RESUMO
The G protein-coupled receptor GPRC6A regulates various physiological processes in response to its interaction with multiple ligands, such as extracellular basic amino acids, divalent cations, testosterone, and the uncarboxylated form of osteocalcin (GluOC). Global ablation of GPRC6A increases the susceptibility of mice to diet-induced obesity and related metabolic disorders. However, given that GPRC6A is expressed in many tissues and responds to a variety of hormonal and nutritional signals, the cellular and molecular mechanisms underlying the development of metabolic disorders in conventional knockout mice have remained unclear. On the basis of our previous observation that long-term oral administration of GluOC markedly reduced adipocyte size and improved glucose tolerance in WT mice, we examined whether GPRC6A signaling in adipose tissue might be responsible for prevention of metabolic disorders. We thus generated adipocyte-specific GPRC6A knockout mice, and we found that these animals manifested increased adipose tissue weight, adipocyte hypertrophy, and adipose tissue inflammation when fed a high-fat and high-sucrose diet compared with control mice. These effects were associated with reduced lipolytic activity because of downregulation of lipolytic enzymes such as adipose triglyceride lipase and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of adipose triglyceride lipase in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.
Assuntos
Inflamação/genética , Obesidade/genética , Osteocalcina/genética , Receptores Acoplados a Proteínas G/genética , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Teste de Tolerância a Glucose , Humanos , Inflamação/patologia , Insulina/genética , Resistência à Insulina/genética , Lipase/genética , Lipólise/genética , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Obesidade/patologiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is detectable in nasopharyngeal specimens for up to 12-20 days regardless of the presence of chronic diseases in patients. We report a case of prolonged SARS-CoV-2 infection that lasted for more than eight weeks. The patient had persistent lymphopenia after receiving six cycles of bendamustine and rituximab (BR) therapy for follicular lymphoma; the last chemotherapy session was completed nine months before admission. The first nasopharyngeal specimen (NPS) for the SARS-CoV-2 polymerase chain reaction assay tested positive for the N501Y variant five weeks before admission. The patient's general and respiratory conditions gradually worsened; therefore, he was admitted to our hospital, and the same SARS-CoV-2 variant was subsequently identified on admission. Treatment for coronavirus disease was initiated, and the patient's condition improved; however, the NPS tested positive on day 15. The patient was discharged on day 28 and was instructed to isolate at home for a month. Hence, possible prolonged SARS-CoV-2 shedding should be considered in patients who receive BR therapy.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Cloridrato de Bendamustina/uso terapêutico , Humanos , Masculino , RNA Viral , Rituximab/efeitos adversos , Eliminação de Partículas ViraisRESUMO
Here, we report a case of ascending colon cancer successfully treated with laparoscopic right hemicolectomy in a 74- year-old man with a medical history of hemophilia A. He was admitted to our hospital because of bloody stool and diagnosed with type 2 ascending colon cancer based on colonoscopy findings. Preoperatively, blood transfusion and administration of recombinant factor â § products were performed. Surgery involved laparoscopic right hemicolectomy plus group 3 lymph node dissection. No complications, such as bleeding, occurred during hospitalization. The patient was discharged on postoperative day 8. There have been a few reports of laparoscopic surgery for patients with hemophilia. However, this case suggests that it can be safely performed with planned factor â § supplementation in the perioperative period.
Assuntos
Coagulantes , Neoplasias do Colo , Hemofilia A , Laparoscopia , Idoso , Colectomia , Colo Ascendente/cirurgia , Neoplasias do Colo/complicações , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/cirurgia , Hemofilia A/complicações , Hemofilia A/tratamento farmacológico , Humanos , MasculinoRESUMO
Pex7p is the cytosolic receptor for peroxisomal matrix proteins harbouring PTS2 (peroxisome-targeting signal type-2). Mutations in the PEX7 gene cause RCDP (rhizomelic chondrodysplasia punctata) type 1, a distinct PTS2-import-defective phenotype of peroxisome biogenesis disorders. The mechanisms by which the protein level and quality of Pex7p are controlled remain largely unknown. In the present study we show that dysfunctional Pex7p, including mutants from RCDP patients, is degraded by a ubiquitin-dependent proteasomal pathway involving the CRL4A (Cullin4A-RING ubiquitin ligase) complex. Furthermore, we demonstrate that the degradation of dysfunctional Pex7p is essential for maintaining normal PTS2 import, thereby suggesting that CRL4A functions as an E3 ligase in the quality control of Pex7p. Our results define a mechanism underlying Pex7p homoeostasis and highlight its importance for regulating PTS2 import. These findings may lead to a new approach to Pex7p-based therapies for the treatment of peroxisome biogenesis disorders such as RCDP.
Assuntos
Proteínas de Transporte/metabolismo , Condrodisplasia Punctata Rizomélica/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mutação , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células CHO , Proteínas de Transporte/genética , Condrodisplasia Punctata Rizomélica/genética , Condrodisplasia Punctata Rizomélica/patologia , Cricetinae , Cricetulus , Proteínas Culina/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Fracionamento Celular , Cricetinae , Cricetulus , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Imunoprecipitação , Fígado/citologia , Fígado/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Receptor 1 de Sinal de Orientação para Peroxissomos , Estabilidade Proteica , Transporte Proteico , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Transfecção , Ubiquitinação , Dedos de ZincoRESUMO
A 65-year-old woman was diagnosed with Coombs-positive autoimmune hemolytic anemia (AIHA) and pure red cell aplasia (PRCA) in May 1992. One month later, her PRCA went into remission following treatment but she developed idiopathic thrombocytopenic purpura and was diagnosed with Evans syndrome. Although her condition resolved with administration of prednisolone and azathioprine, it was necessary to continue treatment with gradual tapering over the following two decades. In October 2012, her hemolytic anemia again worsened, and lymph node swelling, splenomegaly and B symptoms developed. She was diagnosed as having diffuse large B-cell lymphoma (DLBCL) based on lymph node biopsy. However, AIHA was not considered to be the cause of her hemolytic anemia, but rather to be related to DLBCL. This was because a Coombs test and other extensive investigations for Coombs negative-AIHA yielded negative results. The patient underwent CHOP therapy, and all of her symptoms improved. Herein, we report this rare case in which DLBCL developed after the onset of Evans syndrome.
Assuntos
Anemia Hemolítica Autoimune/complicações , Anemia Hemolítica/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma Difuso de Grandes Células B/terapia , Trombocitopenia/complicações , Idade de Início , Idoso , Anemia Hemolítica/complicações , Anemia Hemolítica/diagnóstico , Feminino , Humanos , Linfoma Difuso de Grandes Células B/complicações , Linfoma Difuso de Grandes Células B/diagnóstico , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico , Resultado do TratamentoRESUMO
Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.
Assuntos
Cisteína/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Cisteína/genética , Citosol/metabolismo , Ditiotreitol/farmacologia , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/metabolismo , Células Tumorais CultivadasRESUMO
Periodontal disease is an infectious disease that affects many people worldwide. Disease progression destroys the alveolar bone and causes tooth loss. We have previously shown that alymphoplasia (aly/aly) mice harboring a loss-of-function mutation in the map3k14 gene, which is involved in p100 to p52 processing of the alternative NF-κB pathway, exhibited mild osteopetrosis due to decreased number of osteoclasts, suggesting the alternative NF-κB pathway as a potential drug target for the amelioration of bone disease. In the present study, wild-type (WT) and aly/aly mice were subjected to silk ligation to establish a periodontitis model. Alveolar bone resorption was suppressed in aly/aly mice by decreased numbers of osteoclasts in the alveolar bone in comparison to WT mice. Furthermore, the expression of receptor activator of NF-κB ligand (RANKL) and TNFα (cytokines involved in osteoclast induction in periligative gingival tissue) was decreased. When primary osteoblasts (POBs) and bone marrow cells (BMCs) derived from WT and aly/aly mice were prepared and co-cultured, osteoclasts were induced from WT-derived BMCs, regardless of the origin of the POBs, but hardly formed from aly/aly mouse-derived BMCs. Furthermore, the local administration of an NIK inhibitor, Cpd33, inhibited osteoclast formation and thereby inhibited alveolar bone resorption in the periodontitis model. Therefore, the NIK-mediated NF-κB alternative pathway can be a therapeutic target for periodontal disease.
Assuntos
Doenças Ósseas Metabólicas , Reabsorção Óssea , Doenças Periodontais , Periodontite , Camundongos , Animais , NF-kappa B/metabolismo , InflamaçãoRESUMO
Many cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The predominant form of mammalian GPI contains 1-alkyl-2-acyl phosphatidylinositol (PI), which is generated by lipid remodeling from diacyl PI. The conversion of diacyl PI to 1-alkyl-2-acyl PI occurs in the ER at the third intermediate in the GPI biosynthetic pathway. This lipid remodeling requires the alkyl-phospholipid biosynthetic pathway in peroxisome. Indeed, cells defective in dihydroxyacetone phosphate acyltransferase (DHAP-AT) or alkyl-DHAP synthase express only the diacyl form of GPI-anchored proteins. A defect in the alkyl-phospholipid biosynthetic pathway causes a peroxisomal disorder, rhizomelic chondrodysplasia punctata (RCDP), and defective biogenesis of peroxisomes causes Zellweger syndrome, both of which are lethal genetic diseases with multiple clinical phenotypes such as psychomotor defects, mental retardation, and skeletal abnormalities. Here, we report that GPI lipid remodeling is defective in cells from patients with Zellweger syndrome having mutations in the peroxisomal biogenesis factors PEX5, PEX16, and PEX19 and in cells from patients with RCDP types 1, 2, and 3 caused by mutations in PEX7, DHAP-AT, and alkyl-DHAP synthase, respectively. Absence of the 1-alkyl-2-acyl form of GPI-anchored proteins might account for some of the complex phenotypes of these two major peroxisomal disorders.
Assuntos
Condrodisplasia Punctata Rizomélica/patologia , Glicosilfosfatidilinositóis/metabolismo , Síndrome de Zellweger/patologia , Aciltransferases/genética , Aciltransferases/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Células CHO , Linhagem Celular Transformada , Condrodisplasia Punctata Rizomélica/genética , Condrodisplasia Punctata Rizomélica/metabolismo , Cricetinae , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Receptor 2 de Sinal de Orientação para Peroxissomos , Peroxissomos/genética , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transfecção , Síndrome de Zellweger/genética , Síndrome de Zellweger/metabolismoRESUMO
A 61-year-old man was referred to our hospital for leukocytosis and thrombocytopenia. Bone marrow examination showed hypercellular bone marrow accompanied by dysplasia, and the karyotype of his bone marrow cells was 46,XY, der(5;12)(q10;q10), +mar,inc[3]/46,XY[12]. A diagnosis of myelodysplastic syndrome, unclassifiable, was made. Analysis of major BCR/ABL1 chimeric RNA by real-time polymerase chain reaction method was positive, and then Ph chromosome was observed afterward. His Ph chromosome was seen in der(5;12)-negative cells analyzed by FISH, which suggested the late-appearing Ph chromosome evolved into another clone. Despite treatment containing imatinib, hydroxyurea, and cytosine arabinoside, he died due to respiratory dysfunction 5 months after the initial diagnosis. The autopsy revealed massive pulmonary infiltration by Ph-negative cells, suggesting MDS-derived clones. It has been reported that the late appearance of Ph chromosome associates with leukemic progression. Although the incidence of late-appearing Ph chromosome is estimated to be relatively low, further accumulation of cases is necessary for the evaluation of its impact on prognosis and disease progression.
Assuntos
Medula Óssea/patologia , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 5/genética , Síndromes Mielodisplásicas/genética , Cromossomo Filadélfia , Aberrações Cromossômicas , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnósticoRESUMO
We present three long-term survivors treated with high-dose melphalan with autologous peripheral blood stem cell transplantation(auto PBSCT)for primary AL amyloidosis. Because melphalan toxicity is dose-related, patient age and the extent of organ involvement are very important factors for the success of high-dose melphalan treatment with PBSCT. The patients were therefore given high-dose melphalan using the risk-adapted approach to melphalan dosing. They received 3 courses of a vincristine, doxorubicin and dexamethasone(VAD)regimen, along with high-dose melphalan(100-200mg/m2)with auto PBSCT. There were no serious complications or transplantation-related mortalities. Patients survived for an average of 68 months(22-100 months)in partial response. A risk-adapted approach to melphalan dosing with PBSCT is an effective treatment in patients with primary AL amyloidosis.
Assuntos
Amiloidose/tratamento farmacológico , Melfalan/uso terapêutico , Transplante de Células-Tronco de Sangue Periférico , Amiloidose/patologia , Amiloidose/cirurgia , Biópsia , Terapia Combinada , Feminino , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-IdadeRESUMO
We report the case of a 66-year-old man with primary diffuse large B-cell lymphoma of the prostate presenting with urinary retention and dyschezia as first manifestation. Although a colostomy was needed due to rectal obstruction, rituximab-combined chemotherapy resulted in complete remission. He underwent stoma closure safely and has remained in complete remission for over 3years. Primary prostatic lymphoma is extremely rare, presenting as 0.1% of newly diagnosed lymphomas, but rituximab-containing chemotherapy seems to be as effective as for nodal lymphoma.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Constipação Intestinal/etiologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Próstata/patologia , Neoplasias Retais/tratamento farmacológico , Retenção Urinária/etiologia , Idoso , Anticorpos Monoclonais Murinos/administração & dosagem , Terapia Combinada , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Humanos , Linfoma Difuso de Grandes Células B/complicações , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/cirurgia , Masculino , Prednisona/uso terapêutico , Neoplasias Retais/complicações , Neoplasias Retais/patologia , Neoplasias Retais/cirurgia , Indução de Remissão , Rituximab , Vincristina/uso terapêuticoRESUMO
Menopausal women are susceptible to visceral obesity, which increases the risk of metabolic disorders. However, the mechanisms of menopause-induced visceral fat accumulation are not fully understood. Circulating levels of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) are elevated in an animal model of menopause. RANKL, a multifunctional cytokine, activates the NF-κB pathway, which serves as a pivotal mediator of inflammatory responses. Here, we investigated whether RANKL-induced non-canonical NF-κB pathway activation induces inflammation and lipid accumulation in adipose tissues. RANKL induced Tnfa expression via the non-canonical NF-κB pathway in bone marrow cells. We therefore analyzed aly/aly mice, in which the non-canonical NF-κB pathway is not activated, owing to an inactive form of NF-κB-inducing kinase. A postmenopausal obesity model was generated by ovariectomy and subsequent high-fat and high-sucrose diet feeding. In aly/aly mice with postmenopausal obesity, serum RANKL levels were elevated, and hepatic lipid accumulation and adipocyte hypertrophy were suppressed, resulting in reduced macrophage infiltration and inflammatory cytokine mRNA expression in visceral adipose tissue. Furthermore, aly/aly mice showed protection from glucose intolerance and insulin resistance, which were observed in ovariectomized WT obese mice. These findings indicate that non-canonical NF-κB pathway activation via serum RANKL elevation contributes to postmenopausal obesity.
Assuntos
Resistência à Insulina , NF-kappa B , Animais , Feminino , Humanos , Lipídeos , Camundongos , Camundongos Obesos , NF-kappa B/metabolismo , Obesidade/etiologiaRESUMO
Pex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, 35S-labeled Pex7p was imported into peroxisomes. 35S-Pex7p import was also evident using rat liver peroxisomes. 35S-Pex7p was not imported into peroxisomal remnants from a pex5 ZPG231 defective in PTS2 import and pex2 Z65. When the import of 35S-Pex5pL was inhibited with an excess amount of recombinant Pex5pS, 35S-Pex7p import was concomitantly abrogated, suggesting that Pex5pL was a transporter for Pex7p, unlike a yeast cochaperone, Pex18p. 35S-Pex7p as well as 35S-Pex5p was imported in an ATP-independent manner, whilst the import of PTS1 and PTS2 cargo-proteins was ATP-dependent. Thereby, ATP-independent import of Pex7p implicated that Pex5p export requiring ATP hydrolysis is not a limiting step for its cargo recruitment to peroxisomes. PTS1 protein import was indeed insensitive to N-ethylmaleimide, whereas Pex5p export was N-ethylmaleimide-sensitive. Taken together, the cargo-protein translocation through peroxisomal membrane more likely involves another ATP-requiring step in addition to the Pex5p export. Moreover, upon concurrent import into peroxisomes, 35S-Pex5pL and 35S-Pex7p were detected at mutually distinct ratios in the immunoprecipitates each of the import machinery peroxins including Pex14p, Pex13p, and Pex2p, hence suggesting that Pex7p as well as Pex5p translocated from the initial docking complex to RING complex on peroxisomes.
Assuntos
Sistema Livre de Células , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Acil-CoA Oxidase/metabolismo , Animais , Transporte Biológico/fisiologia , Células CHO , Cricetinae , Cricetulus , Fígado/citologia , Fígado/metabolismo , Complexos Multiproteicos/metabolismo , Receptor 2 de Sinal de Orientação para Peroxissomos , Ratos , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Osteocalcin is a bone-derived hormone that in its uncarboxylated form (GluOC) plays an important role in glucose and energy metabolism by stimulating insulin secretion and pancreatic ß-cell proliferation through its putative receptor GPRC6A. We previously showed that the effect of GluOC on insulin secretion is mediated predominantly by glucagon-like peptide-1 (GLP-1) released from intestinal endocrine cells in response to GluOC stimulation. Moreover, oral administration of GluOC was found to reduce the fasting blood glucose level, to improve glucose tolerance, and to increase the fasting serum insulin concentration and ß-cell area in the pancreas in wild-type mice. We have now examined the effects of oral GluOC administration for at least 4 weeks in GLP-1 receptor-knockout mice. Such administration of GluOC in the mutant mice triggered glucose intolerance, enhanced gluconeogenesis and promoted both lipid accumulation in the liver as well as adipocyte hypertrophy and inflammation in adipose tissue. Furthermore, inactivation of GLP-1 receptor signaling in association with GluOC administration induced activation of the transcription factor FoxO1 and expression of its transcriptional coactivator PGC1α in the liver, likely accounting for the observed upregulation of gluconeogenic gene expression. Our results thus indicate that the beneficial metabolic effects of GluOC are dependent on GLP-1 receptor signaling.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Intolerância à Glucose/metabolismo , Osteocalcina/metabolismo , Animais , Glicemia/metabolismo , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Intolerância à Glucose/genética , Teste de Tolerância a Glucose , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismoRESUMO
Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes.
Assuntos
Receptor 2 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , Animais , Células CHO , Cricetulus , Citosol/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Sinais de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/fisiologia , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
A newly isolated binding protein of peroxisomal targeting signal type 2 (PTS2) receptor Pex7, termed P7BP2, is transported into peroxisomes by binding to the longer isoform of Pex5p, Pex5pL, via Pex7p. The binding to Pex7p and peroxisomal localization of P7BP2 depends on the cleavable PTS2 in the N-terminal region, suggesting that P7BP2 is a new PTS2 protein. By search on human database, three AAA+ domains are found in the N-terminal half of P7BP2. Protein sequence alignment and motif search reveal that in the C-terminal region P7BP2 contains additional structural domains featuring weak but sufficient homology to AAA+ domain. P7BP2 behaves as a monomer in gel-filtration chromatography and the single molecule observed under atomic force microscope shapes a disc-like ring. Collectively, these results suggest that P7BP2 is a novel dynein-type AAA+ family protein, of which domains are arranged into a pseudo-hexameric ring structure.
Assuntos
Adenosina Trifosfatases/metabolismo , Receptor 2 de Sinal de Orientação para Peroxissomos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Peroxissomos/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Sistemas Inteligentes , Células HeLa , Humanos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptor 2 de Sinal de Orientação para Peroxissomos/química , Receptor 2 de Sinal de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Peroxissomos/enzimologia , Domínios e Motivos de Interação entre Proteínas , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteólise , Proteômica/métodos , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
Loss of voltage-dependent anion channel 2 (VDAC2) leads to impaired peroxisome biogenesis in mammalian cells. Knockdown of BAK restores peroxisomal biogenesis in VDAC2-deficient cells, where BAK localization shifts from mitochondria to peroxisomes. Moreover, overexpression of BAK activators in wild-type cells permeabilizes peroxisomes in a BAK-dependent manner. Together, BAK most likely regulates peroxisomal membrane permeability.
RESUMO
Peroxisomal biogenesis disorders (PBDs) are fatal genetic diseases consisting of 14 complementation groups (CGs). We previously isolated a peroxisome-deficient Chinese hamster ovary cell mutant, ZP114, which belongs to none of these CGs. Using a functional screening strategy, VDAC2 was identified as rescuing the peroxisomal deficiency of ZP114 where VDAC2 expression was not detected. Interestingly, knockdown of BAK or overexpression of the BAK inhibitors BCL-XL and MCL-1 restored peroxisomal biogenesis in ZP114 cells. Although VDAC2 is not localized to the peroxisome, loss of VDAC2 shifts the localization of BAK from mitochondria to peroxisomes, resulting in peroxisomal deficiency. Introduction of peroxisome-targeted BAK harboring the Pex26p transmembrane region into wild-type cells resulted in the release of peroxisomal matrix proteins to cytosol. Moreover, overexpression of BAK activators PUMA and BIM permeabilized peroxisomes in a BAK-dependent manner. Collectively, these findings suggest that BAK plays a role in peroxisomal permeability, similar to mitochondrial outer membrane permeabilization.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Peroxissomos/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Citosol/metabolismo , Citosol/fisiologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Transtornos Peroxissômicos/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
To investigate peroxisome assembly and human peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, thirteen different complementation groups (CGs) of Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis have been isolated and established as a model research system. Successful gene-cloning studies by a forward genetic approach utilized a rapid functional complementation assay of CHO cell mutants led to isolation of human peroxin (PEX) genes. Search for pathogenic genes responsible for PBDs of all 14 CGs is now completed together with the homology search by screening the human expressed sequence tag database using yeast PEX genes. Peroxins are divided into three groups: (1) peroxins including Pex3p, Pex16p, and Pex19p, are responsible for peroxisome membrane biogenesis via classes I and II pathways; (2) peroxins that function in matrix protein import; (3) those such as three forms of Pex11p, Pex11pα, Pex11pß, and Pex11pγ, are involved in peroxisome proliferation where DLP1, Mff, and Fis1 coordinately function. In membrane assembly, Pex19p forms complexes in the cytosol with newly synthesized PMPs including Pex16p and transports them to the receptor Pex3p, whereby peroxisomal membrane is formed (Class I pathway). Pex19p likewise forms a complex with newly made Pex3p and translocates it to the Pex3p receptor, Pex16p (Class II pathway). In matrix protein import, newly synthesized proteins harboring peroxisome targeting signal type 1 or 2 are recognized by Pex5p or Pex7p in the cytoplasm and are imported to peroxisomes via translocation machinery. In regard to peroxisome-cytoplasmic shuttling of Pex5p, Pex5p initially targets to an 800-kDa docking complex consisting of Pex14p and Pex13p and then translocates to a 500-kDa RING translocation complex. At the terminal step, Pex1p and Pex6p of the AAA family mediate the export of Pex5p, where Cys-ubiquitination of Pex5p is essential for the Pex5p exit.