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1.
Biochim Biophys Acta ; 1853(9): 2104-14, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25764978

RESUMO

Bax inhibitor-1 (BI-1) is an evolutionarily conserved pH-dependent Ca²âº leak channel in the endoplasmic reticulum and the founding member of a family of six highly hydrophobic mammalian proteins named transmembrane BAX inhibitor motif containing (TMBIM) 1-6 with BI-1 being TMBIM6. Here we compared the structure, subcellular localization, tissue expression and the effect on the cellular Ca²âº homeostasis of all family members side by side. We found that all TMBIM proteins possess the di-aspartyl pH sensor responsible for pH sensing identified in TMBIM6 and its bacterial homologue BsYetJ. TMBIM1-3 and TMBIM4-6 represent two phylogenetically distinct groups that are localized in the Golgi apparatus (TMBIM1-3), endoplasmic reticulum (TMBIM4-6) or mitochondria (TMBIM5) but share a common structure of at least seven transmembrane domains with the last domain being semi-hydrophobic. TMBIM1 is mainly expressed in muscle, TMBIM2 and 3 in the nervous system, TMBIM4 and 5 are ubiquitously expressed and TMBIM6 in skeletal muscle, kidney, liver and spleen. All TMBIM proteins reduce the Ca²âº content of the endoplasmic reticulum, and all but TMBIM5 also reduce the cytosolic resting Ca²âº concentration. These results suggest that the TMBIM family has comparable functions in the maintenance of intracellular Ca²âº homeostasis in a wide variety of tissues. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexo de Golgi/metabolismo , Homeostase/fisiologia , Proteínas de Membrana/biossíntese , Motivos de Aminoácidos , Linhagem Celular , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Humanos , Proteínas de Membrana/genética , Especificidade de Órgãos/fisiologia
2.
J Biol Chem ; 288(52): 37204-15, 2013 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-24240096

RESUMO

Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/fisiologia , Proteínas do Citoesqueleto/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Linhagem Celular Transformada , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos
3.
J Neuroinflammation ; 9: 163, 2012 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-22769044

RESUMO

BACKGROUND: Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate is a promising novel oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. These effects are presumed to originate from a combination of immunomodulatory and neuroprotective mechanisms. We aimed to clarify whether neuroprotective concentrations of dimethyl fumarate have immunomodulatory effects. FINDINGS: We determined time- and concentration-dependent effects of dimethyl fumarate and its metabolite monomethyl fumarate on viability in a model of endogenous neuronal oxidative stress and clarified the mechanism of action by quantitating cellular glutathione content and recycling, nuclear translocation of transcription factors, and the expression of antioxidant genes. We compared this with changes in the cytokine profiles released by stimulated splenocytes measured by ELISPOT technology and analyzed the interactions between neuronal and immune cells and neuronal function and viability in cell death assays and multi-electrode arrays. Our observations show that dimethyl fumarate causes short-lived oxidative stress, which leads to increased levels and nuclear localization of the transcription factor nuclear factor erythroid 2-related factor 2 and a subsequent increase in glutathione synthesis and recycling in neuronal cells. Concentrations that were cytoprotective in neuronal cells had no negative effects on viability of splenocytes but suppressed the production of proinflammatory cytokines in cultures from C57BL/6 and SJL mice and had no effects on neuronal activity in multi-electrode arrays. CONCLUSIONS: These results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.


Assuntos
Fumaratos/farmacologia , Imunomodulação/imunologia , Fármacos Neuroprotetores/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Células Cultivadas , Fumarato de Dimetilo , Feminino , Imunomodulação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Resultado do Tratamento
4.
J Mol Med (Berl) ; 98(6): 849-862, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32394396

RESUMO

Transmembrane BAX inhibitor motif containing 6 (TMBIM6), also known as Bax inhibitor-1, is an evolutionarily conserved protein involved in endoplasmic reticulum (ER) function. TMBIM6 is an ER Ca2+ leak channel and its deficiency enhances susceptibility to ER stress due to inhibition of the ER stress sensor IRE1α. It was previously shown that TMBIM6 overexpression improves glucose metabolism and that TMBIM6 knockout mice develop obesity. We here examined the metabolic alterations underlying the obese phenotype and subjected TMBIM6 knockout mice to indirect calorimetry and euglycemic-hyperinsulinemic tests with stable isotope dilution to gauge tissue-specific insulin sensitivity. This demonstrated no changes in heat production, food intake, activity or hepatic and peripheral insulin sensitivity. TMBIM6 knockout mice, however, featured a higher glucose-stimulated insulin secretion in vivo as assessed by the hyperglycemic clamp test and hepatic steatosis. This coincided with profound changes in glucose-mediated Ca2+ regulation in isolated pancreatic ß cells and increased levels of IRE1α levels but no differences in downstream effects of IRE1α like increased Xbp1 mRNA splicing or Ire1-dependent decay of insulin mRNA in the pancreas. We therefore conclude that lack of TMBIM6 does not affect insulin sensitivity but leads to hyperinsulinemia, which serves to explain the weight gain. TMBIM6-mediated metabolic alterations are mainly caused by its role as a Ca2+ release channel in the ER. KEY MESSAGES: TMBIM6-/- leads to obesity and hepatic steatosis. Food intake and energy expenditure are not changed in TMBIM6-/- mice. No changes in insulin resistance in TMBIM6-/- mice. Increased insulin secretion caused by altered calcium dynamics in ß cells.


Assuntos
Cálcio/metabolismo , Suscetibilidade a Doenças , Secreção de Insulina , Proteínas de Membrana/deficiência , Obesidade/etiologia , Obesidade/metabolismo , Animais , Modelos Animais de Doenças , Ingestão de Alimentos , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica , Genótipo , Glucose/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Camundongos , Camundongos Knockout , Splicing de RNA , Termogênese/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
5.
Gene ; 586(1): 62-8, 2016 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-27040980

RESUMO

Androgen-induced gene 1 (AIG1) is a transmembrane protein implicated with survival (its expression level was shown to correlate with the survival of patients suffering from hepatocellular carcinoma) and Ca(2+) signaling (over-expression of AIG1 increased transcription mediated by the Ca(2+)-dependent nuclear factor of activated T cells). We aimed to shed light on this less-studied protein and investigated its tissue expression, genomic organization, intracellular localization and membrane topology as well as its effects on cell death susceptibility and the Ca(2+) content of the endoplasmic reticulum. Immunoblotting of mouse tissues demonstrated highest expression of AIG1 in the liver, lung and heart. AIG1 has a complex genomic organization and expresses several splice variants in a tissue-dependent manner. Analyzing the topology of AIG1 in the ER membrane using a protease-protection assay suggested that AIG has five transmembrane domains with a luminal N- and cytosolic C-terminus and a hydrophobic stretch between the third and fourth membrane domain that does not cross the membrane. AIG1 over-expression slightly increased susceptibility to oxidative stress, which correlated with an increased ER Ca(2+) concentration in two different cell lines. Together, these results indicate that AIG1 plays a role in the control of the intracellular Ca(2+) concentration and cell death susceptibility.


Assuntos
Cálcio/metabolismo , Morte Celular , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Estresse Oxidativo , Processamento Alternativo , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Expressão Gênica , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Domínios Proteicos , Caracteres Sexuais
6.
Cell Calcium ; 54(3): 186-92, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23867001

RESUMO

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca2+ levels. Recently, we elucidated BI-1's Ca(2+)-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca(2+)-channel pore-dead mutant BI-1 (BI-1(D213R)) was developed. We determined whether BI-1 behaves as a bona fide H+/Ca2+ antiporter or as an ER Ca(2+)-leak channel by investigating the effect of pH on unidirectional Ca(2+)-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1(-/-) cells increased the ER Ca(2+)-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1(D231R) expression in BI-1(-/-), despite its ER localization, did not increase the ER Ca(2+)-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca2+ leak was blocked. Finally, a peptide representing the Ca(2+)-channel pore of BI-1 promoting Ca2+ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca2+ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca(2+)-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Acidose/metabolismo , Acidose/patologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Calcimicina/farmacologia , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Peptídeos/farmacologia , Estrutura Terciária de Proteína
7.
Cell Calcium ; 50(3): 251-60, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21663964

RESUMO

Bax inhibitor-1 (BI-1) was initially identified for its ability to inhibit BAX-induced apoptosis in yeast cells and is the founding member of a family of highly hydrophobic proteins localized in diverse cellular membranes. It is evolutionarily conserved and orthologues from plants can substitute for mammalian BI-1 in regard to its anti-apoptotic function suggesting a high degree of functional conservation. BI-1 interacts with BCL-2 and BCL-XL and, similar to these two anti-apoptotic proteins, the effect of BI-1 on cell death involves changes in the amount of Ca(2+) releasable from intracellular stores. However, BI-1 is also a negative regulator of the endoplasmic reticulum stress sensor IRE1 α, it interacts with G-actin and increases actin polymerization, enhances cancer metastasis by altering glucose metabolism and activating the sodium-hydrogen exchanger, and reduces the production of reactive oxygen species through direct interaction with NADPH-P450 reductase. In this contribution, we summarize what is known about the expression, intracellular localization and structure of BI-1 and specifically illuminate its effects on the intracellular Ca(2+) homeostasis and how this might relate to its other functions. We also present a thorough phylogenetic analysis of BI-1 proteins from major phyla together with paralogues from all BI-1 family members.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Actinas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Morte Celular , Endorribonucleases/metabolismo , Homeostase , Humanos , Mamíferos , Proteínas de Membrana/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Neoplasias/metabolismo , Filogenia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo
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