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1.
Proc Natl Acad Sci U S A ; 111(38): E3966-75, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25201980

RESUMO

The inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP3Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP3 at a distance. Defective allostery in IP3R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP3R type 1 (IP3R1) and negatively regulates IP3R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP3R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.


Assuntos
Autofagia , Sinalização do Cálcio , Proteínas de Ligação ao GTP/metabolismo , Doença de Huntington/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Processamento de Proteína Pós-Traducional , Transglutaminases/metabolismo , Regulação Alostérica/genética , Animais , Modelos Animais de Doenças , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Receptores de Inositol 1,4,5-Trifosfato/genética , Células PC12 , Proteína 2 Glutamina gama-Glutamiltransferase , Estrutura Terciária de Proteína , Ratos , Transglutaminases/genética
2.
Proc Natl Acad Sci U S A ; 108(10): 4230-5, 2011 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-21368109

RESUMO

It has been suggested that the frontal operculum (fO) is a key node in a network for exerting control over cognitive processes. How it exerts this influence, however, has been unclear. Here, using the complementary approaches of functional MRI and transcranial magnetic stimulation, we have shown that the fO regulates increases and decreases of activity in multiple occipitotemporal cortical areas when task performance depended on directing attention to different classes of stimuli held in memory. Only one region, the fO, was significantly more active when subjects selectively attended to a single stimulus so that it determined task performance. The stimuli that guided task performance could belong to three categories--houses, body parts, and faces--associated with three occipitotemporal regions. On each trial, the pattern of functional correlation between the fO and the three occipitotemporal regions became either positive or negative, depending on which stimulus was to be attended and which ignored. Activation of the fO preceded both activity increases and decreases in the occipitotemporal cortex. The causal dependency of the distributed occipitotemporal pattern of activity increases and decreases on the fO was demonstrated by showing that transcranial magnetic stimulation-mediated interference of the fO diminished top-down selective attentional modulation in the occipitotemporal cortex, but it did not alter bottom-up activation of the same areas to the same stimuli when they were presented in isolation. The fO's prominence in cognitive control may stem from a role in regulating the level of activity of representations in posterior brain areas that are relevant or irrelevant, respectively, for response selection.


Assuntos
Lobo Frontal/fisiologia , Análise e Desempenho de Tarefas , Cognição , Humanos
3.
Neuron ; 68(5): 865-78, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-21145001

RESUMO

Deranged Ca(2+) signaling and an accumulation of aberrant proteins cause endoplasmic reticulum (ER) stress, which is a hallmark of cell death implicated in many neurodegenerative diseases. However, the underlying mechanisms are elusive. Here, we report that dysfunction of an ER-resident Ca(2+) channel, inositol 1,4,5-trisphosphate receptor (IP(3)R), promotes cell death during ER stress. Heterozygous knockout of brain-dominant type1 IP(3)R (IP(3)R1) resulted in neuronal vulnerability to ER stress in vivo, and IP(3)R1 knockdown enhanced ER stress-induced apoptosis via mitochondria in cultured cells. The IP(3)R1 tetrameric assembly was positively regulated by the ER chaperone GRP78 in an energy-dependent manner. ER stress induced IP(3)R1 dysfunction through an impaired IP(3)R1-GRP78 interaction, which has also been observed in the brain of Huntington's disease model mice. These results suggest that IP(3)R1 senses ER stress through GRP78 to alter the Ca(2+) signal to promote neuronal cell death implicated in neurodegenerative diseases.


Assuntos
Sinalização do Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Morte Celular/fisiologia , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Metabolismo Energético/fisiologia , Técnicas de Silenciamento de Genes , Receptores de Inositol 1,4,5-Trifosfato/genética , Camundongos , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia
4.
Biochem Biophys Res Commun ; 336(2): 682-91, 2005 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-16143307

RESUMO

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Apoptose/fisiologia , Blástula/metabolismo , Proteínas de Plantas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Adenosilmetionina Descarboxilase/genética , Animais , Blástula/citologia , Caspase 8 , Caspases/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/genética , Xenopus laevis/anatomia & histologia , Xenopus laevis/genética
6.
Cell ; 120(1): 85-98, 2005 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-15652484

RESUMO

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are intracellular channel proteins that mediate Ca(2+) release from the endoplasmic reticulum (ER) and are involved in many biological processes and diseases. IP(3)Rs are differentially regulated by a variety of cytosolic proteins, but their regulation by ER lumenal protein(s) remains largely unexplored. In this study, we found that ERp44, an ER lumenal protein of the thioredoxin family, directly interacts with the third lumenal loop of IP(3)R type 1 (IP(3)R1) and that the interaction is dependent on pH, Ca(2+) concentration, and redox state: the presence of free cysteine residues in the loop is required. Ca(2+)-imaging experiments and single-channel recording of IP(3)R1 activity with a planar lipid bilayer system demonstrated that IP(3)R1 is directly inhibited by ERp44. Thus, ERp44 senses the environment in the ER lumen and modulates IP(3)R1 activity accordingly, which should in turn contribute to regulating both intralumenal conditions and the complex patterns of cytosolic Ca(2+) concentrations.


Assuntos
Canais de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades Proteicas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células COS , Cálcio/metabolismo , Linhagem Celular , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Oxirredução , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Interferência de RNA , Técnicas do Sistema de Duplo-Híbrido
7.
Biochem Biophys Res Commun ; 325(4): 1367-75, 2004 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-15555578

RESUMO

We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [(35)S]procaspase-9, but not [(35)S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Blástula/fisiologia , Caspases/metabolismo , Oócitos/fisiologia , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Adenosilmetionina Descarboxilase/genética , Animais , Apoptose/fisiologia , Blástula/citologia , Caspase 1/genética , Caspase 1/metabolismo , Caspase 9 , Caspases/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Oócitos/citologia , Proteínas Recombinantes/metabolismo
8.
J Biol Chem ; 279(12): 11967-75, 2004 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-14707143

RESUMO

Three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R1, IP(3)R2, and IP(3)R3) Ca(2+) release channel share basic properties but differ in terms of regulation. To what extent they contribute to complex Ca(2+) signaling, such as Ca(2+) oscillations, remains largely unknown. Here we show that HeLa cells express comparable amounts of IP(3)R1 and IP(3)R3, but knockdown by RNA interference of each subtype results in dramatically distinct Ca(2+) signaling patterns. Knockdown of IP(3)R1 significantly decreases total Ca(2+) signals and terminates Ca(2+) oscillations. Conversely, knockdown of IP(3)R3 leads to more robust and long lasting Ca(2+) oscillations than in controls. Effects of IP(3)R3 knockdown are surprisingly similar in COS-7 cells that predominantly (>90% of total IP(3)R) express IP(3)R3, suggesting that IP(3)R3 functions as an anti-Ca(2+)-oscillatory unit without contributing to peak amplitude of Ca(2+) signals, irrespective of its relative expression level. Therefore, differential expression of the IP(3)R subtype is critical for various forms of Ca(2+) signaling, and, particularly, IP(3)R1 and IP(3)R3 have opposite roles in generating Ca(2+) oscillations.


Assuntos
Canais de Cálcio/fisiologia , Isoformas de Proteínas/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Animais , Canais de Cálcio/química , Linhagem Celular , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Dados de Sequência Molecular , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/química , Homologia de Sequência de Aminoácidos
9.
Cell Res ; 13(3): 147-58, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12862315

RESUMO

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due to activation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of the apoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed into tadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpoles obtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cells of the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel at the early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection. We assume that apoptosis is executed in Xenopus early gastrulae as a "fail-safe" mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Apoptose/fisiologia , Embrião não Mamífero/metabolismo , Adenosilmetionina Descarboxilase/genética , Animais , Blástula/efeitos dos fármacos , Blástula/fisiologia , Blástula/ultraestrutura , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Larva/citologia , Larva/efeitos dos fármacos , Larva/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica , Modelos Biológicos , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Xenopus
10.
J Biol Chem ; 278(6): 4048-56, 2003 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-12444087

RESUMO

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) using a yeast two-hybrid system. 4.1N and IP(3)R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP(3)R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP(3)R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573-30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP(3)R1 is necessary and sufficient for the localization of IP(3)R1 at the basolateral membrane domain. A fragment of the IP(3)R1-binding region of 4.1N blocks the localization of co-expressed IP(3)R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP(3)R1 translocation to the basolateral membrane domain in polarized MDCK cells.


Assuntos
Canais de Cálcio/metabolismo , Proteínas do Citoesqueleto , Proteínas de Membrana/fisiologia , Neuropeptídeos , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Sequência de Bases , Membrana Basal/metabolismo , Western Blotting , Encéfalo/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Primers do DNA , Cães , Receptores de Inositol 1,4,5-Trifosfato , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Testes de Precipitina , Ligação Proteica , Transporte Proteico
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