RESUMO
BACKGROUND: The proliferation ability and autophagy level of pulmonary artery endothelial cells (PAECs) play an important role in promoting the development of pulmonary artery hypertension (PAH), and there is still no effective treatment for PAH. Farnesyl diphosphate synthase (FDPS) is a key enzyme in the mevalonate pathway. The intermediate metabolites of this pathway are closely related to the activity of autophagy-associated small G proteins, including Ras-related C3 botulinum toxin substrate 1 (Rac1). Studies have shown that the mevalonate pathway affects the activation levels of different small G proteins, autophagy signaling pathways, vascular endothelial function, and so on. However, the exact relationship between them is still unclear in PAH. METHOD: In vitro, western blotting and mRFP-GFP-LC3 puncta formation assays were used to observe the expression of FDPS and the level of autophagy in PAECs treated with monocrotaline pyrrole (MCTP). In addition, cell proliferation and migration assays were used to assess the effect of FDPS on endothelial function, and Rac1 activity assays were used to evaluate the effect of Rac1 activation on PAEC autophagy via the PI3K/AKT/mTOR signaling pathway. In vivo, the right heart catheterization method, hematoxylin and eosin (H&E) staining and western blotting were used to determine the effect of FDPS on PAEC autophagy and monocrotaline (MCT)-induced PAH. RESULTS: We show that the expression of FDPS is increased in the PAH module in vitro and in vivo, concomitant with the induction of autophagy and the activation of Rac1. Our data demonstrate that inhibition of FDPS ameliorates endothelial function and decreases MCT-induced autophagy levels. Mechanistically, we found that FDPS promotes autophagy, Rac1 activity and endothelial disfunction through the PI3K/AKT/mTOR signaling pathway. CONCLUSION: Our study suggests that FDPS contributes to active small G protein-induced autophagy during MCT-induced PAH, which may serve as a potential therapeutic target against PAH.
Assuntos
Hipertensão Pulmonar , Proteínas Monoméricas de Ligação ao GTP , Hipertensão Arterial Pulmonar , Animais , Autofagia , Proliferação de Células , Células Endoteliais/metabolismo , Geraniltranstransferase/metabolismo , Geraniltranstransferase/farmacologia , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Ácido Mevalônico/farmacologia , Ácido Mevalônico/uso terapêutico , Monocrotalina/efeitos adversos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Proteínas Monoméricas de Ligação ao GTP/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/metabolismoRESUMO
Extra-proliferation and increased migration of vascular smooth cells con-tribute to the formation of atherosclerosis. Ras small G proteins play a critical role in the prolif-eration and migration of a wide range of cells. Mulberry, an economic fruit in Asia, exhibits anti-inflammation, anti-migration, and anti-oxidant properties. The mechanisms of action of mulberry extracts on K-Ras small G protein-induced proliferation and migration of vascular smooth muscle cell have not been extensively investigated. In this study, we explored the effects of mulberry polyphenol extracts (MPE) on the proliferation and migration of K-Ras-overexpressing A7r5 smooth muscle cells. The overexpression of K-Ras enhanced the ex-pression and activity of matrix metalloproteinase (MMP)-2, promoted vascular endothelial growth factor (VEGF) production, and eventually triggered the migration of A7r5 cells. Treatment with MPE attenuated K-Ras-induced phenomenon. In addition, MPE blocked K-Ras-induced actin fibril stress. MPE dose-dependently diminished K-Ras-induced Rho A, Rac1, CDC42, and phosphorylated focal adhesion kinase (FAK) expression. MPE elevated Rho B ex-pression. Phosphorylated AKT and glycogen synthase kinase (GSK) induced by K-Ras were also repressed by MPE treatment. MPE enhanced the interaction of IκB with NFκB. MPE restored the G0/G1 population and p21 and p27 expressions, which were repressed by K-Ras. Finally, MPE triggered the degradation of K-Ras by ubiquitination. MPE inhibited the migration and proliferation of vascular smooth cell through K-Ras-induced pathways and eventually pre-vented atherosclerosis.
Assuntos
Aterosclerose , Proteínas Monoméricas de Ligação ao GTP , Morus , Actinas/metabolismo , Antioxidantes/farmacologia , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Regulação para Baixo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Frutas/metabolismo , Quinases da Glicogênio Sintase/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Músculo Liso Vascular , Miócitos de Músculo Liso , Polifenóis/metabolismo , Polifenóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The experimental protocol of the perfused rat pancreas is commonly used to evaluate ß-cell function. In this context, mathematical models become useful tools through the determination of indexes that allow the assessment of ß-cell function in different experimental groups and the quantification of the effects of antidiabetic drugs, secretagogues, or treatments. However, a minimal model applicable to the isolated perfused rat pancreas has so far been unavailable. In this work, we adapt the C-peptide minimal model applied previously to the intravenous glucose tolerance test to obtain a specific model for the experimental settings of the perfused pancreas. Using the model, it is possible to estimate indexes describing ß-cell responsivity for first (ΦD) and second phase (ΦS, T) of insulin secretion. The model was initially applied to untreated pancreata and afterward used for the assessment of pharmacologically relevant agents (the gut hormone GLP-1, the potent GLP-1 receptor agonist lixisenatide, and a GPR40/FFAR1 agonist, SAR1) to quantify and differentiate their effect on insulin secretion. Model fit was satisfactory, and parameters were estimated with good precision for both untreated and treated pancreata. Model application showed that lixisenatide reaches improvement of ß-cell function similarly to GLP-1 (11.7- vs. 13.1-fold increase in ΦD and 2.3- vs. 2.8-fold increase in ΦS) and demonstrated that SAR1 leads to an additional improvement of ß-cell function in the presence of postprandial GLP-1 levels.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Modelos Biológicos , Receptores de Glucagon/metabolismo , Transdução de Sinais , Algoritmos , Animais , Peptídeo 1 Semelhante ao Glucagon/agonistas , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hipoglicemiantes/agonistas , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Cinética , Masculino , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Peptídeos/farmacologia , Perfusão , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glucagon/agonistas , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: SAMHD1 is a triphosphohydrolase that restricts the replication of HIV-1 and SIV in myeloid cells. In macrophages and dendritic cells, SAMHD1 restricts virus replication by diminishing the deoxynucleotide triphosphate pool to a level below that which supports lentiviral reverse transcription. HIV-2 and related SIVs encode the accessory protein Vpx to induce the proteasomal degradation of SAMHD1 following virus entry. While SAMHD1 has been shown to restrict HIV-1 and SIV, the breadth of its restriction is not known and whether other viruses have a means to counteract the restriction has not been determined. RESULTS: We show that SAMHD1 restricts a wide array of divergent retroviruses, including the alpha, beta and gamma classes. Murine leukemia virus was restricted by SAMHD1 in macrophages yet removal of SAMHD1 did not alleviate the block to infection because of an additional block to viral nuclear import. Prototype foamy virus (PFV) and Human T cell leukemia virus type I (HTLV-1) were the only retroviruses tested that were not restricted by SAMHD1. PFV reverse transcribes predominantly prior to entry and thus is unaffected by the dNTP level in the target cell. It is possible that HTLV-1 has a mechanism to render the virus resistant to SAMHD1-mediated restriction. CONCLUSION: The results suggest that SAMHD1 has broad anti-retroviral activity against which most viruses have not found an escape.
Assuntos
Macrófagos/virologia , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Células Mieloides/virologia , Retroviridae/efeitos dos fármacos , Retroviridae/patogenicidade , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Células Dendríticas/metabolismo , Células Dendríticas/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Células Jurkat , Macrófagos/imunologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células Mieloides/metabolismo , Retroviridae/classificação , Retroviridae/fisiologia , Proteína 1 com Domínio SAM e Domínio HDRESUMO
The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca(2+) channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca(2+) channel beta subunits (Ca(v)beta), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca(2+) channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca(v)beta but not the Gem/Ca(v)beta interaction, and that Gem coimmunoprecipitates with the P/Q channel alpha(1) subunit (Ca(v)alpha(1)) in a Ca(v)beta-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca(v)alpha(1) critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca(v)alpha(1) abolishes Gem inhibition of P/Q channels and confers Ca(v)beta-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca(v)alpha(1) that binds Ca(v)beta). Our results challenge the prevailing view regarding the role of Ca(v)beta in RGK inhibition of high voltage-activated Ca(2+) channels and prompt a paradigm in which Gem directly binds and inhibits Ca(v)beta-primed Ca(v)alpha(1) on the plasma membrane.
Assuntos
Canais de Cálcio Tipo P/fisiologia , Canais de Cálcio Tipo Q/fisiologia , Membrana Celular/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Sequência de Aminoácidos , Animais , Western Blotting , Canais de Cálcio Tipo P/genética , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/genética , Canais de Cálcio Tipo Q/metabolismo , Linhagem Celular , Membrana Celular/fisiologia , Feminino , Humanos , Imunoprecipitação , Cinética , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are agents that mimic the essential posttranslational modifications of G-proteins. It is hypothesized that PCAIs work as anticancer agents by disrupting polyisoprenylation-dependent functional interactions of the G-Proteins. This study tested this hypothesis by determining the effect of the PCAIs on the levels of RAS and related monomeric G-proteins. Following 48 h exposure, we found significant decreases in the levels of KRAS, RHOA, RAC1, and CDC42 ranging within 20-66% after NSL-YHJ-2-27 (5 µM) treatment in all four cell lines tested, A549, NCI-H1299, MDA-MB-231, and MDA-MB-468. However, no significant difference was observed on the G-protein, RAB5A. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, after PCAIs treatment, migration, and invasion of A549 cells were inhibited by 72 and 70%, respectively while the levels of vinculin and fascin dropped by 33 and 43%, respectively. These findings implicate the potential role of PCAIs as anticancer agents through their direct interaction with monomeric G-proteins.
Assuntos
Antineoplásicos , Neoplasias da Mama , Proteínas Monoméricas de Ligação ao GTP , Humanos , Feminino , Movimento Celular , Linhagem Celular Tumoral , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Amidas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Antineoplásicos/farmacologia , Pulmão , Proliferação de CélulasRESUMO
PURPOSE: The main acute and late effects of ionizing radiation on living organisms are the formation of reactive oxygen species (ROS), apoptosis and DNA damage. Since the Rac1 molecule is a subunit of the NADPH oxidase enzyme, it is known to participate in the generation of ROS. The aim of this study was to investigate the role of Rac1 molecule in testicular damage induced by low (0.02 Gy), medium (0.1 Gy) and high (5 Gy) dose irradiation. MATERIAL AND METHOD: In this study, Wistar rats (except the control group) were received whole body X-ray irradiation. Testicular tissues were removed 2 hours, 24 hours and 7 days after radiation exposure. Testicular damage was examined by hematoxylin-eosin staining and Johnsen's score. Immunohistochemical staining and G-LISA method were used to determine Rac1 expression and activation. To evaluate the generation of ROS in the testicular tissues, intracellular ROS, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured. RESULTS: Increases in testicular damage were detected in all radiation exposed groups in a dose- and time-dependent manner. Compared to the control group, Rac1 expression decreased in all irradiated groups, while Rac1 activation increased. In addition, intracellular ROS and MDA levels were increased and SOD activity levels decreased in the irradiated groups compared to the control group. CONCLUSION: Our findings suggest that Rac1 has a role in the increase of intracellular ROS and lipid peroxidation which led to an increase in radiation- induced testicular damage.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Animais , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Estresse Oxidativo/efeitos da radiação , Radiação Ionizante , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/farmacologiaRESUMO
Euphorbia factor L3 (EFL3) is extracted from Euphorbia lathyris and is known for its anti-inflammatory properties. This study focused on the potential anti-inflammatory and therapeutic effects of EFL3 on rheumatoid arthritis (RA) using fibroblast-like synoviocytes (FLSs) and arthritis animal models. Functional analysis showed that EFL3 could ameliorate the inflammatory phenotype of FLSs derived from RA patients, as evidenced by the decreases in cell viability, migration, invasion and cytokine production. Luciferase activity, Western blotting and immunofluorescence assays demonstrated that EFL3 inhibited the nuclear translocation of the p65 subunit and the subsequent activation of the nuclear factor kappa-Β (NF-κB) pathway. Furthermore, the therapeutic effects of EFL3 against arthritic progression were evidenced by decreases in joint swelling, arthritis scores, inflammatory factor production, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) and tumor necrosis factor-α (TNF-α) transgenic (TNF-tg) mouse models. Molecular analysis identified Rac family small GTPase 1 (Rac1) as the potential target that was required for EFL3-mediated suppression of the inflammatory RA FLS phenotype. In summary, this study uncovered the therapeutic potential of EFL3 in RA, which suggests its future clinical use.
Assuntos
Artrite Reumatoide , Euphorbia , Proteínas Monoméricas de Ligação ao GTP , Sinoviócitos , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Euphorbia/metabolismo , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Proteínas Monoméricas de Ligação ao GTP/uso terapêutico , Sinoviócitos/metabolismo , Sinoviócitos/patologiaRESUMO
In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Delta cells also harboring end3Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Delta. Further comparison of ric1Delta and ypt6Delta cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Delta and ypt6Delta cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular , Rede trans-Golgi/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacologia , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/farmacologia , Fatores de Troca do Nucleotídeo Guanina , Membranas Intracelulares/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Fenótipo , Transporte Proteico/efeitos dos fármacos , Proteínas Qb-SNARE , Proteínas SNARE , Saccharomyces cerevisiae/química , Supressão Genética/efeitos dos fármacos , Fatores de Transcrição/genética , Transformação Genética , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismoRESUMO
This study is designed to characterize the signal cascades by which brain-derived neurotrophic factor (BDNF) modulates long-term memory of fear conditioning. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis of tissue homogenates taken from fear-conditioned rats showed an increase in the amygdala of BDNF protein levels and its receptor TrkB phosphorylation. Bilateral administration of a TrkB ligand scavenger TrkB IgG and a Trk-specific tyrosine kinase inhibitor K252a to the amygdala impaired fear memory, as measured with fear-potentiated startle. Fear conditioning resulted in the association of Shc and TrkB, Shc and Ras, the increase in active Ras and phosphorylation of mitogen-activated protein kinase (MAPK). Treatment of amygdala slices with BDNF for 15 min increased the levels of active Ras, and MAPK and Akt phosphorylation. BDNF-induced MAPK phosphorylation was completely abolished by MEK inhibitors, and was partially inhibited by farnesyltransferase or phosphatidylinositol-3 kinase (PI-3 kinase) inhibitors. On the other hand, BDNF-induced Akt phosphorylation was unaffected by farnesyltransferase or MEK inhibitors, but could be blocked by PI-3 kinase inhibitors. Together, these data suggest a requirement of BDNF for fear learning. The memory-enhancing effect of BDNF involves the activation of MAPK and PI-3 kinase. BDNF-induced MAPK phosphorylation in the amygdala is mediated via TrkB and the Shc-binding site. Shc binding to TrkB leads to activation of Ras, Raf, and MEK. In addition, BDNF could induce phosphorylation of MAPK via activation of PI-3 kinase.
Assuntos
Tonsila do Cerebelo/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Condicionamento Psicológico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Comportamento Animal , Western Blotting/métodos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Condicionamento Psicológico/efeitos dos fármacos , Eletrochoque/métodos , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Medo , Expressão Gênica/efeitos dos fármacos , Imunoprecipitação/métodos , Técnicas In Vitro , Masculino , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/fisiologia , Fatores de TempoRESUMO
Activating mutations of Ras have been implicated in approximately 30% of human cancers. In every case, the biochemical consequence of such mutations is to disrupt the GTPase activity of Ras and to render Ras resistant to the actions of GTPase activating proteins. Consequently, oncogenic Ras mutants are "locked" in a GTP-bound active state. We detected a potent activity in Escherichia coli extract that can efficiently convert mutationally activated GTP-bound Ras to the inactive GDP-bound form. Purification of the protein responsible for this activity led to the identification of the enzyme nucleoside diphosphate kinase (Ndk). The human orthologue of Ndk is the NM23 metastasis suppressor, which we found to exhibit a similar activity. Purified Ndk effectively inactivates several of the oncogenic forms of Ras that are seen frequently in human cancers, including RasD12, the most commonly detected Ras mutation. Significantly, Ndk does not detectably affect wild-type Ras or an activated form of the Ras-related Rho GTPase. These results demonstrate that it is possible, through biochemical means, to specifically inactivate oncogenic Ras as a potential therapeutic approach to tumors that harbor Ras mutations. Moreover, the results suggest that the loss of NM23 expression that is commonly observed during tumor progression could lead to increased potency of oncogenic Ras proteins.
Assuntos
Núcleosídeo-Difosfato Quinase/fisiologia , Proteínas ras/genética , Escherichia coli/química , Escherichia coli/enzimologia , Inativação Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Mutagênese Sítio-Dirigida , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/isolamento & purificação , Núcleosídeo-Difosfato Quinase/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Fatores de Transcrição/fisiologia , Proteínas ras/antagonistas & inibidores , Proteínas ras/metabolismoAssuntos
Canais de Cálcio/fisiologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/biossíntese , Expressão Gênica/genética , Expressão Gênica/fisiologia , Humanos , Proteínas Monoméricas de Ligação ao GTP/farmacologiaRESUMO
OBJECTIVE: We previously demonstrated the presence of nucleoside diphosphate kinase NDPK/NM23 in normal human plasma. It also was reported that extracellular NM23 could inhibit differentiation of certain hematopoietic cell lines. We further investigated the extracellular effect of NM23 on hematopoiesis by adding recombinant NM23-H1, NM23-H2, and NM23-H3 proteins to in vitro differentiation assays of normal human hematopoietic progenitors. MATERIALS AND METHODS: To study the effect on the earlier stages of hematopoietic maturation, NM23 was added to serum-free pre-colony-forming unit (pre-CFU) assays starting from immature CD34++CD38- bone marrow cells. Serum-free CFU assays starting from CD34+ CD38+ bone marrow cells were used as a model for terminal hematopoietic differentiation. RESULTS: In pre-CFU assays, none of the NM23 isoforms used significantly changed the expansion of CD34++CD38- cells, nor did NM23 alter the CD34++ CD38- cell lineage commitment. In contrast, terminal differentiation of CD34+CD38+ progenitor cells in CFU assays was significantly altered by addition of NM23 protein. More erythroid burst-forming units and fewer macrophage colonies were observed in cultures containing any of the NM23 isoforms examined. Similar effects were observed using the enzymatically inactive H118N mutant of NM23-H1, strongly suggesting that the observed effect is independent of the nucleoside diphosphate kinase activity of NM23. CONCLUSION: We demonstrated a modulating effect of extracellular NM23 proteins on the terminal stages of normal hematopoietic differentiation. Therefore, the fairly high concentrations of NM23 constitutively present in plasma could have a physiologic role in supporting erythropoiesis and inhibiting excessive macrophage formation.
Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Núcleosídeo-Difosfato Quinase , Fatores de Transcrição/fisiologia , Antígenos CD34/análise , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Eritropoese/fisiologia , Espaço Extracelular/enzimologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Humanos , Macrófagos/citologia , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Mutação , Nucleosídeo NM23 Difosfato Quinases , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologiaRESUMO
The transduction of dopaminergic (DA) neurons with human ras homolog enriched in brain, which has a S16H mutation [hRheb(S16H)] protects the nigrostriatal DA projection in the 6-hydroxydopamine (6-OHDA)-treated animal model of Parkinson's disease (PD). However, it is still unclear whether the expression of active hRheb induces the production of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), which are involved in neuroprotection, in mature neurons. Here, we show that transduction of nigral DA neurons with hRheb(S16H) significantly increases the levels of phospho-cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), GDNF, and BDNF in neurons, which are attenuated by rapamycin, a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1). Moreover, treatment with specific neutralizing antibodies for GDNF and BDNF reduced the protective effects of hRheb(S16H) against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity. These results show that activation of hRheb/mTORC1 signaling pathway could impart to DA neurons the important ability to continuously produce GDNF and BDNF as therapeutic agents against PD.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Neurônios/metabolismo , Neuropeptídeos/farmacologia , Doença de Parkinson/metabolismo , Animais , Humanos , Proteínas Monoméricas de Ligação ao GTP/uso terapêutico , Neurônios/efeitos dos fármacos , Neuropeptídeos/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/prevenção & controle , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismoRESUMO
OBJECTIVE: To investigate the gene expression of some lipopolysaccharide (LPS) receptors after LPS stimulation. METHODS: The total RNA from normal and LPS-challenged mice was extracted by Trizol reagent and the gene expression of Toll-like receptor 2 (TLR2), TLR4, CD(14), LPS-binding protein (LBP) and tumor necrosis factor-alpha(TNF-alpha) were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The gene of TLR2 was expressed in normal lungs and spleen tissues, and TLR2, CD(14), LBP in liver. After challenged by LPS, the expressions of TLR2, TLR4, and TNF-alpha in lungs, TLR2, CD(14), LBP and TNF-alpha in liver, TLR2, TLR4, CD(14), and TNF-alpha in spleen were increased at 1, 3, and 5 hours. CONCLUSION: LPS might alert the ability against pathogen-associated molecules by inducing or enhancing the expression of genes that involved in the LPS signal transduction.
Assuntos
Proteínas de Fase Aguda , Glicoproteínas de Membrana/genética , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Receptores de Superfície Celular/genética , Animais , Proteínas de Transporte/genética , Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/genética , Proteína ran de Ligação ao GTPRESUMO
Some intracellular/membranous factors exert intrinsic immunity against viral pathogens. Most recently, SAMHD1 has been shown to be one of these factors. SAMHD1 is a nucleus-localized protein, and mutations in the gene are associated with Aicardi-Goutières syndrome. As a triphosphohydrolase, it depletes the intracellular pool of dNTPs in myeloid cells, such as macrophages and dendritic cells, to a low level that establishes a precursor-deficient environment for the synthesis of lentiviral cDNA, thereby restricting viral replication in these host cells. However, some viruses evolve Vpx to recruit SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase in the cytoplasm for proteasome-dependent degradation, by which these viruses relieve SAMHD1-mediated restriction of primate lentivirus infection. In this review, we describe the latest knowledge of SAMHD1 biology.
Assuntos
Antivirais/farmacologia , Células Dendríticas/virologia , HIV-1/efeitos dos fármacos , Macrófagos/virologia , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , HIV-1/genética , HIV-1/fisiologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Proteína 1 com Domínio SAM e Domínio HD , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Virais Reguladoras e Acessórias/farmacologiaRESUMO
During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.
Assuntos
Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae , Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Retículo Endoplasmático/ultraestrutura , Guanosina Difosfato/fisiologia , Guanosina Trifosfato/fisiologia , Células HeLa , Humanos , Microinjeções , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas Monoméricas de Ligação ao GTP/administração & dosagem , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Mutação , Proteínas de Transporte VesicularRESUMO
Karyopherins (Kaps) transport cargo across the nuclear pore complex (NPC) by interacting with nucleoporins that contain phenylalanine-glycine (FG) peptide repeats (FG Nups). As a test of the "affinity gradient" model for Kap translocation, we measured the apparent affinity of Kap95p to FG Nups representing three distinct regions of the S. cerevisiae NPC. We find that the affinity of Kap95p-Kap60p-cargo complexes to Nup1p (a nuclear basket Nup) is 225-fold higher than to Nup100p (a central scaffold Nup) and 4000-fold higher than to Nup42p (a cytoplasmic filament Nup), revealing a steep gradient of affinity for Kap95p complexes along the yeast NPC. A high affinity binding site for a Kap95p import complex was mapped to the C terminus of Nup1p, and, surprisingly, deletion of all FG repeats in that region did not eliminate binding of the complex. Instead, a 36-amino acid truncation of the C terminus of Nup1p reduced its affinity for the Kap95p import complex by 450-fold. Mutant yeast that express Nup1pDelta36 instead of full-length Nup1p display specific defects in Kap95p localization and Kap95p-mediated nuclear import. We conclude that a high affinity binding site for Kap95p at the nuclear basket increases the translocation efficiency of Kap95p import complexes across the NPC.
Assuntos
Transporte Ativo do Núcleo Celular , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Sítios de Ligação , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Poro Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas Nucleares/farmacologia , Ligação ProteicaRESUMO
The Golgi apparatus is partitioned during mitosis in animal cells by a process of fragmentation, dispersal, and reassembly in each daughter cell. We fractionated the Golgi apparatus in vivo using the drug brefeldin A or a dominant-negative mutant of the Sar1p protein. After these treatments, Golgi enzymes moved back to the endoplasmic reticulum, leaving behind a matrix of Golgi structural proteins. Under these conditions, cells still entered and exited mitosis normally, and their Golgi matrix partitioned in a manner very similar to that of the complete organelle. Thus, the matrix may be the partitioning unit of the Golgi apparatus and may carry the Golgi enzyme-containing membranes into the daughter cells.