RESUMO
A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RIIα. Binding is abrogated upon dephosphorylation of RIIα. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post-natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development.
Assuntos
Proteínas de Transporte/metabolismo , Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Feminino , Humanos , Imunoprecipitação , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Nucleares/química , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismoRESUMO
Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α-actinin and vinculin. Here we examine the roles and expression of the α and ß forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell-cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α-synemin preferentially localized to cell-cell junctions, whereas ß-synemin was primarily at the level of Z disks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell-cell junctions, with no effect on sarcomeric organization. Solubility assays showed that ß-synemin was soluble and interacted with sarcomeric α-actinin by coimmunoprecipitation, while α-synemin and desmin were insoluble. We conclude that ß-synemin mediates the association of desmin IFs with Z disks, whereas α-synemin stabilizes junctional complexes between cardiomyocytes.
Assuntos
Desmina/fisiologia , Junções Intercelulares/fisiologia , Proteínas de Filamentos Intermediários/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Actinina/metabolismo , Animais , Animais Recém-Nascidos , Adesão Celular/fisiologia , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/genética , Filamentos Intermediários/fisiologia , Isomerismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Sarcômeros/fisiologia , Solubilidade , Vinculina/metabolismoRESUMO
Maintaining cellular lipid homeostasis is crucial to oxidative tissues, and it becomes compromised in obesity. Lipid droplets (LD) play a central role in lipid homeostasis by mediating fatty acid (FA) storage in the form of triglyceride, thereby lowering intracellular levels of lipids that mediate cellular lipotoxicity. LDs and mitochondria have interconnected functions, and anecdotal evidence suggests they physically interact. However, the mechanisms of interaction have not been identified. Perilipins are LD-scaffolding proteins and potential candidates to play a role in their interaction with mitochondria. We examined the contribution of LD perilipin composition to the physical and metabolic interactions between LD and mitochondria using multiple techniques: confocal imaging, electron microscopy (EM), and lipid storage and utilization measurements. Using neonatal cardiomyocytes, reconstituted cell culture models, and rodent heart tissues, we found that perilipin 5 (Plin5) recruits mitochondria to the LD surface through a C-terminal region. Compared with control cells, Plin5-expressing cells show decreased LD hydrolysis, decreased palmitate ß-oxidation, and increased palmitate incorporation into triglycerides in basal conditions, whereas in stimulated conditions, LD hydrolysis inhibition is lifted and FA released for ß-oxidation. These results suggest that Plin5 regulates oxidative LD hydrolysis and controls local FA flux to protect mitochondria against excessive exposure to FA during physiological stress.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Bovinos , Linhagem Celular , Humanos , Hidrólise , Metabolismo dos Lipídeos , Lipídeos/química , Masculino , Camundongos , Dados de Sequência Molecular , Oxirredução , Palmitatos/metabolismo , Estrutura Terciária de Proteína , Ratos , Triglicerídeos/metabolismoRESUMO
A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Miosinas/metabolismo , Regeneração Nervosa/fisiologia , Nervo Isquiático/metabolismo , Neuropatia Ciática/metabolismo , Animais , Transporte Axonal/fisiologia , Axônios/ultraestrutura , Modelos Animais de Doenças , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Ratos , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/fisiopatologiaRESUMO
Targeting of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) contributes to high specificity of PKA signaling pathways. PKA phosphorylation of myofilament and cytoskeletal proteins may regulate myofibrillogenesis and myocyte remodeling during heart disease; however, known cardiac AKAPs do not localize to these regions. To identify novel AKAPs which target PKA to the cytoskeleton or myofilaments, a human heart cDNA library was screened and the intermediate filament (IF) protein, synemin, was identified as a putative RII (PKA regulatory subunit type II) binding protein. A predicted RII binding region was mutated and resulted in loss of RII binding. Furthermore, synemin co-localized with RII in SW13/cl.1-vim+ cells and co-immunoprecipitated with RII from adult rat cardiomyocytes. Synemin was localized at the level of Z-lines with RII and desmin in adult hearts, however, neonatal cardiomyocytes showed differential synemin and desmin localization. Quantitative Western blots also showed significantly more synemin was present in failing human hearts. We propose that synemin provides temporal and spatial targeting of PKA in adult and neonatal cardiac myocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ventrículos do Coração/metabolismo , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/metabolismo , Células Musculares/metabolismo , Proteínas de Ancoragem à Quinase A , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Células Cultivadas , Humanos , Proteínas de Filamentos Intermediários/genética , Dados de Sequência Molecular , Ligação ProteicaRESUMO
With a sample of 120 Gulf War veterans, the present study investigated the main effects of childhood and lifetime trauma, combat exposure, and coping strategies on posttraumatic stress disorder (PTSD), as well as combat exposure's moderating effects on the other variables' relationships with PTSD. Logistic regression results indicated correct classification of PTSD diagnosis for 88% of the participants, with combat exposure and avoidant coping making significant contributions to this classification. Multiple regression results indicated that lifetime trauma, combat exposure, and avoidant coping were strongly related to PTSD symptoms. Multiple regression results also revealed that combat exposure moderated the strength and direction of PTSD's relationships with childhood trauma and avoidant coping. Study findings have implications for longitudinal investigation of PTSD development and preventive interventions.
Assuntos
Síndrome do Golfo Pérsico/epidemiologia , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Veteranos/psicologia , Veteranos/estatística & dados numéricos , Adaptação Psicológica , Adulto , Criança , Maus-Tratos Infantis/psicologia , Maus-Tratos Infantis/estatística & dados numéricos , Feminino , Humanos , Acontecimentos que Mudam a Vida , Masculino , Estudos Retrospectivos , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Transtornos de Estresse Pós-Traumáticos/psicologia , Inquéritos e QuestionáriosRESUMO
We have examined spinal motor neurons in Sprague-Dawley rats to further characterize a mechanoenzyme, myosin-Igamma (myr4), which is found in high concentration during axon tract formation in neonates. We raised an antibody to myr4 and made riboprobes for in situ hybridization. Myr4 mRNA was abundant in spinal cord motor neurons (particularly during axon regrowth). Nerves undergoing Wallerian degeneration (from a crush 7 days earlier) showed anti-myr4 labeling of the axolemma and SER--after microtubules, neurofilaments, and F-actin had already been degraded--which is consistent with a described lipid-binding domain in the tail region of myosin-Is. Newly synthesized myr4 was carried in axons by the slow component (SC) of axonal transport at 1-8 mm/day, whereas, none was carried by the fast component (FC). We conclude that SC delivers myr4 to the cytoplasmic surfaces of stationary axonal membranes (SER and axolemma). This positioning would anchor the tail domain of myr4 and leave the catalytic head domain free to interact with F-actin.
Assuntos
Axônios/metabolismo , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Animais , Transporte Axonal , Western Blotting , Imuno-Histoquímica , Hibridização In Situ , Masculino , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Spinal motor neurons of Sprague-Dawley rats were examined to determine which of the neuronal isoforms of actin (beta or gamma) upregulate following axon injury. In situ hybridization studies showed greater beta-actin mRNA levels but no change in gamma-actin mRNA levels-suggesting that axon regrowth utilizes beta-actin. We radiolabeled the newly synthesized actin and tubulin that are subsequently transported in the axon to the site of an axotomizing injury. This allowed us to evaluate changes in polymerization as new cytoskeletal elements approach the injury site. Previous studies had shown that the rate of the most rapid subcomponent of actin and tubulin transport (called SCb) accelerates following axotomy (J. Jacob and I. McQuarrie, J. Neurobiol. 22: 570-583, 1991). This rate increase is associated with an increased proportion of SCb tubulin and actin in polymer (vs monomer) form (J. Jacob and I. McQuarrie, J. Neurosci, Res. 43: 412-419, 1996). However, in that study newly synthesized proteins were radiolabeled at 7 days after axotomy-which is at the peak of increased protein synthesis. This time-course did not examine actin and tubulin that were already in transit in axons when the injury occurred. This actin and tubulin would enter the regrowing axons first. Here, we have radiolabeled newly synthesized proteins 3 days prior to axotomy. For beta-tubulin, the ratio of monomer to polymer was unaffected. For actin, the equilibrium shifted strongly toward polymerization. We conclude that the acceleration of axonal outgrowth seen after the second of two serial axotomies (the "conditioning lesion effect") is related to the ability of actin that is already in transit to polymerize in response to the first axotomy.
Assuntos
Actinas/metabolismo , Axônios/metabolismo , Neurônios Motores/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Transporte Axonal/fisiologia , Axônios/fisiologia , Axotomia , Vértebras Lombares/metabolismo , Masculino , Regeneração Nervosa/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Previously, we identified calcium/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) mRNA in spinal motor neurons with 372 bp inserted in what corresponds to the "association" domain of the protein. This was interesting because known additions and deletions to CaMKIIbeta mRNA are usually less than 100 bp in size and found in the "variable" region. Changes in the association domain of CaMKIIbeta could influence substrate specificity, activity or intracellular targeting. We show that three variations of this insert are found in CNS neurons or sciatic motor neurons of Sprague-Dawley rats. We used PCR and nucleic acid sequencing to identify inserts of 114, 243, or 372 bases. We also show that addition of the 372 bases is associated with outgrowth of the axon (the standard CaMKIIbeta downregulates when axon outgrowth occurs). Radiolabeling, immunoblots, and 2D PAGE identified this larger CaMKIIbeta as part of the group of soluble proteins moving at the slowest rate of axonal transport (SCa) in sciatic motor neurons (similar1 mm/day). This group is composed mainly of structural proteins (e.g., tubulin) used to assemble the cytoskeleton of regrowing axons.