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Catalytic hydrothermal processing is a promising technology for the production of biofuels used in transportation to alleviate the energy crisis. An important challenge for these processes is the need for an external supply of hydrogen gas to accelerate the deoxygenation of fatty acids or lipids. It follows that in situ-produced hydrogen can improve process economics. This study reports on the use of various alcohol and carboxylic acid amendments as sources for in situ hydrogen production to accelerate Ru/C-catalyzed hydrothermal deoxygenation of stearic acid. Addition of these amendments significantly increases yields of liquid hydrocarbon products, including the major product heptadecane, from stearic acid conversion at subcritical conditions (330 °C, 14-16 MPa during the reaction). This research provided guidance for simplifying the catalytic hydrothermal process of biofuel production, making the production of the desired biofuel in one pot possible without the need for an external H2 supply.
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BACKGROUND: Osteoporosis (OP) is a systemic disease with bone loss and microstructural deterioration. Numerous noncoding RNAs (ncRNAs) have been proved to participate in various diseases, especially circular RNAs (circRNAs). However, the expression profile and mechanisms underlying circRNAs in male osteoporosis have not yet been explored. METHODS: The whole transcriptome expression profile and differences in mRNAs, circRNAs, and microRNAs (miRNAs) were investigated in peripheral blood samples of patients with osteoporosis and healthy controls consisting of males ≥ 60-years-old. RESULTS: A total of 398 circRNAs, 51 miRNAs, and 642 mRNAs were significantly and differentially expressed in osteoporosis compared to healthy controls. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the host genes of significantly differentially expressed circRNAs were mainly enriched in the regulation of cell cycle process: biological process (BP), organelle part cellular components (CC), protein binding molecular function (MF), Toll-like receptor signaling pathway, tumor necrosis factor (TNF) signaling pathway, and thyroid hormone signaling pathway. circRNA-miRNA-mRNA regulatory network was constructed using the differentially expressed RNAs. Moreover, key circRNAs (hsa_circ_0042409) in osteoporosis were discovered and validated by qPCR. CONCLUSIONS: The key cicrRNAs plays a major role in the pathogenesis of osteoporosis and could be used as potential biomarkers or targets in the diagnosis and treatment of osteoporosis.
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Private sector participation in the healthcare market via public-private partnership (PPP) could be considered an available approach to narrow down the medical resource gap and improve the operational efficiency of healthcare facilities. Accordingly, this study aims to examine the influence and relative importance among critical factors for the intention and behaviour of the private sector towards participation in Chinese healthcare market (CHM) via PPP. We defined five hypotheses from previous literature and built a theoretical model based on modified theory of planned behaviour. Then, covariance-based structural equation modelling was applied to analyse the questionnaires provided by 248 respondents from construction companies, real estate developers, pharmaceutical companies, private hospitals, asset management companies, and medical industry property investment companies in China. Results indicated that attitude towards behaviour (ß = 0.466, P < 0.001), subjective norm (ß = 0.167, P < 0.05), perceived behavioural control (ß = 0.231, P < 0.01), and facilitating conditions (ß = 0.305, P < 0.001) are positively significant to behavioural intention; behavioural intention also shows a strong linkage with behaviour (ß = 0.931, P < 0.001). Findings provide reference for governments and public authorities to exert additional efforts in implementing appropriate measures that will stimulate the private sector's motivation to participate in CHM via PPP.
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Atención a la Salud/organización & administración , Intención , Sector Privado , Asociación entre el Sector Público-Privado , China , Femenino , Humanos , Masculino , Evaluación de Programas y Proyectos de Salud , Encuestas y CuestionariosRESUMEN
Osteoporosis (OP) is a disease characterized by bone loss, imbalance of bone metabolism and destruction of trabecular microstructure, and associated with menopause. Studies have shown that immune related lymphocytes are involved in bone metabolism. However, the molecular mechanisms hidden in the interaction of lymphocytes with OP need to be further studied. In the present study, we investigated the expression profiles and differences of lncRNAs, mRNAs, circRNAs and miRNAs in peripheral blood lymphocytes of patients with postmenopausal OP using Illumina-based complementary DNA (cDNA) deep sequencing (RNA-seq). 70 lncRNAs, 475 mRNAs, 260 circRNAs and 13 miRNAs were differentially expressed in patients with postmenopausal osteoporosis (OP group) compared with healthy controls (NC group). The functions of differentially expressed lncRNAs, circRNA, miRNA and potential targeting genes were predicted by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Complex lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA regulatory networks were constructed based on differentially expressed RNAs. Taken together, our study indicated that lncRNAs, mRNA, circRNAs and miRNA could associate with the occurrence of postmenopausal OP and may be as possible biomarkers and target genes in lymphocytes for OP.
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We investigated the effects of tumor suppressor candidate 3 (TUSC3) on autophagy in human non-small cell lung cancer (NSCLC) cells. A total of 118 NSCLC patients (88 males and 30 females) who underwent surgery at our institute were enrolled in the study. Immunohistochemical analysis revealed that TUSC3 protein expression was lower in NSCLC specimens than adjacent normal tissue. Correspondingly, there was greater methylation of TUSC3 in NSCLC than adjacent normal tissue. After transient transfection of A549 NSCLC cells with constructs designed to up-regulate or down-regulate TUSC3 expression, we analyzed the effects of inhibiting the Wnt pathway (XAV939) and autophagy (chloroquine, CQ) on the behavior of NSCLC cells. We also performed TOP/FOP-Flash reporter assays, MTT assays, Annexin V-FITC/propidium iodide staining, and acridine orange staining to evaluate Wnt/ß-catenin signaling, cell proliferation, apoptosis, and autophagy, respectively. Expression of Wnt/ß-catenin pathway components and autophagy-related proteins was analyzed using qRT-PCR and Western blotting. We found that TUSC3 inhibited cell proliferation and promoted both apoptosis and autophagy in A549 cells. In addition, TUSC3 increased expression of autophagy-related proteins. It also increased expression of Wnt/ß-catenin signaling pathway components and promoted nuclear transfer of ß-catenin, resulting in activation of Wnt/ß-catenin signaling. TUSC3 thus induces autophagy in human NSCLC cells through activation of the Wnt/ß-catenin signaling pathway.
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OBJECTIVE: Infrarenal aortic cross-clamping (IAC) is a common procedure during infrarenal vascular operations. It often causes ischemia-reperfusion injury to lower limbs, resulting in systemic inflammation response and damage to remote organs (particularly lungs). Hydrogen sulfide (H2S) is a gaseous mediator that has been shown to have a protective effect against lung injury. METHODS: Wistar rats underwent IAC for 2 hours, followed by 4 hours of reperfusion. GYY4137 (a slow-releasing H2S donor) and dl-propargylglycine (PAG, an inhibitor of cystathionine γ-lyase) were preadministered to rats 1 hour before IAC, and their effects on severity of lung injury and related mechanisms were investigated. RESULTS: IAC induced a significant increase in plasma levels of H2S, H2S-synthesizing activity, and cystathionine γ-lyase expression in lung tissues compared with sham operation. Administration of GYY4137 significantly increased the levels of H2S but had little effect on H2S-synthesizing activity, whereas PAG reduced H2S levels and H2S-synthesizing activity. Preadministration of GYY4137 significantly attenuated acute lung injury induced by IAC, evidenced by reduced histologic scores and wet lung contents; improved blood gas parameters; reduced cell counts and protein amounts in bronchoalveolar lavage fluids; and reduced myeloperoxidase activity in lung tissues and plasma levels of tumor necrosis factor α, interleukin 6, and interleukin 1ß. However, PAG further aggravated the severity of lung injury and displayed opposite effects to GYY4137. In exploration of the mechanisms, we found that IAC increased the release of angiopoietin 2 (Ang2) and its expression in lung tissues. GYY4137 attenuated the increase of Ang2 release and expression and increased the phosphorylation of Akt and the activation of its downstream factors, glycogen synthase kinase 3ß and ribosomal protein S6 kinase; PAG showed opposite effects. CONCLUSIONS: The study indicates that H2S may play a protective role in IAC-induced acute lung injury in rats by inhibiting inflammation and Ang2 release.
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Lesión Pulmonar Aguda/prevención & control , Angiopoyetina 2/metabolismo , Antiinflamatorios/farmacología , Aorta Abdominal/cirugía , Sulfuro de Hidrógeno/farmacología , Pulmón/efectos de los fármacos , Morfolinas/farmacología , Compuestos Organotiofosforados/farmacología , Neumonía/prevención & control , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Alquinos/farmacología , Animales , Antiinflamatorios/metabolismo , Constricción , Cistationina gamma-Liasa/antagonistas & inhibidores , Cistationina gamma-Liasa/metabolismo , Citoprotección , Modelos Animales de Enfermedad , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Sulfuro de Hidrógeno/metabolismo , Pulmón/metabolismo , Pulmón/patología , Liasas/metabolismo , Masculino , Morfolinas/metabolismo , Compuestos Organotiofosforados/metabolismo , Neumonía/etiología , Neumonía/metabolismo , Neumonía/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas WistarRESUMEN
Breast cancer is one of the most common malignant tumors of women. Modern combinatorial therapeutic regimens can reduce patient tumor burdens to undetectable levels, yet in many cases these tumors will relapse. Understanding of breast cancer biology, developing more potent therapeutic approaches, and overcoming resistance are of great importance. WNT5A is a non-canonical signaling member of the WNT family. Its role in breast cancer still remains unclear. Most of the evidence shows that WNT5A is a suppressor in breast cancer and loss of its expression is associated with poor prognosis, while some evidence suggests the tumorigenicity of WNT5A. WNT signaling molecules are potent targets for treatment of cancer. Therefore, understanding the role of WNT5A in breast cancer may provide new ideas and methods for breast cancer treatment. We review the evidence concerning WNT5A and breast cancer involving the signaling pathways and the molecular-targeted therapy of WNT5A. Our results show that the role WNT5A plays depends on the availability of key receptors and intercellular interactions among different cell types.
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Neoplasias de la Mama/metabolismo , Proteínas Wnt/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Transducción de Señal , Proteínas Wnt/genéticaRESUMEN
We aimed to explore the possible mechanism of microRNA-196a (miR-196a) inhibition and reversion of drug resistance to cisplatin (DDP) of the A549/DDP non-small-cell lung cancer (NSCLC) cell line. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expression differences of miR-196a in the drug-resistant A549/DDP NLCLC cell line and the parental A549 cell line, and expressions of miR-196a in the A549/DDP NLCLC cell line transfected with miR-196a inhibitor (anti-miR-196a group) and the miR-196a negative control (miR-NC) group and blank group (without transfection). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was applied in examining the cell viability of A549/DDP cell line before and after transfection. Clonogenic assay was used to detect cell proliferation ability. Flow cytometry was applied in detecting apoptosis rate of assayed tumor cell and rhodamine-123 changes in cells. Western blot was applied in detecting proteins of drug-resistant related gene in A549/DDP cell line. Significantly higher expression of miR-196a was detected in the drug-resistant A549/DDP cell line than that in the parental A549 cell line (P < 0.05). However, miR-196a expression in the anti-miR-196a group decreased obviously compared to that in the blank group and the miR-NC group (both P < 0.05); The value of IC50 in the anti-miR-196a group showed remarkably lower than that in the blank group and the miR-NC group (both P < 0.05); Rh-123 absorbing ability in the anti-miR-196a group increased 2.51 times and 2.49 times respectively compared to that in the blank group and the miR-NC group (both P < 0.05). No statistical differences in the apoptosis rate of A549/DDP cell line in the early stage were found among the three groups (all P > 0.05), but the late-stage apoptosis rate in the anti-miR-196a group was significantly higher than that in the blank group and the miR-NC group (both P < 0.05); The expressions of human multidrug resistance 1 (MDR1), multidrug resistance protein 1 (MRP1), excision repair cross-complementation 1 (ERCC1), survivin, and B cell lymphoma 2 (Bcl-2) decreased significantly while RhoE increased significantly in the anti-miR-196a group than the blank group and the miR-NC group (all P < 0.05). Inhibition of miR-196a could reverse cisplatin resistance of A549/DDP cell lines, which might relate with inhibition of drug efflux, down-regulation of drug-resistant protein expression, cell apoptosis, and cell proliferation suppression.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Cisplatino/farmacocinética , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , MicroARNs/genética , Células A549 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Transfección/métodosRESUMEN
Hydrogen sulfide (H(2)S) displays an anti-apoptotic activity against myocardial ischemia reperfusion (MIR). Apoptosis repressor with caspase recruitment domain (ARC) is constitutively expressed in the heart and inhibits cell apoptosis when it is phosphorylated. Here, we investigated whether H(2)S could inhibit apoptosis by affecting ARC phosphorylation using cultured rat cardiomyocytes and a rat model of MIR. Primary cardiomyocytes were prepared from hearts of newborn rats and were pre-incubated with NaHS, a donor of H(2)S, for 60 min. Cardiomyocytes were subjected to hypoxia for 4 h, followed by reoxygenation for 2 h. The hypoxia and subsequent reoxygenation (H/R) significantly induced cell apoptosis, increased expression levels of Fas and FasL proteins, enhanced release of cytochrome c from mitochondria, and elevated caspase-3 activity, while H/R reduced ARC phosphorylation and increased the activity of calcineurin that dephosphorylates ARC. Pre-incubation with NaHS significantly attenuated the above effects through promoting ARC phosphorylation by reducing calcineurin activity and by increasing the activity of protein kinase casein kinase II (CK2) that phosphorylates ARC. In fact, TBB, a specific inhibitor of CK2, abolished the effects of NaHS. In rats undergoing MIR, NaHS significantly reduced the myocardial infarct size, cell apoptosis, calcineurin activity, and the expression levels of Fas, FasL and cleaved caspase-3 proteins, while NaHS increased ARC phosphorylation. In contrast, DL-propargylglycine, an inhibitor of cystathionine γ-lyase, the main enzyme for H(2)S production in hearts, showed opposite effects to NaHS. The results indicate that H(2)S inhibits apoptosis of cardiomyocytes induced by MIR through enhancing ARC phosphorylation.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Cardiotónicos/farmacología , Sulfuro de Hidrógeno/farmacología , Proteínas Musculares/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Quinasa de la Caseína II/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/citología , Miocitos Cardíacos/enzimología , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Ratas , Ratas WistarRESUMEN
BACKGROUND: Hydrogen sulfide (H(2)S) displays anti-inflammatory and cytoprotective activities to attenuate myocardial ischemia-reperfusion (MIR)-induced injury, but its role in MIR in diabetics is not known. This study was undertaken to investigate whether H(2)S plays a protective role in MIR in diabetic rats. METHODS: Diabetes was induced by streptozocin in Wistar rats, which were subjected to myocardial ischemia by blocking the left circumflex artery for 30 min, followed by 2h reperfusion. dl-propargylglycine (PAG) and sodium hydrosulfide (NaHS) were administered to the rats to investigate their effects on severity of MIR-induced injury. RESULTS: Diabetic rats had smaller myocardial infarct sizes and higher serum levels of H(2)S (both P < 0.05) than non-diabetics when they underwent MIR. MIR significantly increased the serum level of H(2)S (49.5 ± 7.1 µM), H(2)S-synthesizing activity (7.4 ± 1.6 nmol/mg) and the myocardial infarct size (44.0 ± 7.2%), compared with sham-operated diabetic rats (21.7 ± 2.1 µM, 0.15 ± 0.4 nmol/mg and 1.2 ± 0.4%, respectively). Administration of NaHS increased the H(2)S level (65.8 ± 6.9 µM) and had little effect on H(2)S production activity (6.5 ± 2.2 nmol/mg), while PAG reduced both the H(2)S level (29.2 ± 5.0 µM) and H(2)S-synthesizing activity (2.2 ± 1.8 nmol/mg). NaHS significantly reduced the myocardial infarct size (31.2 ± 4.7%), inhibited the production of lipid peroxidation, MPO activity, and cell apoptosis, and downregulated expression of caspase-3, Fas, FasL, and TNF-α, which had been elevated by MIR, while PAG further increased the myocardial infarct size (58.3 ± 5.9%), and displayed opposite effects. CONCLUSIONS: The study indicates that H(2)S may play a protective role in MIR-induced myocardial injury in diabetics by its anti-apoptotic, anti-oxidative and anti-inflammatory activities.
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Diabetes Mellitus Experimental , Sulfuro de Hidrógeno/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Alquinos/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Sulfuro de Hidrógeno/sangre , Peroxidación de Lípido/efectos de los fármacos , Masculino , Infarto del Miocardio/patología , Peroxidasa/metabolismo , Ratas , Ratas Wistar , Índice de Severidad de la Enfermedad , Sulfuros/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/metabolismoRESUMEN
Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like beta-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF beta-chain (HGF beta) and competitively inhibited binding to Met with an IC(50) of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC(50) of approximately 20 microM. The 2D (1)H-NMR structure of HB10 revealed a beta-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF beta mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF beta domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.
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Factor de Crecimiento de Hepatocito/metabolismo , Biblioteca de Péptidos , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/metabolismo , Soluciones , Relación Estructura-ActividadRESUMEN
OBJECTIVE: To compare the expression of human leukocyte antigen-G (HLA-G) in endometrial samples from patients with and without adenomyosis. DESIGN: Laboratory study using human tissue. SETTING: University hospital. PATIENT(S): Thirty-four patients with adenomyosis and 11 with myoma of the uterus. INTERVENTION(S): Human leukocyte antigen-G protein expression and localization with immunohistochemistry. MAIN OUTCOME MEASURE(S): Quantitative analysis of HLA-G protein expression, according to H-score. RESULT(S): A statistically significant difference was noted in HLA-G expression between the glandular cells of eutopic and ectopic endometrium (Mann-Whitney test, 412). The H-score of the negative control was 0.05 +/- 0.03 (mean +/- SD). In stromal cells, we observed a statistically significantly higher expression of HLA-G in eutopic endometrium than in ectopic endometrium (Mann-Whitney test, 169), whereas the H-score of the negative control was 0.05 +/- 0.02. A statistically significant correlation was found between HLA-G expression in ectopic and eutopic endometrium in patients with adenomyosis, both in glands (r = 0.89) and in stroma (r = 0.78). CONCLUSION(S): Patients with adenomyosis had HLA-G expression in eutopic and ectopic endometrial cells. This could be an explanation for the cells' ability to escape from the hosts' immunosurveillance and to survive without being eliminated by the immune system.
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Endometriosis/inmunología , Endometrio/inmunología , Antígenos HLA/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Adulto , Estudios de Casos y Controles , Femenino , Antígenos HLA-G , Humanos , Inmunohistoquímica , Leiomioma/inmunología , Persona de Mediana Edad , Células del Estroma/inmunología , Neoplasias Uterinas/inmunologíaRESUMEN
Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its alpha/beta heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the alpha-chain binds Met with high affinity. Recently, we reported that the protease-like HGF beta-chain binds to Met with low affinity (Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. (2004) EMBO J. 23, 2325-2335). Here we demonstrate that the zymogen-like form of HGF beta also binds Met, albeit with 14-fold lower affinity than the protease-like form, suggesting optimal interactions result from conformational changes upon cleavage of the single-chain form. Extensive mutagenesis of the HGF beta region corresponding to the active site and activation domain of serine proteases showed that 17 of the 38 purified two-chain HGF mutants resulted in impaired cell migration or Met phosphorylation but no loss in Met binding. However, reduced biological activities were well correlated with reduced Met binding of corresponding mutants of HGF beta itself in assays eliminating dominant alpha-chain binding contributions. Moreover, the crystal structure of HGF beta determined at 2.53 A resolution provides a structural context for the mutagenesis data. The functional Met binding site is centered on the "active site region" including "triad" residues Gln(534) [c57], Asp(578) [c102], and Tyr(673) [c195] and neighboring "activation domain" residues Val(692), Pro(693), Gly(694), Arg(695), and Gly(696) [c214-c219]. Together they define a region that bears remarkable resemblance to substrate processing regions of serine proteases. Models of HGF-dependent Met receptor activation are discussed.
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Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Neoplasias de la Mama , Células CHO , Línea Celular Tumoral/citología , Movimiento Celular/fisiología , Cricetinae , Cristalografía , Dimerización , Factor de Crecimiento de Hepatocito/genética , Humanos , Insectos , Datos de Secuencia Molecular , Mutagénesis , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Serina Endopeptidasas/metabolismoRESUMEN
The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF-dependent signaling requires proteolysis from an inactive single-chain precursor into an active alpha/beta-heterodimer. We show that the serine protease-like HGF beta-chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven-bladed beta-propeller, where the bottom face of blades 2 and 3 binds to the HGF beta-chain 'active site region'. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF beta binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF beta-chains brings two HGF beta:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.
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Factor de Crecimiento de Hepatocito/química , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , Cartilla de ADN , Factor de Crecimiento de Hepatocito/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
Because of the apparently greater conformational flexibility of yeast versus muscle actin and the ability of other members in the actin protein superfamily to efficiently use both ATP and GTP, we assessed the ability of yeast actin to function with GTP. Etheno-ATP exchange studies showed that the binding of GTP to yeast actin is about 1/9 as tight as that of ATP in contrast to the 1/1,240 ratio for muscle actin. Proteolysis of GTP-bound G-yeast actin suggests that the conformation of subdomain 2 is very much like that of ATP-bound actin, but CD studies show that GTP-bound actin is less thermostable than ATP-bound actin. GTP-actin polymerizes with an apparent critical concentration of 1.5 microm, higher than that of ATP-actin (0.3 microm) although filament structures observed by electron microscopy were similar. Yeast actin hydrolyzes GTP in a polymerization-dependent manner, and GTP-bound F-actin decorates with the myosin S1. Conversion of Phe(306) in the nucleotide binding site to the Tyr found in muscle actin raised the nucleotide discrimination ratio from the 1/9 of wild-type actin to 1/125. This result agrees with modeling that predicts that removal of the Tyr hydroxyl will create a space for the C2 amino group of the GTP guanine.
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Actinas/metabolismo , Guanosina Trifosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Actinas/química , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Cationes Bivalentes/metabolismo , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Modelos Moleculares , Unión ProteicaRESUMEN
His(73) participates in the regulation of the nucleotide binding cleft conformation in yeast actin. Earlier molecular dynamics studies suggested that Asp(184) interacts with His(73) thereby stabilizing a "closed-cleft" G-actin. However, beta-actin in the open-cleft state shows a closer interaction of His(73) with Asp(179) than with Asp(184). We have thus assessed the relative importance of Asp(184) and Asp(179) on yeast actin stability and function. Neutral substitutions at 184 or 179 alone had little adverse effect on the monomer and polymerization behavior of actin. Arg or His at 184 in H73E actin partially rescued the monomeric properties of H73E actin, as demonstrated by near-normal thermostability and wild-type (WT)-like protease digestion patterns. ATP exchange was still considerably faster than with WT-actin although slower than that of H73E alone. However, polymerization of H73E/D184R and H73E/D184H is worse than with H73E alone. Conversely, D179R rescued all monomeric properties of H73E to near WT values and largely restored polymerization rate and filament thermostability. These results and new simulations of G-actin in the "open" state underscore the importance of the His(73)-Asp(179) interaction and suggest that the open and not the closed state of yeast actin may be favored in the absence of the methyl group of His(73).