RESUMO
Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated.
Assuntos
Proteína 4 Homóloga a Disks-Large/metabolismo , Proteína GAP-43/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Processamento de Proteína Pós-Traducional , Rede trans-Golgi/metabolismo , Acilação , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células CHO , Sequência Conservada , Cricetulus , Proteína 4 Homóloga a Disks-Large/química , Proteína 4 Homóloga a Disks-Large/genética , Proteína GAP-43/química , Proteína GAP-43/genética , Interações Hidrofóbicas e Hidrofílicas , Eletricidade Estática , Fatores de Tempo , Rede trans-Golgi/genéticaRESUMO
The aim of this study was to investigate whether exogenous retinoic acid (RA) can upregulate the mRNA and protein expression of growth-associated protein 43 (GAP-43), thereby promoting brain functional recovery in a rat distal middle cerebral artery occlusion (MCAO) model of ischemia. A total of 216 male Sprague Dawley rats weighing 300-320 g were divided into 3 groups: sham-operated group, MCAO+vehicle group and MCAO+RA group. Focal cortical infarction was induced with a distal MCAO model. The expression of GAP-43 mRNA and protein in the ipsilateral perifocal region was assessed using qPCR and immunocytochemistry at 1, 3, 7, 14, 21, and 28 days after distal MCAO. In addition, an intraperitoneal injection of RA was given 12 h before MCAO and continued every day until the animal was sacrificed. Following ischemia, the expression of GAP-43 first increased considerably and then decreased. Administration of RA reduced infarction volume, promoted neurological functional recovery and upregulated expression of GAP-43. Administration of RA can ameliorate neuronal damage and promote nerve regeneration by upregulating the expression of GAP-43 in the perifocal region after distal MCAO.
Assuntos
Proteína GAP-43/metabolismo , Expressão Gênica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Isquemia Encefálica/prevenção & controle , Proteína GAP-43/genética , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
The aim of this study was to investigate whether exogenous retinoic acid (RA) can upregulate the mRNA and protein expression of growth-associated protein 43 (GAP-43), thereby promoting brain functional recovery in a rat distal middle cerebral artery occlusion (MCAO) model of ischemia. A total of 216 male Sprague Dawley rats weighing 300–320 g were divided into 3 groups: sham-operated group, MCAO+vehicle group and MCAO+RA group. Focal cortical infarction was induced with a distal MCAO model. The expression of GAP-43 mRNA and protein in the ipsilateral perifocal region was assessed using qPCR and immunocytochemistry at 1, 3, 7, 14, 21, and 28 days after distal MCAO. In addition, an intraperitoneal injection of RA was given 12 h before MCAO and continued every day until the animal was sacrificed. Following ischemia, the expression of GAP-43 first increased considerably and then decreased. Administration of RA reduced infarction volume, promoted neurological functional recovery and upregulated expression of GAP-43. Administration of RA can ameliorate neuronal damage and promote nerve regeneration by upregulating the expression of GAP-43 in the perifocal region after distal MCAO.
Assuntos
Animais , Masculino , Proteína GAP-43/metabolismo , Expressão Gênica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos , Isquemia Encefálica/prevenção & controle , Proteína GAP-43/genética , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Growth-associated protein 43 (GAP-43) is a neuronal phosphoprotein associated with initial axonal outgrowth and synaptic remodeling and recent work also suggests its involvement in cell cycle control. The complex expression of GAP-43 features transcriptional and posttranscriptional components. However, in some conditions, GAP-43 gene expression is controlled primarily by the interaction of stabilizing or destabilizing RNA-binding proteins (RBPs) with adenine and uridine (AU)-rich instability elements (AREs) in its 3'UTR. Like GAP-43, many proteins involved in cell proliferation are encoded by ARE-containing mRNAs, some of which codify cell-cycle-regulating proteins including cyclin D1. Considering that GAP-43 and cyclin D1 mRNA stabilization may depend on similar RBPs, this study evaluated the participation of GAP-43 in cell cycle control and its underlying mechanisms, particularly the possible role of its 3'UTR, using GAP-43-transfected NIH-3T3 fibroblasts. Our results show an arrest in cell cycle progression in the G0/G1 phase. This arrest may be mediated by the competition of GAP-43 3'UTR with cyclin D1 3'UTR for the binding of Hu proteins such as HuR, which may lead to a decrease in cyclin D1 expression. These results might lead to therapeutic applications involving the use of sequences in the B region of GAP-43 3'UTR to slow down cell cycle progression.
Assuntos
Proteína GAP-43/metabolismo , Regiões 3' não Traduzidas , Animais , Adesão Celular , Ciclo Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Ativação Enzimática , Proteína GAP-43/genética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células NIH 3T3 , Proteínas de Ligação a RNA/metabolismoRESUMO
The aim of this study was to explore the changes in gene and protein expressions of tyrosine hydroxylase (TH) and growth-associated protein 43 (GAP43) in aging atrial fibrillation patients of Xinjiang Uygur and Han nationality, and the significance of the changes. Real-time polymerase chain reaction and Western blot analysis were used to detect gene and protein expressions of TH and GAP43 in atrial tissues of 54 patients with valvular heart disease. mRNA and protein expressions of GAP43 and TH were significantly different between the sinus rhythm and atrial fibrillation groups (P < 0.05). Protein expressions of GAP43 and TH of both nationalities differed significantly between the sinus rhythm group and the atrial fibrillation group (P < 0.05), whereas there was no statistical difference between the two nationalities within each group (P > 0.05). Protein expressions of GAP43 and TH differed significantly among different age groups of different nationalities in the sinus rhythm and atrial fibrillation groups (P < 0.05); only protein expression of GAP43 differed significantly in different age groups in the atrial fibrillation group (P < 0.05). The changes of mRNA and protein expressions of TH and GAP43 played a vital role in the process of maintaining the atrial fibrillation. Therefore, increased expression of TH and GAP43 might be a molecular mechanism for left atrial myoelectricity remodeling of aging atrial fibrillation patients, which might be potential therapeutic targets of atrial fibrillation.
Assuntos
Fibrilação Atrial/genética , Etnicidade/genética , Proteína GAP-43/genética , Expressão Gênica , Tirosina 3-Mono-Oxigenase/genética , Adulto , Idoso , Fibrilação Atrial/metabolismo , Estudos de Casos e Controles , China , Feminino , Proteína GAP-43/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of ß-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of ß-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na(+) current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required ß-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.
Assuntos
Catepsina L/antagonistas & inibidores , Diferenciação Celular , Cistatina C/fisiologia , Células-Tronco Embrionárias/fisiologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Calpaína/antagonistas & inibidores , Catepsina L/metabolismo , Linhagem Celular , Extensões da Superfície Celular/metabolismo , Técnicas de Cocultura , Cistatina C/metabolismo , Cistatina C/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Ligação a DNA , Corpos Embrioides/citologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/enzimologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/enzimologia , Proteínas do Olho/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Potenciais da Membrana , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Proteínas de Neurofilamentos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismoRESUMO
An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.
Assuntos
Membrana Celular/metabolismo , Proteína GAP-43/metabolismo , Lisofosfolipase/metabolismo , Tioléster Hidrolases/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Biocatálise , Western Blotting , Células CHO , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteína GAP-43/genética , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisofosfolipase/genética , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/genéticaRESUMO
In the Wobbler mouse, a mutation in the Vps54 gene is accompanied by motoneuron degeneration and astrogliosis in the cervical spinal cord. Previous work has shown that these abnormalities are greatly attenuated by progesterone treatment of clinically afflicted Wobblers. However, whether progesterone is effective at all disease stages has not yet been tested. The present work used genotyped (wr/wr) Wobbler mice at three periods of the disease: early progressive (1-2 months), established (5-8 months) or late stages (12 months) and age-matched wildtype controls (NFR/NFR), half of which were implanted with a progesterone pellet (20 mg) for 18 days. In untreated Wobblers, degenerating vacuolated motoneurons were initially abundant, experienced a slight reduction at the established stage and dramatically diminished during the late period. In motoneurons, the cholinergic marker choline acetyltransferase (ChAT) was reduced at all stages of the Wobbler disease, whereas hyperexpression of the growth-associated protein (GAP43) mRNA preferentially occurred at the early progressive and established stages. Progesterone therapy significantly reduced motoneuron vacuolation, enhanced ChAT immunoreactive perikarya and reduced the hyperexpression of GAP43 during the early progressive and established stages. At all stage periods, untreated Wobblers showed high density of glial fibrillary acidic protein (GFAP)+ astrocytes and decreased number of glutamine synthase (GS) immunostained cells. Progesterone treatment down-regulated GFAP+ astrocytes and up-regulated GS+ cell number. These data reinforced the usefulness of progesterone to improve motoneuron and glial cell abnormalities of Wobbler mice and further showed that therapeutic benefit seems more effective at the early progressive and established periods, rather than on advance stages of spinal cord neurodegeneration.
Assuntos
Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Progesterona/farmacologia , Doenças da Medula Espinal/patologia , Medula Espinal/patologia , Animais , Células do Corno Anterior/efeitos dos fármacos , Células do Corno Anterior/enzimologia , Células do Corno Anterior/patologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Contagem de Células , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Feminino , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/metabolismo , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Neurônios Motores/enzimologia , Neuroglia/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Doenças da Medula Espinal/enzimologiaRESUMO
Experimental autoimmune encephalomyelitis (EAE), an induced model of Multiple Sclerosis presents spinal cord demyelination, axonal pathology and neuronal dysfunction. Previous work has shown that progesterone attenuated the clinical severity, demyelination and neuronal dysfunction of EAE mice (Garay et al., J. Steroid Biochem. Mol. Biol., 2008). Here we studied if progesterone also prevented axonal damage, a main cause of neurological disability. To this end, some axonal parameters were compared in EAE mice pretreated with progesterone a week before immunization with MOG(40-54) and in a group of steroid-free EAE mice. On day 16th after EAE induction, we determined in both groups and in control mice: a) axonal density in semithin sections of the spinal cord ventral funiculus; b) appearance of amyloid precursor protein (APP) immunopositive spheroids as an index of damaged axons; c) levels of the growth associated protein GAP43 mRNA and immunopositive cell bodies, as an index of aberrant axonal sprouting. Steroid-naive EAE mice showed decreased axonal density, shrunken axons, abundance of irregular vesicular structures, degenerating APP+ axons, increased expression of GAP43 mRNA and immunoreactive protein in motoneurons. Instead, EAE mice receiving progesterone treatment showed increased axonal counts, high proportion of small diameter axons, reduced APP+ profiles, and decreased GAP43 expression. In conclusion, progesterone enhanced axonal density, decreased axonal damage and prevented GAP43 hyperexpression in the spinal cord of EAE mice. Thus, progesterone also exerts protective effects on the axonal pathology developing in EAE mice.
Assuntos
Axônios/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Progesterona/farmacologia , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Biomarcadores/análise , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Contagem de Células , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Proteína GAP-43/genética , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Fármacos Neuroprotetores/metabolismo , Progesterona/metabolismo , Progestinas/metabolismo , Progestinas/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Resultado do Tratamento , Degeneração Walleriana/tratamento farmacológico , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologiaRESUMO
GAP-43 (growth-associated protein-43) is a dually palmitoylated protein, at cysteine residues at positions 3 and 4, that mostly localizes in plasma membrane both in neural and non-neural cells. In the present study, we have examined membrane association, subcellular distribution and intracellular trafficking of GAP-43 in CHO (Chinese hamster ovary)-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrated that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane and TGN (trans-Golgi network). Pharmacological inhibition of newly synthesized GAP-43 acylation or double mutation of Cys3 and Cys4 of GAP-43 completely disrupts TGN, plasma membrane and recycling endosome association. A combination of selective photobleaching techniques and time-lapse fluorescence microscopy reveals a dynamic association of GAP-43 with recycling endosomes in equilibrium with the plasma membrane pool. Newly synthesized GAP-43 is found mainly associated with the TGN, but not with the pericentriolar recycling endosome, and traffics to the plasma membrane by a brefeldin A-insensitive pathway. Impairment of plasma membrane fusion and internalization by treatment with tannic acid does affect the trafficking of GAP-43 from plasma membrane to recycling endosomes which reveals a vesicle-mediated retrograde trafficking of GAP-43. Here, we also show that internalization of GAP-43 is regulated by Arf (ADP-ribosylation factor) 6. Taken together, these results demonstrate that dual acylation is required for sorting of peripheral membrane-associated GAP-43 to recycling endosome via an Arf6-associated endocytic vesicular pathway.