RESUMEN
Myelin basic protein (MBP) is an oligodendrocyte-specific protein essential for oligodendrocyte morphogenesis at late stages of cell differentiation. There is evidence that the morphogenetic function of MBP is mediated by MBP interaction with the cytoskeleton. Thus, an MBP/cytoplasmic microtubule association has been reported, and MBP has Ca(2+)/calmodulin-regulated microtubule cold-stabilizing activity in vitro. However, the unambiguous demonstration of a microtubule-stabilizing activity for MBP in cells has been difficult because oligodendrocytes contain variants of STOP (stable tubule only polypeptide) proteins, which are responsible for microtubule cold stability in different cell types. Herein, we have used genetic mouse models and RNA interference to assay independently the microtubule cold-stabilizing activities of MBP and of STOP in developing oligodendrocytes. In wild-type oligodendrocytes, microtubules were cold stable throughout maturation, which is consistent with the presence of STOP proteins from early stages of differentiation. In contrast, in oligodendrocytes from STOP-deficient mice, microtubules were cold labile in the absence of MBP expression or when MBP expression was restricted to the cell body and became stable in fully differentiated oligodendrocytes, where MBP is expressed in cell extensions. The suppression of MBP by RNA interference in STOP-deficient oligodendrocytes suppressed microtubule cold stability. Additionally, STOP suppression in oligodendrocytes derived from shiverer mice that lack MBP led to the complete suppression of microtubule cold stability at all stages of cell differentiation. These results demonstrate that both STOP and MBP function as microtubule-stabilizing proteins in differentiating oligodendrocytes and could be important for the morphogenetic function of MBP.
Asunto(s)
Diferenciación Celular/fisiología , Sistema Nervioso Central/crecimiento & desarrollo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteína Básica de Mielina/metabolismo , Oligodendroglía/metabolismo , Animales , Técnicas de Cultivo de Célula , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Regulación hacia Abajo/genética , Ratones , Ratones Mutantes Neurológicos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/ultraestructura , Proteína Básica de Mielina/genética , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Oligodendroglía/ultraestructura , Interferencia de ARNRESUMEN
Many cell types contain subpopulations of microtubules that resist depolymerizing conditions, such as exposure to cold or to the drug nocodazole. This stabilization is due mainly to polymer association with STOP proteins. In mouse, neurons express two major variants of these proteins, N-STOP and E-STOP (120 kDa and 79 kDa, respectively), whereas fibroblasts express F-STOP (42 kDa) and two minor variants of 48 and 89 kDa. N- and E-STOP induce microtubule resistance to both cold and nocodazole exposure, whereas F-STOP confers microtubule stability only to the cold. Here, we investigated the expression of STOP proteins in oligodendrocytes and astrocytes in culture. We found that STOP proteins were expressed in precursor cells, in immature and mature oligodendrocytes, and in astrocytes. We found that oligodendrocytes express a major STOP variant of 89 kDa, which we called O-STOP, and two minor variants of 42 and 48 kDa. The STOP variants expressed by oligodendrocytes induce microtubule resistance to the cold and to nocodazole. For astrocytes, we found the expression of two STOP variants of 42 and 48 kDa and a new STOP isoform of 60 kDa, which we called A-STOP. The STOP variants expressed by astrocytes induce microtubule resistance to the cold but not to nocodazole, as fibroblast variants. In conclusion, astrocytes and oligodendrocytes express different isoforms of STOP protein, which show different microtubule-stabilizing capacities.
Asunto(s)
Astrocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Oligodendroglía/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/efectos de la radiación , Biomarcadores/metabolismo , Western Blotting/métodos , Encéfalo/citología , Encéfalo/metabolismo , Células Cultivadas , Frío , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Ratones , Proteínas Asociadas a Microtúbulos/clasificación , Microtúbulos/fisiología , Células 3T3 NIH/metabolismo , Nocodazol/farmacología , Antígenos O/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/efectos de la radiación , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismoRESUMEN
Apotransferrin injected intracranially into young rats has been shown in our laboratories to induce an early differentiation of oligodendroglial cells and an increased deposition of myelin. The expression of some myelin-specific proteins such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and of their mRNAs were significantly increased in these animals. Also, in the cytoskeleton obtained from isolated myelin, it was found that several microtubule associated proteins (MAPs), particularly the stable tubule only peptide (STOP) and MAP 1B, as well as actin and tubulin were markedly increased. In the present paper, we compare the changes in expression of brain and myelin cytoskeletal proteins in a newly generated transferrin transgenic mouse (Tg), overexpressing the human transferrin gene, with the results obtained in aTf-injected rats. In the myelin cytoskeletal fraction of Tg mice there was a significant increase in the expression of MBP, tubulin, tau and STOP, similarly to what was previously found in the aTf-injected rats. Immunohistochemical studies showed that a variance with what occurs in the aTf-injected model, in which the above mentioned changes were limited to the corpus callosum, in the Tg mice the changes in expression of cytoskeletal proteins were observed in the various anatomical areas studied such as cerebral cortex, brain stem and cerebellum. There was also an increased expression of neurofilaments in the Tg animals, in contrast with results obtained in aTf-injected rats, suggesting that in the Tg mice, the continuous overexpression of Tf might also induce some neuronal changes. Changes in tau, total and acetylated tubulin and MAP 1B were observed in both neurons and OLGc. The increase in STOP was more significant in OLGc while the changes in MAP2 were exclusively found in neurons.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Vaina de Mielina/metabolismo , Transferrina/genética , Animales , Encéfalo/efectos de los fármacos , Proteínas del Citoesqueleto/efectos de los fármacos , Proteínas del Citoesqueleto/genética , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/genética , Proteína Básica de Mielina/biosíntesis , Proteína Básica de Mielina/efectos de los fármacos , Proteína Básica de Mielina/genética , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/genética , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Ratas , Ratas Wistar , Transferrina/farmacologíaRESUMEN
We have previously shown that in rat pups intracranially injected with a single dose of apotransferrin (aTf), there is an early oligodendroglial cell OLGc differentiation. The expression of the mRNAs of myelin basic proteins and of 2',3' cyclic nucleotide 3'-phosphodiesterase and the amount of the corresponding proteins, as well as myelin glycolipids and phospholipids, were significantly increased in these animals at 10 and 17 days of age. Microtubules and myelin basic proteins appear to be closely associated in OLGc and it has been shown that the mRNAs of myelin basic proteins are concentrated in the OLGc processes. The aim of this work was to clarify if the accelerated myelination produced by aTf could be linked to changes in certain cytoskeletal elements present in the myelin fraction such as tubulin, actin, and different microtubule-associated proteins (MAPs). A significant increase in the expression of the mRNA of tubulin and actin was observed in the brain of the aTf-treated animals. Several MAPs, particularly MAP 1B and stable tubule only peptide as well as actin and tubulin, were markedly increased in the Triton X-100 insoluble pellet obtained from the myelin fraction of these animals. The changes that we have previously described in the myelin of aTf intracranially injected rats, could be the consequence of its action on the cytoskeletal network of the OLGc. An enlargement of this structure would result in a more efficient and faster movement of the different components that are normally transported to the myelin by the cytoskeleton of this cell.
Asunto(s)
Apoproteínas/farmacología , Proteínas del Citoesqueleto/genética , Citoesqueleto/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Básica de Mielina/genética , Oligodendroglía/fisiología , Transferrina/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Animales , Animales Recién Nacidos , Apoproteínas/administración & dosificación , Citoesqueleto/metabolismo , Glucolípidos/metabolismo , Microinyecciones , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Fosfolípidos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Transcripción Genética/efectos de los fármacos , Transferrina/administración & dosificaciónRESUMEN
The aim of this study was to analyze the N-terminal post-translational incorporation of arginine into cytosolic proteins from cultured cells and the in vitro incorporation of arginine into soluble proteins of PC12 cells after serum deprivation. Arginine incorporation was measured in the presence of protein synthesis inhibitors. None of the inhibitors used affected significantly the arginylation reaction while the novo synthesis of protein was reduced by 98%. Under these conditions, we found that of the total [14C]arginine incorporated into the proteins, around 20% to 40% was incorporated into the N-terminal position of soluble proteins by a post-translational mechanism. These results suggest that this post-translational aminoacylation may be a widespread reaction in neuronal and non-neuronal cells. We also found that in PC12 cells, the in vitro post-translational arginylation was 60% higher in apoptotic cells with respect to control cells. These findings suggest that the post-translational arginylation of proteins may be involved in programmed cell death.
Asunto(s)
Arginina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Apoptosis , Química Encefálica , Radioisótopos de Carbono , Células Cultivadas , Embrión de Pollo , Fragmentación del ADN , Células PC12 , RatasRESUMEN
We have previously reported the posttranslational addition of [14C]-arginine in the N-terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule-associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway. Since this degradative via is exacerbated as a response to stress, we studied in vitro the posttranslational [14C]-arginylation of cytosolic brain proteins of rats subjected to hyperthermia in vivo. Immediately after subjecting the animals to hyperthermia, a minor reduction (16%) in the acceptor capacity of [14C]-arginine into proteins was observed in comparison with animals maintained at 28 degrees C. However, in the animals allowed to recover for 3 h, an increase (46%) in the arginylation was observed concomitantly with a significant accumulation of the heat shock protein (70 kDa; hsp 70) when compared to the control animals. These findings suggest that the posttranslational arginylation of proteins participate in the heat shock response. The STOP protein of the soluble brain fraction of control animals, which in Western blot appears as a doublet band (125 and 130 kDa, respectively), is seen, after the hyperthermic treatment, as a single band of 125 kDa. The amount of 125 kDa protein, as well as the in vitro incorporation of [14C]-arginine, increases after hyperthermia in comparison with control animals. Following hyperthermic treatment, we observed a decrease in the amount of in vivo [35S]-methionine-labeled brain proteins. We speculate that, as observed for STOP protein, the increase in the degradation of protein that occurs in hyperthermia, would produce an increase in the amount of arginine acceptor proteins.
Asunto(s)
Arginina/metabolismo , Encéfalo/metabolismo , Hipertermia Inducida , Proteínas de Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Radioisótopos de Carbono , Citosol/metabolismo , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Masculino , Metionina/metabolismo , Proteínas de Microtúbulos/biosíntesis , Proteínas de Microtúbulos/aislamiento & purificación , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/aislamiento & purificación , Ratas , Ratas Wistar , Radioisótopos de AzufreRESUMEN
The knowledge of brain protein metabolism is important in understanding nervous system brain function. Protein synthesis rates are high in young brain, decline rapidly at adult stages, and thereafter continue falling slowly with age. The breakdown of protein appears to follow a similar rate (1). Protein synthesis and degradation however, are only the two extremes of a complex phenomena which includes a variety of other protein modifications. Proteolytic cleavage is the most common covalent modification of proteins; probably all proteins that have been isolated were modified by proteolysis, since only few are found with the starting amino acid (methionine) attached. This suggests that most proteins were subject to one or more co- and/or posttranslational modifications (2). One of these posttranslational modifications is the arginylation of proteins, described 30 years ago, which now is being recognized as a widespread modification of proteins. In this review, the current status of posttranslational arginylation of brain proteins is discussed.
Asunto(s)
Arginina/metabolismo , Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Procesamiento Proteico-Postraduccional , Envejecimiento , Péptidos beta-Amiloides/metabolismo , Animales , Proteínas Asociadas a Microtúbulos/metabolismo , Ubiquitinas/metabolismoRESUMEN
The beta-amyloid peptide (beta AP1-40) inhibited the in vitro post-translational incorporation of [14C]arginine at the N-terminus of brain soluble proteins and was labelled by the incorporation of [14C]arginine. Addition of arginine at the N-terminal position of beta AP1-40 is predicted to increase the probability of an alpha-helix structure being formed on the first residues with a higher hydrophilic characteristic, increasing the possibility of these residues being exposed to the aqueous environment. Unmodified beta AP1-40 has a low alpha-helix content and a higher probability of beta-turn formation. Accumulation of beta AP1-40 in Alzheimer's disease may therefore be due to a reduced arginylation reaction and consequently to a decrease in its normal degradation by the ubiquitin pathway.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Arginina/química , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , ProbabilidadRESUMEN
Properties so far studied of the 125-kDa 14C-arginylated protein from rat brain show remarkable similarities with those of the STOP (stable tubule only polypeptide) protein. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the 125-kDa 14C-arginylated protein moves to the same position as the STOP protein. The 125-kDa 14C-arginylated protein was immunoprecipitated by the monoclonal Mab 296 antibody specific for neuronal STOP protein. The 125-kDa 14C-arginylated protein was retained by a calmodulin column like STOP protein. As occurs with the STOP protein, the 125-kDa 14C-arginylated protein is found in higher proportion in cold-stable than in cold-labile microtubules. However, the modified protein associates with microtubules in a lower proportion than the STOP protein. We conclude that the STOP protein incorporates arginine by a posttranslational reaction but that only a small fraction of the STOP protein shows acceptor capacity in vitro.
Asunto(s)
Arginina/metabolismo , Química Encefálica , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Encéfalo/ultraestructura , Calmodulina/metabolismo , Frío , Electroforesis en Gel de Poliacrilamida , Técnicas de Inmunoadsorción , Proteínas Asociadas a Microtúbulos/inmunología , Microtúbulos/metabolismo , RatasRESUMEN
The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.