RESUMO
Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.
Assuntos
Átrios do Coração , Peixe-Zebra , Camundongos , Animais , Peixe-Zebra/genética , Átrios do Coração/metabolismo , Ventrículos do Coração , Miosinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Pentachloropseudilin (PClP) is a reversible and allosteric inhibitor of typeâ 1 myosin. Here, we addressed the impact of PClP treatment of Trypanosoma cruzi and mammalian host cell on the parasite migration, cell adhesion and invasion. We observed that PClP was not toxic to either T.â cruzi or host cell. Moreover, treatment of T.â cruzi with PClP inhabited parasite motility, host cell adhesion and invasion. Treatment of host cell with PClP also impaired parasite invasion probably by decreasing lysosome migration to the entry site of the parasite. Therefore, PClP treatment impaired fundamental processes necessary for a successful T.â cruzi infection.
Assuntos
Hidrocarbonetos Clorados , Trypanosoma cruzi , Animais , Lisossomos , Mamíferos , Miosinas/metabolismo , Pirróis/metabolismoRESUMO
Molecular motors are microscopic machines that use energy from adenosine triphosphate (ATP) hydrolysis to generate movement. While kinesins and dynein are molecular motors associated with microtubule tracks, myosins bind to and move on actin filaments. Mammalian cells express several myosin motors. They power cellular processes such as endo- and exocytosis, intracellular trafficking, transcription, migration, and cytokinesis. As viruses navigate through cells, they may take advantage or be hindered by host components and machinery, including the cytoskeleton. This review delves into myosins' cell roles and compares them to their reported functions in viral infections. In most cases, the previously described myosin functions align with their reported role in viral infections, although not in all cases. This opens the possibility that knowledge obtained from studying myosins in viral infections might shed light on new physiological roles for myosins in cells. However, given the high number of myosins expressed and the variety of viruses investigated in the different studies, it is challenging to infer whether the interactions found are specific to a single virus or can be applied to other viruses with the same characteristics. We conclude that the participation of myosins in viral cycles is still a largely unexplored area, especially concerning unconventional myosins.
Assuntos
Miosinas , Viroses , Animais , Humanos , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Cinesinas , Viroses/metabolismo , Actinas/metabolismo , Mamíferos/metabolismoRESUMO
Myosin 1g (Myo1g) is a mechanoenzyme associated with actin filaments, expressed exclusively in hematopoietic cells, and involved in various cellular functions, including cell migration, adhesion, and membrane trafficking. Despite the importance of Myo1g in distinct functions, there is currently no monoclonal antibody (mAb) against Myo1g. mAbs are helpful tools for the detection of specific antigens in tumor cells and other tissues. The development of mAbs against targeted dysregulated molecules in cancer cells remains a crucial tool for aiding in the diagnosis and the treatment of patients. Using hybridoma technology, we generated a panel of hybridomas specific for Myo1g. ELISA, immunofluorescence, and Western blot assay results revealed the recognition of Myo1g by these novel monoclonal antibodies in normal and transformed T and B cells. Here, we report the development and application of new monoclonal antibodies against Myo1g for their potential use to detect its overexpression in acute lymphoblastic leukemia (ALL) patients.
Assuntos
Anticorpos Monoclonais , Leucemia Linfoide , Miosinas , Anticorpos Monoclonais/metabolismo , Criança , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas/metabolismo , Leucemia Linfoide/genética , Leucemia Linfoide/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Miosinas/genética , Miosinas/metabolismoRESUMO
Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.
Assuntos
Endocitose , Miosinas/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/fisiologia , Miosinas/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
To gain insight on the impact of preventive exercise during pulmonary arterial hypertension (PAH), we evaluated the gene expression of myosins and gene-encoding proteins associated with the extracellular matrix remodeling of right hypertrophied ventricles. We used 32 male Wistar rats, separated in four groups: Sedentary Control (S, n = 8); Control with Training (T, n = 8); Sedentary with Pulmonary Arterial Hypertension (SPAH, n = 8); and Pulmonary Arterial Hypertension with Training (TPAH, n = 8). All rats underwent a two-week adaptation period; T and TPAH group rats then proceeded to an eight-week training period on a treadmill. At the beginning of the 11th week, S and T groups received an intraperitoneal injection of saline, and SPAH and TPAH groups received an injection of monocrotaline (60 mg/kg). Rats in the T and TPAH groups then continued with the training protocol until the 13th week. We assessed exercise capacity, echocardiography analysis, Fulton's index, cross-sectional areas of cardiomyocytes, collagen content and types, and fractal dimension (FD). Transcript abundance of myosins and extracellular matrix genes were estimated through reverse transcription-quantitative PCR (RT-qPCR). When compared to the SPAH group, the TPAH group showed increases in functional capacity and pulmonary artery acceleration time/pulmonary ejection time ratio and decreases in Fulton's index and cross-sectional areas of myocyte cells. However, preventive exercise did not induce alterations in col1a1 and myh7 gene expression. Our findings demonstrate that preventive exercise improved functional capacity, reduced cardiac hypertrophy, and attenuated PH development without interfering in mRNA-encoding myosin and collagen expression during PAH.
Assuntos
Hipertensão Arterial Pulmonar , Animais , Colágeno/metabolismo , Hipertensão Pulmonar , Masculino , Miosinas/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Remodelação VentricularRESUMO
Trypanosomatids are a monophyletic group of parasitic flagellated protists belonging to the order Kinetoplastida. Their cytoskeleton is primarily made up of microtubules in which no actin microfilaments have been detected. Although all these parasites contain actin, it is widely thought that their actin cytoskeleton is reduced when compared to most eukaryotic organisms. However, there is increasing evidence that it is more complex than previously thought. As in other eukaryotic organisms, trypanosomatids encode for a conventional actin that is expected to form microfilament-like structures, and for members of three conserved actin-related proteins probably involved in microfilament nucleation (ARP2, ARP3) and in gene expression regulation (ARP6). In addition to these canonical proteins, also encode for an expanded set of actins and actin-like proteins that seem to be restricted to kinetoplastids. Analysis of their amino acid sequences demonstrated that, although very diverse in primary sequence when compared to actins of model organisms, modelling of their tertiary structure predicted the presence of the actin fold in all of them. Experimental characterization has been done for only a few of the trypanosomatid actins and actin-binding proteins. The most studied is the conventional actin of Leishmania donovani (LdAct), which unusually requires both ATP and Mg2+ for polymerization, unlike other conventional actins that do not require ATP. Additionally, polymerized LdAct tends to assemble in bundles rather than in single filaments. Regulation of actin polymerization depends on their interaction with actin-binding proteins. In trypanosomatids, there is a reduced but sufficient core of actin-binding proteins to promote microfilament nucleation, turnover and stabilization. There are also genes encoding for members of two families of myosin motor proteins, including one lineage-specific. Homologues to all identified actin-family proteins and actin-binding proteins of trypanosomatids are also present in Paratrypanosoma confusum (an early branching trypanosomatid) and in Bodo saltans (a closely related free-living organism belonging to the trypanosomatid sister order of Bodonida) suggesting they were all present in their common ancestor. Secondary losses of these genes may have occurred during speciation within the trypanosomatids, with salivarian trypanosomes having lost many of them and stercorarian trypanosomes retaining most.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/química , Proteínas dos Microfilamentos/química , Miosinas/química , Proteínas de Protozoários/química , Trypanosomatina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/classificação , Actinas/genética , Actinas/metabolismo , Animais , Sítios de Ligação , Expressão Gênica , Humanos , Proteínas dos Microfilamentos/classificação , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Miosinas/classificação , Miosinas/genética , Miosinas/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosomatina/classificação , Trypanosomatina/genéticaRESUMO
The myofibrillar fragmentation index (MFI) is an indicative trait for meat tenderness. Longissimus thoracis muscle samples from the 20 most extreme bulls (out of 80 bulls set) for MFI (high (n = 10) and low (n = 10) groups) trait were used to perform transcriptomic analysis, using RNA Sequencing (RNA-Seq). An average of 24.616 genes was expressed in the Nellore muscle transcriptome analysis. A total of 96 genes were differentially expressed (p value ≤ 0.001) between the two groups of divergent bulls for MFI. The HEBP2 and BDH1 genes were overexpressed in animals with high MFI. The MYBPH and MYL6, myosin encoders, were identified. The differentially expressed genes were related to increase mitochondria efficiency, especially in cells under oxidative stress conditions, and these also were related to zinc and calcium binding, membrane transport, and muscle constituent proteins, such as actin and myosin. Most of those genes were involved in metabolic pathways of oxidation-reduction, transport of lactate in the plasma membrane, and muscle contraction. This is the first study applying MFI phenotypes in transcriptomic studies to identify and understand differentially expressed genes for beef tenderness. These results suggest that differences detected in gene expression between high and low MFI animals are related to reactive mechanisms and structural components of oxidative fibers under the condition of cellular stress. Some genes may be selected as positional candidate genes to beef tenderness, MYL6, MYBPH, TRIM63, TRIM55, TRIOBP, and CHRNG genes. The use of MFI phenotypes could enhance results of meat tenderness studies.
Assuntos
Bovinos/genética , Músculo Esquelético/metabolismo , Característica Quantitativa Herdável , Carne Vermelha/normas , Transcriptoma , Animais , Bovinos/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Perfilação da Expressão Gênica , Proteínas Ligantes de Grupo Heme/genética , Proteínas Ligantes de Grupo Heme/metabolismo , Masculino , Miosinas/genética , Miosinas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismoRESUMO
The inclusion of lymphocytes in high endothelial venules and their migration to the lymph nodes are critical steps in the immune response. Cell migration is regulated by the actin cytoskeleton and myosins. Myo1e is a long-tailed class I myosin and is highly expressed in B cells, which have not been studied in the context of cell migration. By using intravital microscopy in an in vivo model and performing in vitro experiments, we studied the relevance of Myo1e for the adhesion and inclusion of activated B cells in high endothelial venules. We observed reduced expression of integrins and F-actin in the membrane protrusions of B lymphocytes, which might be explained by deficiencies in vesicular trafficking. Interestingly, the lack of Myo1e reduced the phosphorylation of focal adhesion kinase (FAK; also known as PTK2), AKT (also known as AKT1) and RAC-1, disturbing the FAK-PI3K-RAC-1 signaling pathway. Taken together, our results indicate a critical role of Myo1e in the mechanism of B-cell adhesion and migration.
Assuntos
Miosina Tipo I , Miosinas , Actinas/metabolismo , Linfócitos B/metabolismo , Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal , Linfonodos/metabolismo , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Miosinas/genética , Miosinas/metabolismo , FosforilaçãoRESUMO
Sequences derived from parvoviruses (family Parvoviridae) are relatively common in animal genomes, but the functional significance of these endogenous parvoviral element (EPV) sequences remains unclear. In this study, we used a combination of in silico and molecular biological approaches to investigate a fusion gene carried by guinea pigs (genus Cavia) that is partially derived from an EPV. This gene, named enRep-M9l, encodes a predicted polypeptide gene product comprising a partial myosin9-like (M9l) gene fused to a 3' truncated, EPV-encoded replicase. We examined the genomic and phylogenetic characteristics of the EPV locus (enRep) that encodes the viral portions of enRep-M9l, revealing that it derives from an ancient dependoparvovirus (genus Dependoparvovirus) that was incorporated into the guinea pig germ line between approximately 22 and 35 million years ago (MYA). Despite these ancient origins, the regions of the enRep locus that are expressed in the enRep-M9l gene are conserved across multiple species in the family Caviidae (guinea pigs and cavies), consistent with a potential function at the amino acid level. Using molecular biological approaches, we further demonstrated that (i) enRep-M9l mRNA is broadly transcribed in guinea pig cells, (ii) the cloned enRep-M9l transcript can express a protein of the expected size in guinea pig cells in vitro, and (iii) the expressed protein localizes to the cytosol. Our findings demonstrate that, consistent with a functional role, the enRep-M9l fusion gene is evolutionarily conserved, broadly transcribed, and capable of expressing protein.IMPORTANCE DNA from viruses has been "horizontally transferred" to mammalian genomes during evolution, but the impact of this process on mammalian biology remains poorly understood. The findings of our study indicate that a novel gene has evolved in guinea pigs through fusion of host and virus genes.