ABSTRACT
Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Myosin Heavy Chains/metabolism , Myosin Type V/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Binding Sites , Centrosome/metabolism , Cilia/genetics , Cilia/metabolism , Conserved Sequence , Humans , Models, Molecular , Myosin Heavy Chains/chemistry , Myosin Heavy Chains/genetics , Myosin Type V/chemistry , Myosin Type V/genetics , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant ProteinsABSTRACT
Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Here, we demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Immobilized MVaf interacted directly with DLCs, but complexed MVaf/DLCs did not interact with Bmf. Overexpression of DLC2 attenuated MVaf-induced apoptosis. Thus, we suggest that, MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. Murine embryonic fibroblasts (MEFs) lacking Bim and Bmf or Bax and Bak were less sensitive to apoptosis caused by MVaf expression than wild-type MEFs, strengthening the putative role of the intrinsic apoptotic pathway in this response. Finally, MVaf expression attenuated B16-F10 solid tumor growth in mice, suggesting that this peptide may be useful as an apoptosis-inducing tool for basic and translational studies.
Subject(s)
Cytoplasmic Dyneins/genetics , Gene Expression Regulation, Neoplastic/drug effects , Melanoma/genetics , Myosin Heavy Chains/genetics , Myosin Type V/genetics , Peptide Fragments/genetics , Skin Neoplasms/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Bcl-2-Like Protein 11 , Cell Line, Tumor , Cytoplasmic Dyneins/metabolism , DNA Fragmentation/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Melanoma/metabolism , Melanoma/pathology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Myosin Heavy Chains/metabolism , Myosin Type V/metabolism , Neoplasm Transplantation , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Binding , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Signal Transduction/drug effects , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , bcl-2 Homologous Antagonist-Killer Protein/deficiency , bcl-2 Homologous Antagonist-Killer Protein/geneticsABSTRACT
Apolipoprotein E (apoE - e2, e3, e4 alleles) plays a role in the regulation of lipid metabolism, with the e4 considered to be a risk factor for coronary artery disease (CAD). We aimed to evaluate the apoE polymorphisms in Brazilians with CAD and their influence on the lipid profile and other risk factors (hypertension, diabetes mellitus, smoking). Two hundred individuals were examined: 100 patients with atherosclerosis confirmed by coronary angiography and 100 controls. Blood samples were drawn to determine apoE polymorphisms and lipid profile. As expected, the e3 allele was prevalent in the CAD (0.87) and non-CAD groups (0.81; P = 0.099), followed by the e4 allele (0.09 and 0.14, respectively; P = 0.158). The e3/3 (76 and 78%) and e3/4 (16 and 23%) were the most common genotypes for patients and controls, respectively. The lipid profile was altered in patients compared to controls (P < 0.05), independently of the e4 allele. However, in the controls this allele was prevalent in individuals with elevated LDL-cholesterol levels only (odds ratio = 2.531; 95% CI = 1.028-6.232). The frequency of risk factors was higher in the CAD group (P < 0.05), but their association with the lipid profile was not demonstrable in e4 carriers. In conclusion, the e4 allele is not associated with CAD or lipid profile in patients with atherosclerosis. However, its frequency in the non-CAD group is associated with increased levels of LDL-cholesterol, suggesting an independent effect of the e4 allele on lipid profile when the low frequency of other risk factors in this group is taken into account.
Subject(s)
Apolipoproteins E/genetics , Coronary Artery Disease/blood , Polymorphism, Genetic , Adult , Aged , Alleles , Case-Control Studies , Coronary Artery Disease/genetics , Female , Genotype , Humans , Lipids/blood , Male , Middle Aged , Risk FactorsABSTRACT
Apolipoprotein E (apoE - e2, e3, e4 alleles) plays a role in the regulation of lipid metabolism, with the e4 considered to be a risk factor for coronary artery disease (CAD). We aimed to evaluate the apoE polymorphisms in Brazilians with CAD and their influence on the lipid profile and other risk factors (hypertension, diabetes mellitus, smoking). Two hundred individuals were examined: 100 patients with atherosclerosis confirmed by coronary angiography and 100 controls. Blood samples were drawn to determine apoE polymorphisms and lipid profile. As expected, the e3 allele was prevalent in the CAD (0.87) and non-CAD groups (0.81; P = 0.099), followed by the e4 allele (0.09 and 0.14, respectively; P = 0.158). The e3/3 (76 and 78 percent) and e3/4 (16 and 23 percent) were the most common genotypes for patients and controls, respectively. The lipid profile was altered in patients compared to controls (P < 0.05), independently of the e4 allele. However, in the controls this allele was prevalent in individuals with elevated LDL-cholesterol levels only (odds ratio = 2.531; 95 percent CI = 1.028-6.232). The frequency of risk factors was higher in the CAD group (P < 0.05), but their association with the lipid profile was not demonstrable in e4 carriers. In conclusion, the e4 allele is not associated with CAD or lipid profile in patients with atherosclerosis. However, its frequency in the non-CAD group is associated with increased levels of LDL-cholesterol, suggesting an independent effect of the e4 allele on lipid profile when the low frequency of other risk factors in this group is taken into account.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Apolipoproteins E/genetics , Coronary Artery Disease/blood , Coronary Artery Disease/genetics , Lipids/blood , Alleles , Case-Control Studies , Genotype , Polymorphism, Genetic , Risk FactorsABSTRACT
Os autores apresentam um caso de cisticercose do terceiro ventriculo, que determinou coma subito. Foi realizado tratamento cirurgico (seccao da porcao anterior do fornix) e obtida cura completa de hipertensao intracraniana. Sao feitos comentarios sobre a investigacao neurologica utilizada, a possibilidade de abordagem cirurgica da porcao media do terceiro ventriculo, assim como os avancos mais recentes sobre o estudo liquorico e possivel tratamento quimico desta enfermidade
