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1.
Cell Mol Life Sci ; 77(12): 2367-2386, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31471680

RESUMO

Selenoprotein P (SELENOP), secreted from the liver, functions as a selenium (Se) supplier to other tissues. In the brain, Se homeostasis is critical for physiological function. Previous studies have reported that SELENOP co-localizes with the apolipoprotein E receptor 2 (ApoER2) along the blood-brain barrier (BBB). However, the mechanism underlying SELENOP transportation from hepatocytes to neuronal cells remains unclear. Here, we found that SELENOP was secreted from hepatocytes as an exosomal component protected from plasma kallikrein-mediated cleavage. SELENOP was interacted with apolipoprotein E (ApoE) through heparin-binding sites of SELENOP, and the interaction regulated the secretion of exosomal SELENOP. Using in vitro BBB model of transwell cell culture, exosomal SELENOP was found to supply Se to brain endothelial cells and neuronal cells, which synthesized selenoproteins by a process regulated by ApoE and ApoER2. The regulatory role of ApoE in SELENOP transport was also observed in vivo using ApoE-/- mice. Exosomal SELENOP transport protected neuronal cells from amyloid ß (Aß)-induced cell death. Taken together, our results suggest a new delivery mechanism for Se to neuronal cells by exosomal SELENOP.


Assuntos
Apolipoproteínas E/metabolismo , Exossomos/metabolismo , Transporte Proteico/fisiologia , Selenoproteína P/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Células HEK293 , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
2.
Biochim Biophys Acta ; 1863(1): 10-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26474786

RESUMO

Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein.


Assuntos
Proteínas 14-3-3/metabolismo , Estresse Oxidativo/fisiologia , Selenoproteína W/metabolismo , Proteínas 14-3-3/genética , Motivos de Aminoácidos , Ditiotreitol/farmacologia , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Células MCF-7 , Mutação de Sentido Incorreto , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Selenoproteína W/genética
3.
Biochem Biophys Res Commun ; 486(2): 444-450, 2017 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-28315680

RESUMO

Amyloid beta precursor protein (APP) is normally cleaved by α-secretase, but can also be cleaved by ß-secretase (BACE1) to produce C99 fragments in the endoplasmic reticulum (ER) membrane. C99 is subsequently cleaved to amyloid ß (Aß), the aggregation of which is known to cause Alzheimer's disease. Therefore, C99 removing is for preventing the disease. Selenoprotein S (SelS) is an ER membrane protein participating in endoplasmic reticulum-associated degradation (ERAD), one of the stages in resolving ER stress of misfolded proteins accumulated in the ER. ERAD has been postulated as one of the processes to degrade C99; however, it remains unclear if the degradation depends on SelS. In this study, we investigated the effect of SelS on C99 degradation. We observed that both SelS and C99 were colocalized in the membrane fraction of mouse neuroblastoma Neuro2a (N2a) cells. While the level of SelS was increased by ER stress, the level of C99 was decreased. However, despite the induction of ER stress, there was no change in the amount of C99 in SelS knock-down cells. The interaction of C99 with p97(VCP), an essential component of the ERAD complex, did not occur in SelS knock-down cells. The ubiquitination of C99 was decreased in SelS knock-down cells. We also found that the extracellular amount of Aß1-42 was relatively higher in SelS knock-down cells than in control cells. These results suggest that SelS is required for C99 degradation through ERAD, resulting in inhibition of Aß production.


Assuntos
Adenosina Trifosfatases/genética , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Proteínas de Ciclo Celular/genética , Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana/genética , Fragmentos de Peptídeos/genética , Selenoproteínas/genética , Adenosina Trifosfatases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Cinética , Proteínas de Membrana/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteólise , Selenoproteínas/metabolismo , Transgenes , Ubiquitinação , Proteína com Valosina
4.
J Biol Chem ; 290(50): 29941-52, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26504085

RESUMO

Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Selenoproteínas/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Ligação Proteica , Proteína com Valosina
5.
J Biol Chem ; 289(20): 13758-68, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24700463

RESUMO

During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro(178) and Pro(183) of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Prolina/metabolismo , Selenoproteínas/química , Selenoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Estresse do Retículo Endoplasmático , Inativação Gênica , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Selenoproteínas/deficiência , Selenoproteínas/genética , Proteína com Valosina
6.
Biochim Biophys Acta ; 1843(7): 1356-64, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24726955

RESUMO

Selenoprotein W (SelW) is expressed in various tissues, particularly in skeletal muscle. We have previously reported that SelW is up-regulated during C2C12 skeletal muscle differentiation and inhibits binding of 14-3-3 to its target proteins. 14-3-3 reduces myogenic differentiation by inhibiting nuclear translocation of transcriptional co-activator with PDZ-binding motif (TAZ). Phosphorylation of TAZ at Ser89 is required for binding to 14-3-3, leading to cytoplasmic retention of TAZ and a delay in myogenic differentiation. Here, we show that myogenic differentiation was delayed in SelW-knockdown C2C12 cells. Down-regulation of SelW also increased TAZ binding to 14-3-3, which eventually resulted in decreasing translocation of TAZ to the nucleus. However, phosphorylation of TAZ at Ser89 was not affected. Although phosphorylation of TAZ at Ser89 was sustained by the phosphatase inhibitor okadaic acid, nuclear translocation of TAZ was increased by ectopic expression of SelW. This result was due to decreased binding of TAZ to 14-3-3. We also found that the interaction between TAZ and MyoD was increased by ectopic expression of SelW. Taken together, these findings strongly demonstrate that SelW enhances C2C12 cell differentiation by inhibiting TAZ binding to 14-3-3.


Assuntos
Proteínas 14-3-3/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Selenoproteína W/metabolismo , Fatores de Transcrição/metabolismo , Proteínas 14-3-3/genética , Aciltransferases , Animais , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Ácido Okadáico/farmacologia , Fosforilação , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Selenoproteína W/antagonistas & inibidores , Selenoproteína W/genética , Transdução de Sinais , Fatores de Transcrição/genética
7.
Biochim Biophys Acta ; 1833(10): 2135-42, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23680186

RESUMO

14-3-3 reduces cell proliferation by inhibiting the activity of proteins involved in the signaling pathway that includes Akt kinase. Activation of Akt is enhanced by activating the mammalian target of rapamycin complex 2 (mTORC2). 14-3-3 is also a negative regulator of the mTORC2/Akt pathway, by interacting with a component of mTORC2. Recently, we reported that selenoprotein W (SelW) regulated the interaction between 14-3-3 and its target protein, CDC25B. Here, we show that the binding of Rictor, a component of mTORC2, to 14-3-3, is regulated by the interaction of 14-3-3 with SelW. When SelW was down-regulated, mTORC2-dependent phosphorylation of Akt at Ser473 was decreased. However, the phosphorylation of Thr308 was not affected. The interaction of Rictor with 14-3-3 was increased in SelW-knockdown cells, as compared to control cells. SelW-knockdown cells were also more sensitive to DNA damage induced by etoposide, than control cells. This phenomenon was due to the decreased phosphorylation of Akt at Ser473. We also found that ectopic expression of SelW(U13C) reduced the interaction between Rictor and 14-3-3, leading to Akt phosphorylation at Ser473. Taken together, these findings demonstrate that SelW activates the mTORC2/Akt pathway for Akt phosphorylation at Ser473, by interrupting the binding of Rictor to 14-3-3.


Assuntos
Proteínas 14-3-3/metabolismo , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Pulmonares/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Selenoproteína W/metabolismo , Serina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas 14-3-3/antagonistas & inibidores , Proteínas 14-3-3/genética , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proliferação de Células , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Proteína Companheira de mTOR Insensível à Rapamicina , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Selenoproteína W/genética , Serina/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Cicatrização
8.
J Immunol ; 188(11): 5665-73, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22539796

RESUMO

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow-derived mast cells. This cleavage event was inhibited when intracellular Ca(2+) signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A(2) (cPLA(2)) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA(2) interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA(2) mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA(2) and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA(2) and production of eicosanoids during mast-cell activation.


Assuntos
Anexina A1/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Proteólise , Animais , Anexina A1/fisiologia , Linhagem Celular , Células Cultivadas , Eicosanoides/biossíntese , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Mastócitos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipases A2 Citosólicas/fisiologia , Fosforilação/imunologia
9.
Free Radic Biol Med ; 141: 362-371, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299423

RESUMO

Mouse selenoprotein W (SELENOW) is a small protein containing a selenocysteine (Sec, U) and four cysteine (Cys, C) residues. The Sec residue in SELENOW is located within the conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin (Trx). It is known that glutathione (GSH) binds to SELENOW and that this binding is involved in protecting cells from oxidative stress. However, the regulatory mechanisms controlling the glutathionylation of SELENOW in oxidative stress are unclear. In this study, using purified recombinant SELENOW in which Sec13 was changed to Cys, we found that SELENOW was glutathionylated at Cys33 and that this S-glutathionylation was enhanced by oxidative stress. We also found that the S-glutathionylation of SELENOW at Cys33 in HEK293 cells was due to glutathione S-transferase Pi (GSTpi) and that this modification was reversed by glutaredoxin1 (Grx1). In addition to the disulfide bond between the Cys10 and Cys13 of SELENOW, a second disulfide bond was formed between Cys33 and Cys87 under oxidative stress conditions. The second disulfide bond was reduced by Trx1, but the disulfide bond between Cys10 and Cys13 was not. The second disulfide bond was also reduced by glutathione, but the disulfide bond in the CXXC motif was not. The second disulfide bond of the mutant SELENOW, in which Cys37 was replaced with Ser, was formed at a much lower concentration of hydrogen peroxide than the wild type. We also observed that Cys37 was required for S-glutathionylation, and that S-glutathionylated SELENOW containing Cys37 protected the cells from oxidative stress. Furthermore, the SELENOW (C33, 87S) mutant, which could not form the second disulfide bond, also showed antioxidant activity. Taken together, these results indicate that GSTpi-mediated S-glutathionylation of mouse SELENOW at Cys33 is required for the protection of cells in conditions of oxidative stress, through inhibition of the formation of the second disulfide bond.


Assuntos
Dissulfetos/metabolismo , Glutationa S-Transferase pi/genética , Estresse Oxidativo/genética , Selenoproteína W/genética , Animais , Sítios de Ligação/genética , Morte Celular/genética , Cisteína/genética , Dissulfetos/antagonistas & inibidores , Glutarredoxinas/genética , Glutationa/genética , Glutationa/metabolismo , Células HEK293 , Humanos , Camundongos , Oxirredução , Ligação Proteica/genética , Selenocisteína/genética , Selenoproteína W/metabolismo
10.
Cell Death Differ ; 26(6): 1007-1023, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082770

RESUMO

Adipocyte differentiation is known to be related with endoplasmic reticulum (ER) stress. We have reported that selenoprotein S (SelS) and selenoprotein K (SelK) have a function in the regulation of ER stress and ER-associated degradation. However, the association between adipocyte differentiation and the ER-resident selenoproteins, SelS and SelK, is unclear. In this study, we found that the levels of SelS and SelK were decreased during adipocyte differentiation and were inversely related to the levels of peroxisome proliferator-activated receptor γ (PPARγ), a central regulator of adipogenesis. It has been recently reported that PPARγ has E3 ubiquitin ligase activity. Here, we report that PPARγ directly interacts with both SelS and SelK via its ligand-binding domain to induce ubiquitination and degradation of the selenoproteins. Lysine residues at the 150th position of SelS and the 47th and 48th positions of SelK were the target sites for ubiquitination by PPARγ. We also found that adipocyte differentiation was inhibited when either SelS or SelK was not degraded by PPARγ. Thus, these data indicate that PPARγ-mediated ubiquitination and degradation of SelS and SelK is required for adipocyte differentiation.


Assuntos
Adipócitos/metabolismo , Proteínas de Membrana/metabolismo , PPAR gama/metabolismo , Selenoproteínas/metabolismo , Ubiquitina/metabolismo , Animais , Diferenciação Celular , Células HT29 , Humanos , Células MCF-7 , Proteínas de Membrana/genética , Camundongos , Camundongos Obesos , Mutação , PPAR gama/genética , Selenoproteínas/genética , Células Tumorais Cultivadas , Ubiquitinação
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