27307584

RESUMO

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Galinhas , Proteínas do Citoesqueleto/genética , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Miócitos Cardíacos/metabolismo , Ligação Proteica , Domínios Proteicos , Sarcômeros/metabolismo , Tropomodulina/genética , Tropomodulina/metabolismo , Tropomiosina/metabolismo
10.
Cytoskeleton (Hoboken) ; 73(6): 316-28, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27126680

RESUMO

Tropomodulins (Tmods) cap F-actin pointed ends and have altered expression in the brain in neurological diseases. The function of Tmods in neurons has been poorly studied and their role in neurological diseases is entirely unknown. In this article, we show that Tmod1 and Tmod2, but not Tmod3, are positive regulators of dendritic complexity and dendritic spine morphology. Tmod1 increases dendritic branching distal from the cell body and the number of filopodia/thin spines. Tmod2 increases dendritic branching proximal to the cell body and the number of mature dendritic spines. Tmods utilize two actin-binding sites and two tropomyosin (Tpm)-binding sites to cap F-actin. Overexpression of Tmods with disrupted Tpm-binding sites indicates that Tmod1 and Tmod2 differentially utilize their Tpm- and actin-binding sites to affect morphology. Disruption of Tmod1's Tpm-binding sites abolished the overexpression phenotype. In contrast, overexpression of the mutated Tmod2 caused the same phenotype as wild type overexpression. Proximity ligation assays indicate that the mutated Tmods are shuttled similarly to wild type Tmods. Our data begins to uncover the roles of Tmods in neural development and the mechanism by which Tmods alter neural morphology. These observations in combination with altered Tmod expression found in several neurological diseases also suggest that dysregulation of Tmod expression may be involved in the pathology of these diseases. © 2016 Wiley Periodicals, Inc.


Assuntos
Dendritos/metabolismo , Tropomodulina/metabolismo , Animais , Dendritos/genética , Mutação , Células PC12 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Tropomodulina/genética
11.
Biochim Biophys Acta ; 1864(5): 523-30, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26873245

RESUMO

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein-protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7-41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.


Assuntos
Cardiomiopatia Dilatada/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Tropomodulina/metabolismo , Tropomiosina/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Sequência de Aminoácidos/genética , Sítios de Ligação , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Dicroísmo Circular , Humanos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Estriado/química , Músculo Estriado/metabolismo , Músculo Estriado/patologia , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Tropomodulina/química , Tropomodulina/genética , Tropomiosina/química , Tropomiosina/genética
12.
Int J Oncol ; 48(2): 607-12, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26718916

RESUMO

Tropomodulin1 (TMOD1), which regulates the length and depolymerization of actin filaments by binding to the pointed end of the actin filament, has been reported to be a powerful diagnostic marker for ALK-negative anaplastic large-cell lymphoma; however, little is known about the relevance of TMOD1 in the behavior of oral squamous cell carcinoma (OSCC). We evaluated TMOD1 expression in OSCC-derived cell lines and primary OSCC samples (n=200) using quantitative reverse transcriptase-polymerase chain reaction, immunoblotting and semi-quantitative immunohistochemistry. We also analyzed the clinical correlation between TMOD1 expression status and clinical parameters in patients with OSCC and performed a prospective study using 40 primary OSCC samples. TMOD1 expression was upregulated significantly (p<0.05) in OSCC in vitro and in vivo compared with normal counterparts. TMOD1 expression also was correlated significantly (p=0.0199 and p=0.0064, respectively) with regional lymph node metastasis (RLNM) and 5-year survival rates. This prospective study also showed that high TMOD1 expression was seen in 12 (75%) of 16 cases in RLNM-positive patients and 9 (37.5%) of 24 cases in RLNM-negative patients. The current data provide the first evidence that TMOD1 expression is a critical biomarker for RLNM and prognosis of patients with OSCC.


Assuntos
Linfonodos/patologia , Metástase Linfática/genética , Metástase Linfática/patologia , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Tropomodulina/genética , Citoesqueleto de Actina/genética , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Masculino , Prognóstico , Estudos Prospectivos
13.
Nat Commun ; 6: 8314, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26370058

RESUMO

How proteins sharing a common fold have evolved different functions is a fundamental question in biology. Tropomodulins (Tmods) are prototypical actin filament pointed-end-capping proteins, whereas their homologues, Leiomodins (Lmods), are powerful filament nucleators. We show that Tmods and Lmods do not compete biochemically, and display similar but distinct localization in sarcomeres. Changes along the polypeptide chains of Tmods and Lmods exquisitely adapt their functions for capping versus nucleation. Tmods have alternating tropomyosin (TM)- and actin-binding sites (TMBS1, ABS1, TMBS2 and ABS2). Lmods additionally contain a C-terminal extension featuring an actin-binding WH2 domain. Unexpectedly, the different activities of Tmods and Lmods do not arise from the Lmod-specific extension. Instead, nucleation by Lmods depends on two major adaptations-the loss of pointed-end-capping elements present in Tmods and the specialization of the highly conserved ABS2 for recruitment of two or more actin subunits. The WH2 domain plays only an auxiliary role in nucleation.


Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas Musculares/química , Tropomodulina/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Tropomodulina/genética , Tropomodulina/metabolismo
14.
PLoS One ; 10(8): e0136607, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26308647

RESUMO

The membrane skeleton of mature erythrocyte is formed during erythroid differentiation. Fluid shear stress is one of the main factors that promote embryonic hematopoiesis, however, its effects on erythroid differentiation and cytoskeleton remodeling are unclear. Erythrocyte tropomodulin of 41 kDa (E-Tmod41) caps the pointed end of actin filament (F-actin) and is critical for the formation of hexagonal topology of erythrocyte membrane skeleton. Our study focused on the regulation of E-Tmod41 and its role in F-actin cytoskeleton remodeling during erythroid differentiation induced by fluid shear stress. Mouse erythroleukemia (MEL) cells and embryonic erythroblasts were subjected to fluid shear stress (5 dyn/cm2) and erythroid differentiation was induced in both cells. F-actin content and E-Tmod41 expression were significantly increased in MEL cells after shearing. E-Tmod41 overexpression resulted in a significant increase in F-actin content, while the knockdown of E-Tmod41 generated the opposite result. An E-Tmod 3'UTR targeting miRNA, miR-23b-3p, was found suppressed by shear stress. When miR-23b-3p level was overexpressed / inhibited, both E-Tmod41 protein level and F-actin content were reduced / augmented. Furthermore, among the two alternative promoters of E-Tmod, PE0 (upstream of exon 0), which mainly drives the expression of E-Tmod41, was found activated by shear stress. In conclusion, our results suggest that fluid shear stress could induce erythroid differentiation and F-actin cytoskeleton remodeling. It upregulates E-Tmod41 expression through miR-23b-3p suppression and PE0 promoter activation, which, in turn, contributes to F-actin cytoskeleton remodeling.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Eritropoese/fisiologia , Regulação da Expressão Gênica , MicroRNAs/genética , Estresse Mecânico , Tropomodulina/metabolismo , Animais , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Eritrócitos/citologia , Eritrócitos/metabolismo , Feminino , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tropomodulina/genética
15.
Gene Expr Patterns ; 17(1): 45-55, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25721257

RESUMO

Full-length erythrocyte tropomodulin (E-Tmod or Tmod1) isoform of 41 kDa is an actin nucleation protein and caps the pointed end of tropomyosin-coated actin filaments. It participates in the length control of short actin protofilaments in the erythrocyte membrane skeletal network as well as the organization of microfilaments in non-erythroid cells. Recently we discovered and characterized a truncated isoform of 29 kDa, which lacks the N-terminal sequence encoded by exons 1 and 2 required for nucleation and capping. Thus, it is important to study the expression pattern of solely the E-Tmod41 isoform in tissues. We utilized our exon 1 knockout (KO) mouse model with a knock-in lacZ reporter gene which reports the expression of E-Tmod41, but not E-Tmod29. Because this homozygous isoform-specific KO is an embryonic lethal mutation, we used heterozygous mice. X-gal staining localized specific signals at the single cell level and revealed a timed expression during embryonic development and restricted expression in adult mice. Our results showed that E-Tmod41 expressing cells include developing and young erythroid cells, developing somites, young fiber cells in the lens, certain subtype(s) of tubular cells in the kidney, smooth muscle cells in various tissues, and horizontal cells in the retina. A comparison with previous studies revealed that most if not all tissues known to express E-Tmod contained lacZ-expressing cells. Interestingly, some tubular cells were lacZ-positive while others in the same renal tubule were not, indicating heterogeneity within the tubular cells. Combined with double immunocytochemistry, we further localized E-Tmod41 to dendritic spines of horizontal cells. These timed and cell-type restricted expressions of E-Tmod41 suggest a role of actin nucleation and/or short actin protofilaments in these cell types and sub-cellular structures.


Assuntos
Óperon Lac/genética , Tropomodulina/genética , Tropomodulina/metabolismo , Animais , Células Dendríticas/metabolismo , Eritrócitos/citologia , Éxons , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Especificidade de Órgãos , Isoformas de Proteínas/metabolismo , Retina/metabolismo
16.
Cancer Res ; 75(1): 62-72, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25398440

RESUMO

Triple-negative breast cancers (TNBC), which include the basal-like and claudin-low disease subtypes, are aggressive malignancies for which effective therapeutic targets are lacking. NF-κB activation has an established role in breast malignancy, and it is higher in TNBC than other breast cancer subtypes. On this basis, we hypothesized that proteins derived from NF-κB target genes might be molecular targets for TNBC therapy. In this study, we conducted a microarray-based screen for novel NF-κB-inducible proteins as candidate therapeutic targets, identifying tropomodulin 1 (TMOD1) as a lead candidate. TMOD1 expression was regulated directly by NF-κB and was significantly higher in TNBC than other breast cancer subtypes. TMOD1 elevation is associated with enhanced tumor growth in a mouse tumor xenograft model and in a 3D type I collagen culture. TMOD1-dependent tumor growth was correlated with MMP13 induction, which was mediated by TMOD1-dependent accumulation of ß-catenin. Overall, our study highlighted a novel TMOD1-mediated link between NF-κB activation and MMP13 induction, which accounts in part for the NF-κB-dependent malignant phenotype of TNBC.


Assuntos
NF-kappa B/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Tropomodulina/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/genética , Transdução de Sinais , Análise Serial de Tecidos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Tropomodulina/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Cell Sci ; 128(2): 239-50, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25431137

RESUMO

Precise regulation of thin filament length is essential for optimal force generation during muscle contraction. The thin filament capping protein tropomodulin (Tmod) contributes to thin filament length uniformity by regulating elongation and depolymerization at thin filament ends. The leiomodins (Lmod1-3) are structurally related to Tmod1-4 and also localize to actin filament pointed ends, but in vitro biochemical studies indicate that Lmods act instead as robust nucleators. Here, we examined the roles of Tmod4 and Lmod3 during Xenopus skeletal myofibrillogenesis. Loss of Tmod4 or Lmod3 resulted in severe disruption of sarcomere assembly and impaired embryonic movement. Remarkably, when Tmod4-deficient embryos were supplemented with additional Lmod3, and Lmod3-deficient embryos were supplemented with additional Tmod4, sarcomere assembly was rescued and embryonic locomotion improved. These results demonstrate for the first time that appropriate levels of both Tmod4 and Lmod3 are required for embryonic myofibrillogenesis and, unexpectedly, both proteins can function redundantly during in vivo skeletal muscle thin filament assembly. Furthermore, these studies demonstrate the value of Xenopus for the analysis of contractile protein function during de novo myofibril assembly.


Assuntos
Embrião não Mamífero , Desenvolvimento Muscular/genética , Proteínas Musculares/biossíntese , Tropomodulina/biossíntese , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Regulação da Expressão Gênica no Desenvolvimento , Contração Muscular/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Sarcômeros/genética , Sarcômeros/ultraestrutura , Tropomodulina/genética , Xenopus laevis/embriologia , Xenopus laevis/genética
18.
Dis Model Mech ; 7(12): 1407-15, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25288681

RESUMO

Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, five encode components of striated muscle sarcomeres. In a genetic zebrafish screen, the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganised and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerize to form thin filaments in the skeletal muscle of tmod4(trg) mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion and early death. Myofibrils of tmod4(trg) mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods, together with the reactivity of rods with phalloidin and an antibody against actinin, is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4(trg) mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicateTMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4(trg) mutant will be a valuable tool to study human muscle disorders.


Assuntos
Mutação , Tropomodulina/genética , Tropomodulina/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Actinina/química , Actinas/química , Alelos , Animais , Animais Geneticamente Modificados , Citoplasma/metabolismo , Modelos Animais de Doenças , Ligação Genética , Masculino , Músculos/patologia , Doenças Musculares/genética , Doenças Musculares/patologia , Miofibrilas/metabolismo , Doenças Neuromusculares/metabolismo , Faloidina/química , Fenótipo , Sarcômeros/metabolismo , Peixe-Zebra
19.
Science ; 345(6195): 463-7, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-25061212

RESUMO

Proteins that cap the ends of the actin filament are essential regulators of cytoskeleton dynamics. Whereas several proteins cap the rapidly growing barbed end, tropomodulin (Tmod) is the only protein known to cap the slowly growing pointed end. The lack of structural information severely limits our understanding of Tmod's capping mechanism. We describe crystal structures of actin complexes with the unstructured amino-terminal and the leucine-rich repeat carboxy-terminal domains of Tmod. The structures and biochemical analysis of structure-inspired mutants showed that one Tmod molecule interacts with three actin subunits at the pointed end, while also contacting two tropomyosin molecules on each side of the filament. We found that Tmod achieves high-affinity binding through several discrete low-affinity interactions, which suggests a mechanism for controlled subunit exchange at the pointed end.


Assuntos
Citoesqueleto de Actina/química , Actinas/química , Tropomodulina/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Coelhos , Tropomodulina/genética
20.
Cytoskeleton (Hoboken) ; 71(7): 395-411, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24922351

RESUMO

Tropomodulins (Tmods) interact with tropomyosins (TMs) via two TM-binding sites and cap the pointed ends of TM-coated actin filaments. To study the functional interplay between TM binding and TM-actin filament capping by Tmods, we introduced disabling mutations into the first, second, or both TM-binding sites of full-length Tmod1 (Tmod1-L27G, Tmod1-I131D, and Tmod1-L27G/I131D, respectively) and full-length Tmod3 (Tmod3-L29G, Tmod3-L134D, and Tmod3-L29G/L134D, respectively). Tmod1 and Tmod3 showed somewhat different TM-binding site utilization, but nearly all TM binding was abolished in Tmod1-L27G/I131D and Tmod3-L29G/L134D. Disruption of Tmod-TM binding had a modest effect on Tmod1's ability and no effect on Tmod3's ability to stabilize TM-actin pointed ends against latrunculin A-induced depolymerization. However, disruption of Tmod-TM binding did significantly impair the ability of Tmod3 to reduce elongation rates at pointed ends with α/ßTM, albeit less so with TM5NM1, and not at all with TM5b. For Tmod1, disruption of Tmod-TM binding only slightly impaired its ability to reduce elongation rates with α/ßTM and TM5NM1, but not at all with TM5b. Thus, Tmod-TM binding has a greater influence on Tmods' ability to inhibit subunit association as compared to dissociation from TM-actin pointed ends, particularly for α/ßTM, with Tmod3's activity being more dependent on TM binding than Tmod1's activity. Nevertheless, disruption of Tmod1-TM binding precluded Tmod1 targeting to thin filament pointed ends in cardiac myocytes, suggesting that the functional effects of Tmod-TM binding on TM-coated actin filament capping can be significantly modulated by the in vivo conformation of the pointed end or other factors in the intracellular environment.


Assuntos
Mutação/genética , Tropomodulina/genética , Tropomodulina/metabolismo , Tropomiosina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Embrião de Galinha , Humanos , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteínas Mutantes/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Polimerização/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Coelhos , Ratos , Tiazolidinas/farmacologia
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