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1.
Mol Cell ; 80(4): 621-632.e6, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33152269

RESUMO

Mitochondria are highly dynamic organelles that continuously grow, divide, and fuse. The division of mitochondria is crucial for human health. During mitochondrial division, the mechano-guanosine triphosphatase (GTPase) dynamin-related protein (Drp1) severs mitochondria at endoplasmic reticulum (ER)-mitochondria contact sites, where peripheral ER tubules interact with mitochondria. Here, we report that Drp1 directly shapes peripheral ER tubules in human and mouse cells. This ER-shaping activity is independent of GTP hydrolysis and located in a highly conserved peptide of 18 amino acids (termed D-octadecapeptide), which is predicted to form an amphipathic α helix. Synthetic D-octadecapeptide tubulates liposomes in vitro and the ER in cells. ER tubules formed by Drp1 promote mitochondrial division by facilitating ER-mitochondria interactions. Thus, Drp1 functions as a two-in-one protein during mitochondrial division, with ER tubulation and mechano-GTPase activities.


Assuntos
Dinaminas/metabolismo , Dinaminas/fisiologia , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mitocôndrias/metabolismo , Animais , Dinaminas/genética , Retículo Endoplasmático/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial , Oligopeptídeos/farmacologia
2.
J Biol Chem ; 299(2): 102822, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36563856

RESUMO

RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.


Assuntos
Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/isolamento & purificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ligação Proteica , Mitofagia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Quinase I-kappa B/metabolismo
3.
EMBO J ; 39(24): e105074, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33200421

RESUMO

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin-related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin-related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1-knockout cells. Re-activation of Opa1 and Mitofusin 1 in Drp1-knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.


Assuntos
Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Estresse Fisiológico/fisiologia , Animais , Dinaminas/genética , Dinaminas/metabolismo , Metabolismo Energético , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Metaloproteases/metabolismo , Camundongos , Mitocôndrias/genética , Dinâmica Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico/genética , Transcriptoma
4.
Mol Cell ; 63(6): 1034-43, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27635761

RESUMO

Mitochondria divide to control their size, distribution, turnover, and function. Dynamin-related protein 1 (Drp1) is a critical mechanochemical GTPase that drives constriction during mitochondrial division. It is generally believed that mitochondrial division is regulated during recruitment of Drp1 to mitochondria and its oligomerization into a division apparatus. Here, we report an unforeseen mechanism that regulates mitochondrial division by coincident interactions of Drp1 with the head group and acyl chains of phospholipids. Drp1 recognizes the head group of phosphatidic acid (PA) and two saturated acyl chains of another phospholipid by penetrating into the hydrophobic core of the membrane. The dual phospholipid interactions restrain Drp1 via inhibition of oligomerization-stimulated GTP hydrolysis that promotes membrane constriction. Moreover, a PA-producing phospholipase, MitoPLD, binds Drp1, creating a PA-rich microenvironment in the vicinity of a division apparatus. Thus, PA controls the activation of Drp1 after the formation of the division apparatus.


Assuntos
Dinaminas/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/genética , Testículo/metabolismo , Animais , Sítios de Ligação , Dinaminas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Masculino , Camundongos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/metabolismo , Fosfolipase D/metabolismo , Ligação Proteica , Transdução de Sinais , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Testículo/ultraestrutura
5.
Gastric Cancer ; 26(2): 307-316, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36695982

RESUMO

BACKGROUND: Surgical resection of oligo-metastasis in gastric cancer (GC) is weakly recommended for patients without other incurable factors in the Japanese GC Treatment Guidelines. While post-operative chemotherapy is the standard treatment in patients with stage II or III GC, its efficacy for resected stage IV GC is unclear. This study aimed to evaluate the efficacy of post-operative chemotherapy after curative resection of GC with oligo-metastasis. METHODS: We retrospectively reviewed the medical records of patients with GC who were diagnosed with synchronous oligo-metastasis at 20 institutions in Japan between 2007 and 2012. The selection criteria were: adenocarcinoma, stage IV with oligo-metastasis at liver or lymph node without other distant metastasis, curative resection including synchronous oligo-metastasis, and no prior treatment of GC before surgery. RESULTS: A total of 110 patients were collected. Of the 94 eligible patients, 84 underwent gastrectomy with surgical resection of oligo-metastasis (39 [41%] liver metastasis and 55, [59%] distant lymph node metastasis), followed by post-operative chemotherapy with S-1 (S1: n = 55), S1 plus cisplatin (CS: n = 22), or Others (n = 7). Moreover, 10 patients did not receive post-operative chemotherapy (Non-Cx). The median overall survival (OS) was 35.2 and 11.1 months in the post-operative chemotherapy and Non-Cx groups (hazard ratio, 3.56; 95% confidence interval, 1.74-7.30; p < 0.001), respectively. In multivariable analysis, Non-Cx and age over 70 years were identified as poor prognostic factors for OS (p < 0.05). CONCLUSIONS: Curative resection followed by post-operative chemotherapy in patients with GC with synchronous oligo-metastasis showed favorable survival.


Assuntos
Neoplasias Gástricas , Humanos , Idoso , Neoplasias Gástricas/patologia , Estudos Retrospectivos , Cisplatino , Linfonodos/patologia , Excisão de Linfonodo , Gastrectomia , Prognóstico , Estadiamento de Neoplasias
6.
Br J Nutr ; 126(11): 1642-1650, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33551001

RESUMO

BACKGROUND: Previous research has suggested that curcumin potentially induces mitochondrial biogenesis in skeletal muscle via increasing cyclic AMP (cAMP) levels. However, the regulatory mechanisms for this phenomenon remain unknown. The purpose of the present study was to clarify the mechanism by which curcumin activates cAMP-related signalling pathways that upregulate mitochondrial biogenesis and respiration in skeletal muscle. METHODS: The effect of curcumin treatment (i.p., 100 mg/kg-BW/d for 28 d) on mitochondrial biogenesis was determined in rats. The effects of curcumin and exercise (swimming for 2 h/d for 3 d) on the cAMP signalling pathway were determined in the absence and presence of phosphodiesterase (PDE) or protein kinase A (PKA) inhibitors. Mitochondrial respiration, citrate synthase (CS) activity, cAMP content and protein expression of cAMP/PKA signalling molecules were analysed. RESULTS: Curcumin administration increased cytochrome c oxidase subunit (COX-IV) protein expression, and CS and complex I activity, consistent with the induction of mitochondrial biogenesis by curcumin. Mitochondrial respiration was not altered by curcumin treatment. Curcumin and PDE inhibition tended to increase cAMP levels with or without exercise. In addition, exercise increased the phosphorylation of phosphodiesterase 4A (PDE4A), whereas curcumin treatment strongly inhibited PDE4A phosphorylation regardless of exercise. Furthermore, curcumin promoted AMP-activated protein kinase (AMPK) phosphorylation and PPAR gamma coactivator (PGC-1α) deacetylation. Inhibition of PKA abolished the phosphorylation of AMPK. CONCLUSION: The present results suggest that curcumin increases cAMP levels via inhibition of PDE4A phosphorylation, which induces mitochondrial biogenesis through a cAMP/PKA/AMPK signalling pathway. Our data also suggest the possibility that curcumin utilises a regulatory mechanism for mitochondrial biogenesis that is distinct from the exercise-induced mechanism in skeletal muscle.


Assuntos
Curcumina , Biogênese de Organelas , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Curcumina/farmacologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos , Fatores de Transcrição/metabolismo
7.
Gastric Cancer ; 24(3): 701-709, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33179192

RESUMO

BACKGROUND: Gastric cancer (GC) patients with peritoneal metastasis are defined as stage IV in the Japanese classification of GC. For patients with peritoneal metastasis limited to positive peritoneal lavage cytology (CY1) and/or localized peritoneal metastasis (P1a), gastrectomy followed by S1 monotherapy is one of the most widely accepted therapeutic strategy in Japan. This study investigated the efficacy of preoperative chemotherapy as initial treatment in GC patients with CY1 and/or P1a. METHODS: We retrospectively reviewed GC patients diagnosed with CY1 and/or P1a at 34 institutions in Japan between 2008 and 2012. Selection criteria were: adenocarcinoma, no distant metastasis except CY1 or P1a, and no prior treatment. The subjects were divided into an Initial-Chemotherapy group and an Initial-Surgery group, according to the initial treatment. RESULTS: A total of 824 patients were collected and 713 eligible patients were identified for this study. As the initial treatment, 150 patients received chemotherapy (Initial-Cx), and 563 patients underwent surgery (Initial-Sx). Initial-Cx regimens were cisplatin plus S1/docetaxel plus cisplatin plus S1/others (n = 90/37/23). Both overall survival (OS) and progression-free survival (PFS) were similar between the Initial-Cx and Initial-Sx groups (median OS 24.8 and 24.0 months, HR 1.07, 95% CI 0.87-1.3; median PFS 14.9 and 13.9 months, HR 1.04, 95% CI 0.85-1.27). The 5-year OS rates were 22.3% in the Initial-Cx group and 21.5% in the Initial-Sx group. CONCLUSIONS: Although, the preoperative chemotherapy did not show a survival benefit for GC patients with CY1 and/or P1a, initial-Cx showed favorable survival in patients who converted to P0 and CY0.


Assuntos
Recidiva Local de Neoplasia/terapia , Neoplasias Peritoneais/terapia , Neoplasias Gástricas/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Gastrectomia , Humanos , Japão , Masculino , Prontuários Médicos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Metástase Neoplásica , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Lavagem Peritoneal , Neoplasias Peritoneais/mortalidade , Neoplasias Peritoneais/secundário , Estudos Retrospectivos , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise de Sobrevida , Resultado do Tratamento , Adulto Jovem
8.
Gan To Kagaku Ryoho ; 48(13): 1969-1971, 2021 Dec.
Artigo em Japonês | MEDLINE | ID: mdl-35045463

RESUMO

We have experienced 3 cases of solitary brain metastasis after radical surgery in advanced gastric cancer. All of them have similar characteristics such as, upper third location, solitary metastasis to the cerebellum, and no other organ metastasis. As there is a risk of brain hernia, resection have been underwent first, and radiotherapy administered after surgery. One case has been provided over 2-year survival.


Assuntos
Neoplasias Encefálicas , Neoplasias Gástricas , Neoplasias Encefálicas/cirurgia , Gastrectomia , Humanos , Neoplasias Gástricas/cirurgia
9.
Trends Biochem Sci ; 41(3): 209-210, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26879678

RESUMO

Mitochondrial division apparatuses are generally thought to form by oligomerization of Drp1 at pre-determined sites on mitochondria. A recent study by Ji et al. now shows that the Drp1 oligomers on mitochondria move, merge, and mature into a functional division apparatus.


Assuntos
Mitocôndrias , Proteínas Mitocondriais , GTP Fosfo-Hidrolases , Dinâmica Mitocondrial
10.
Lab Invest ; 100(12): 1575-1588, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32801334

RESUMO

SOX2 is recognized as an oncogene in human small cell lung cancer (SCLC), which is an aggressive neuroendocrine (NE) tumor. However, the role of SOX2 in SCLC is not completely understood, and strategies to selectively target SOX2 in SCLC cells remain elusive. Here, we show, using next-generation sequencing, that SOX2 expressed in the ASCL1-high SCLC (SCLC-A) subtype cell line is dependent on ASCL1, which is a lineage-specific transcriptional factor, and is involved in NE differentiation and tumorigenesis. ASCL1 recruits SOX2, which promotes INSM1 and WNT11 expression. Immunohistochemical studies revealed that SCLC tissue samples expressed SOX2, ASCL1, and INSM1 in 18 out of the 30 cases (60%). Contrary to the ASCL1-SOX2 signaling axis controlling SCLC biology in the SCLC-A subtype, SOX2 targets distinct genes such as those related to the Hippo pathway in the ASCL1-negative, YAP1-high SCLC (SCLC-Y) subtype. Although SOX2 knockdown experiments suppressed NE differentiation and cell proliferation in the SCLC-A subtype, they did not sufficiently impair the growth of the SCLC-Y subtype cell lines in vitro and ex vivo. The present results support the importance of the ASCL1-SOX2 axis as a main subtype of SCLC, and suggest the therapeutic potential of targeting the ASCL1-SOX2 axis.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Humanos , Pulmão/química , Neoplasias Pulmonares/química , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/genética , Masculino , Camundongos , Fenótipo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/genética , Carcinoma de Pequenas Células do Pulmão/química , Carcinoma de Pequenas Células do Pulmão/classificação , Carcinoma de Pequenas Células do Pulmão/genética
11.
Histochem Cell Biol ; 153(6): 443-456, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32170367

RESUMO

ASCL1 is one of the master transcription factors of small cell lung carcinoma (SCLC). To investigate the significance of ASCL1 in pulmonary neuroendocrine carcinoma, we performed 2 comparative RNA-seq studies between H69 (ASCL1-positive, classical type SCLC) and H69AR (ASCL1-negative, variant type SCLC) and between ASCL1-transfected A549 adenocarcinoma cell lines (A549(ASCL1+) cell lines) and A549(control) cell lines. RNA-seq analyses revealed that 940 genes were significantly different between the H69 and H69AR cell lines, and 728 between the A549(ASCL1+) and A549(control) cell lines. In total, 120 common genes between these analyses were selected as candidate ASCL1-related genes, and included genes with various cellular functions, such as neural development, secretion, growth, and morphology. Their expression degrees in three classical and two variant SCLC cell lines, two A549(ASCL1+) and two A549(control) cell lines were subjected to quantitative PCR analyses. Since the candidate ASCL1-related genes were strongly expressed in the classical SCLC and A549(ASCL1+) cell lines and more weakly expressed in the variant SCLC and A549(control) cell lines, the ASCL1-related 7 molecules INSM1, ISL1, SYT4, KCTD16, SEZ6, MS4A8, and COBL were further selected. These molecules suggested diverse functions for A549(ASCL1+): INSM1 and ISL1 are transcription factors associated with neuroendocrine differentiation, while SYT4, KTCD16, and SEZ6 may be related to neurosecretory functions and MS4A8 and COBL to cell growth and morphology. An immunohistochemistry of these seven molecules was performed on lung carcinoma tissues and the xenotransplanted tumors of A549(ASCL1+), and they were preferentially and positively stained in ASCL1-postive tumor tissues.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Neuroendócrino/genética , Neoplasias Pulmonares/genética , Análise de Sequência de RNA , Carcinoma de Pequenas Células do Pulmão/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia
12.
Ann Surg Oncol ; 27(1): 284-292, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31535301

RESUMO

BACKGROUND: Gastric cancer (GC) patients with positive peritoneal lavage cytology (CY1) and/or localized peritoneum metastasis (P1a) are defined as stage IV in the 15th edition of the Japanese Classification of Gastric Cancer. In Japan, the most common treatment for patients with CY1 and/or P1a is gastrectomy followed by postoperative chemotherapy. PATIENTS AND METHODS: Subjects in this multi-institutional retrospective study were GC patients with CY1 and/or P1a who received surgical resection that leaves no macroscopically visible disease. Patients were selected from 34 institutions in Japan between 2007 and 2012. Selection criteria included adenocarcinoma, no distant metastasis except CY1 and P1a, and no prior treatment for GC before surgery. RESULTS: Among 824 patients registered, 506 were identified as eligible, with a background of P0CY1, P1aCY0, or P1aCY1 (72.5%, 16.0%, and 11.5% of subjects, respectively). Sixty-two patients had not received postoperative chemotherapy (no-Cx), whereas 444 patients had received postoperative chemotherapy: S-1 monotherapy (S-1; n = 267, 52.7%), cisplatin plus S-1 (CS; n = 114, 22.5%), and others (n = 63, 12.6%). Overall survival (OS) was 29.5, 24.7, 25.4 and 9.9 months in the S-1, CS, 'others', and no-Cx groups, respectively [CS vs. S-1: hazard ratio (HR) 1.15, 95% confidence interval (CI) 0.89-1.50; p = 0.275]. In multivariate analysis, OS was similar between the S-1 and CS groups (CS vs. S-1: HR 1.19, 95% CI 0.92-1.55; p = 0.18). CONCLUSIONS: Postoperative chemotherapy after gastrectomy that leaves no macroscopically visible disease may have some survival benefits for GC patients with CY1 and/or P1a. In contrast, S-1 plus cisplatin seems to have no additional benefit over S-1 treatment alone.


Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cisplatino/administração & dosagem , Ácido Oxônico/administração & dosagem , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/tratamento farmacológico , Tegafur/administração & dosagem , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Intervalo Livre de Doença , Combinação de Medicamentos , Feminino , Gastrectomia , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Cavidade Peritoneal/citologia , Lavagem Peritoneal , Período Pós-Operatório , Estudos Retrospectivos , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Taxa de Sobrevida , Adulto Jovem
13.
J Biol Chem ; 293(24): 9292-9300, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29735527

RESUMO

Defects in phosphatase and tensin homolog (PTEN) are associated with neurological disorders and tumors. PTEN functions at two primary intracellular locations: the plasma membrane and the nucleus. At the membrane, PTEN functions as a phosphatidylinositol (3,4,5)-trisphosphate phosphatase and suppresses PI 3-kinase signaling that drives cell growth and tumorigenesis. However, the in vivo function of nuclear PTEN is unclear. Here, using CRISPR/Cas9, we generated a mouse model in which PTEN levels in the nucleus are decreased. Nuclear PTEN-deficient mice were born with microcephaly and maintained a small brain during adulthood. The size of neuronal soma was significantly smaller in the cerebellum, cerebral cortex, and hippocampus. Also, these mice were prone to seizure. No changes in PI 3-kinase signaling were observed. By contrast, the size of other organs was unaffected. Therefore, nuclear PTEN is essential for the health of the brain by promoting the growth of neuronal soma size during development.


Assuntos
Sistemas CRISPR-Cas , Núcleo Celular/genética , Microcefalia/genética , Neurônios/patologia , PTEN Fosfo-Hidrolase/genética , Convulsões/genética , Substituição de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Tamanho Celular , Feminino , Edição de Genes , Masculino , Camundongos , Microcefalia/complicações , Microcefalia/patologia , Mutação , Neurônios/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Convulsões/complicações , Convulsões/patologia , Transdução de Sinais
14.
J Biol Chem ; 293(30): 11809-11822, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29853636

RESUMO

Dynamin-related protein 1 (Drp1) constricts mitochondria as a mechanochemical GTPase during mitochondrial division. The Drp1 gene contains several alternative exons and produces multiple isoforms through RNA splicing. Here we performed a systematic analysis of Drp1 transcripts in different mouse tissues and identified a previously uncharacterized isoform that is highly enriched in the brain. This Drp1 isoform is termed Drp1ABCD because it contains four alterative exons: A, B, C, and D. Remarkably, Drp1ABCD is located at lysosomes, late endosomes, and the plasma membrane in addition to mitochondria. Furthermore, Drp1ABCD is concentrated at the interorganelle interface between mitochondria and lysosomes/late endosomes. The localizations of Drp1ABCD at lysosomes, late endosomes, and the plasma membrane require two exons, A and B, that are present in the GTPase domain. Drp1ABCD assembles onto these membranes in a manner that is regulated by its oligomerization and GTP hydrolysis. Experiments using lysosomal inhibitors show that the association of Drp1ABCD with lysosomes/late endosomes depends on lysosomal pH but not their protease activities. Thus, Drp1 may connect mitochondria to endosomal-lysosomal pathways in addition to mitochondrial division.


Assuntos
Encéfalo/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Dinaminas/análise , Camundongos , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo
15.
Lab Invest ; 99(11): 1622-1635, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31231131

RESUMO

The involvement of Wnt signaling in human lung cancer remains unclear. This study investigated the role of Wnt11 in neuroendocrine (NE) differentiation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) in human small-cell lung cancer (SCLC). Immunohistochemical staining of resected specimens showed that Wnt11 was expressed at higher levels in SCLCs than in non-SCLCs; 58.8% of SCLC, 5.2% of adenocarcinoma (ADC), and 23.5% of squamous cell carcinoma tissues stained positive for Wnt11. A positive relationship was observed between Achaete-scute complex homolog 1 (Ascl1) and Wnt11 expression in SCLC cell lines, and this was supported by transcriptome data from SCLC tissue. The expression of Wnt11 and some NE markers increased after the transfection of ASCL1 into the A549 ADC cell line. Knockdown of Ascl1 downregulated Wnt11 expression in SCLC cell lines. Ascl1 regulated Wnt11 expression via lysine H3K27 acetylation at the enhancer region of the WNT11 gene. Wnt11 controlled NE differentiation, cell proliferation, and E-cadherin expression under the regulation of Ascl1 in SCLC cell lines. The phosphorylation of AKT and p38 mitogen-activated protein kinase markedly increased after transfection of WNT11 into the SBC3 SCLC cell line, which suggests that Wnt11 promotes cell proliferation in SCLC cell lines. Ascl1 plays an important role in regulating the Wnt signaling pathway and is one of the driver molecules of Wnt11 in human SCLC. Ascl1 and Wnt11 may employ a cooperative mechanism to control the biology of SCLC. The present results indicate the therapeutic potential of targeting the Ascl1-Wnt11 signaling axis and support the clinical utility of Wnt11 as a biological marker in SCLC.


Assuntos
Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caderinas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Proteínas Wnt/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Antígenos CD/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Elementos Facilitadores Genéticos , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Histonas/metabolismo , Humanos , Imidas/farmacologia , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Knockout , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/patologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , Carcinoma de Pequenas Células do Pulmão/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genética , Via de Sinalização Wnt/efeitos dos fármacos
16.
J Chem Inf Model ; 59(8): 3533-3544, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31282659

RESUMO

An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by approximately -4 kcal/mol. Here, we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.


Assuntos
Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-mdm2/química , Especificidade por Substrato , Termodinâmica , Proteína Supressora de Tumor p53/química
17.
Surg Today ; 48(9): 841-847, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29858668

RESUMO

PURPOSE: Splenic infarction may occur if the splenic branches are injured or ligated accidentally during gastrectomy. We used three-dimensional computed tomography (3D-CT) imaging to distinguish the vascular anatomy of the splenic hilum in individual patients, focusing on the splenic polar branches and the gastric branches. METHODS: The subjects of this study were 104 patients who underwent computed tomography (CT) with intravenous contrast before gastrectomy. SYNAPSE 3D® (Fujifilm Medical, Tokyo, Japan) was used to generate the 3D-CT images. The total spleen volume and the area supplied by the superior polar artery (SPA) in each patient were estimated using the "liver analysis" function. RESULTS: The SPA without the gastric branch (supplying only the spleen), the SPA with the gastric branch (supplying both the stomach and the spleen), and the posterior gastric artery (supplying only the stomach) were present in 14, 45, and 18% of the patients, respectively. The SPA supplied 12% of the total spleen volume on average; however, it supplied over 30% in two patients. CONCLUSION: We identified the vascular anatomy around the splenic hilum in over 100 patients. Based on our findings, we recommend preservation of the SPA when it is supplying a large area of the spleen. Preoperative 3D-CT analysis provides useful information to optimize safe gastrectomy.


Assuntos
Gastrectomia , Imageamento Tridimensional , Baço/irrigação sanguínea , Artéria Esplênica/anatomia & histologia , Artéria Esplênica/diagnóstico por imagem , Veia Esplênica/anatomia & histologia , Veia Esplênica/diagnóstico por imagem , Neoplasias Gástricas/cirurgia , Tomografia Computadorizada por Raios X , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Complicações Intraoperatórias/prevenção & controle , Masculino , Pessoa de Meia-Idade , Tratamentos com Preservação do Órgão , Infarto do Baço/prevenção & controle
18.
Am J Pathol ; 186(11): 2798-2802, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27640145

RESUMO

Mitochondria are dynamic organelles that divide and fuse to modulate their number and shape. We have previously reported that the loss of dynamin-related protein 1 (Drp1), which mediates mitochondrial division, leads to the degeneration of cerebellar Purkinje cells in mice. Because Drp1 has been shown to be important for apoptosis and necroptosis, it is puzzling how Purkinje neurons die in the absence of Drp1. In this study, we tested whether neurodegeneration involves necrotic cell death by generating Purkinje cell-specific Drp1-knockout (KO) mice that lack the receptor-interacting protein kinase 3 (Rip3), which regulates necroptosis. We found that the loss of Rip3 significantly delays the degeneration of Drp1-KO Purkinje neurons. In addition, before neurodegeneration, mitochondrial tubules elongate because of unopposed fusion and subsequently become large spheres as a result of oxidative damage. Surprisingly, Rip3 loss also helps Drp1-KO Purkinje cells maintain the elongated morphology of the mitochondrial tubules. These data suggest that Rip3 plays a role in neurodegeneration and mitochondrial morphology in the absence of mitochondrial division.


Assuntos
Dinaminas/genética , Doenças Neurodegenerativas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Apoptose , Modelos Animais de Doenças , Dinaminas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Necrose/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/patologia , Oxirredução , Células de Purkinje/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
19.
Gan To Kagaku Ryoho ; 44(12): 1114-1116, 2017 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-29394551

RESUMO

We herein report 2 cases of laparoscopic total gastrectomy(LTG)in patient with multiple gastric neuroendocrine tumor (NET)related to multiple endocrine neoplasia type 1(MEN1). Case 1: A 66-year-old female was diagnosed with multiple gastric NET. There was no finding of any other tumor, and parathyroid function was normal. She underwent LTG. Case 2: A 58-year-old female was diagnosed with multiple gastric NET. The patient had a previous history of surgery for pituitary gland tumor. There was no finding of any other tumor, and parathyroid function was normal. She underwent LTG. In our cases, we could perform complete resection of gastric NET by laparoscopic surgery. Multiple gastric NET is a good indication of laparoscopic gastrectomy.


Assuntos
Neoplasia Endócrina Múltipla Tipo 1/cirurgia , Tumores Neuroendócrinos/cirurgia , Neoplasias Gástricas/cirurgia , Idoso , Feminino , Gastrectomia , Humanos , Laparoscopia , Pessoa de Meia-Idade , Resultado do Tratamento
20.
Biophys J ; 111(10): 2086-2098, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27851934

RESUMO

Developments in atomic force microscopy have opened up a new path toward single-molecular phenomena; in particular, during the process of pulling a membrane protein out of a lipid bilayer. However, the characteristic features of the force-distance (F-D) curve of a bacteriorhodopsin in purple membrane, for instance, have not yet been fully elucidated in terms of physicochemical principles. To address the issue, we performed a computer simulation of bacteriorhodopsin with, to our knowledge, a novel coarse-grained (C-G) model. Peptide planes are represented as rigid spheres, while the surrounding environment consisting of water solvents and lipid bilayers is represented as an implicit continuum. Force-field parameters were determined on the basis of auxiliary simulations and experimental values of transfer free energy of each amino acid from water to membrane. According to Popot's two-stage model, we separated molecular interactions involving membrane proteins into two parts: I) affinity of each amino acid to the membrane and intrahelical hydrogen bonding between main chain peptide bonds; and II) interhelix interactions. Then, only part I was incorporated into the C-G model because we assumed that the part plays a dominant role in the forced unfolding process. As a result, the C-G simulation has successfully reproduced the key features, including peak positions, of the experimental F-D curves in the literature, indicating that the peak positions are essentially determined by the residue-lipid and intrahelix interactions. Furthermore, we investigated the relationships between the energy barrier formation on the forced unfolding pathways and the force peaks of the F-D curves.


Assuntos
Bacteriorodopsinas/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Bacteriorodopsinas/metabolismo , Cinética , Conformação Proteica , Termodinâmica , Viscosidade
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