RESUMO
Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly.
RESUMO
Abscisic acid-, stress-, and ripening-induced (ASR) proteins in plants play a significant role in plant response to diverse abiotic stresses. However, the functions of ASR genes in maize remain unclear. In the present study, we identified a novel drought-induced ASR gene in maize (ZmASR1) and functionally characterized its role in mediating drought tolerance. The transcription of ZmASR1 was upregulated under drought stress and abscisic acid (ABA) treatment, and the ZmASR1 protein was observed to exhibit nuclear and cytoplasmic localization. Moreover, ZmASR1 knockout lines generated with the CRISPR-Cas9 system showed lower ROS accumulation, higher ABA content, and a higher degree of stomatal closure than wild-type plants, leading to higher drought tolerance. Transcriptome sequencing data indicated that the significantly differentially expressed genes in the drought treatment group were mainly enriched in ABA signal transduction, antioxidant defense, and photosynthetic pathway. Taken together, the findings suggest that ZmASR1 negatively regulates drought tolerance and represents a candidate gene for genetic manipulation of drought resistance in maize.
Assuntos
Ácido Abscísico , Secas , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Zea mays , Zea mays/genética , Zea mays/metabolismo , Zea mays/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Estresse Fisiológico/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Programed cell death (PCD) plays fundamental roles in plant development and responses to environmental stresses. Here, we report a protein, SICKLE (SIC), which represses PCD. In Arabidopsis (Arabidopsis thaliana), the loss-of-function mutant of SIC, sic-4, hyperaccumulated lariat intronic RNAs (lariRNAs) and exhibited PCD. The gene encoding an RNA debranching enzyme 1 (DBR1), a rate-limiting enzyme for lariRNAs decay, was overexpressed to reduce the level of lariRNAs in the sic-4 mutant, which led to suppression of PCD. Meanwhile, another lariRNAs hyper-accumulating mutant, dbr1-2, also exhibited PCD, further indicating that sic-4 PCD is caused by hyper-accumulation of lariRNAs. Transcriptional profiling analyses revealed that the sic-4 mutation disturbed alternative splicing and decay of mRNAs associated with salicylic acid (SA) homeostasis, a well-known molecule functioning in PCD regulation. Moreover, SA is dramatically increased in sic-4 and the disruption of SA biosynthesis and signaling suppressed PCD in the mutant, demonstrating that SA functions downstream of sic-4. Taken together, our results demonstrate that SIC is involved in regulating SA-triggered PCD.
Assuntos
Processamento Alternativo , Apoptose , Proteínas de Arabidopsis , Arabidopsis , Estabilidade de RNA , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , RNA Mensageiro/genética , Ácido Salicílico/metabolismoRESUMO
Singlet oxygen (1O2) has been recently identified as a key molecule against toxic Aß aggregation, which is associated with the currently incurable Alzheimer's disease (AD). However, limited research has studied its efficiency against tau protein aggregation, the other major hallmark of AD. Herein, we designed and synthesized boron-dipyrromethene (BODIPY)-ruthenium conjugates and isolated three isomers. Under visible-light irradiation, the ε isomer can be photoactivated and efficiently generate singlet oxygen. Particularly, the complex demonstrated successful results in attenuating tauopathyâan appreciable decrease to 43 ± 2% at 100 nM. The photosensitizer was further found to remarkably promote neurite outgrowth and significantly increased the length and number of neurites in nerve cells. As a result of effective photoinduced singlet oxygen generation and proactive neurite outgrowth, the hybrid design has great potential for therapeutics for Alzheimer's disease.
Assuntos
Doença de Alzheimer , Rutênio , Humanos , Doença de Alzheimer/tratamento farmacológico , Boro/farmacologia , Crescimento Neuronal , Fármacos Fotossensibilizantes/farmacologia , Agregados Proteicos , Rutênio/farmacologia , Oxigênio Singlete/metabolismo , Proteínas tau/metabolismoRESUMO
Ribosome biogenesis is a process of making ribosomes that is tightly linked with plant growth and development. Here, through a suppressor screen for the smo2 mutant, we found that lack of a ribosomal stress response mediator, ANAC082 partially restored growth defects of the smo2 mutant, indicating SMO2 is required for the repression of nucleolar stress. Consistently, the smo2 knock-out mutant exhibited typical phenotypes characteristic of ribosome biogenesis mutants, such as pointed leaves, aberrant leaf venation, disrupted nucleolar structure, abnormal distribution of rRNA precursors, and enhanced tolerance to aminoglycoside antibiotics that target ribosomes. SMO2 interacted with ROOT INITIATION DEFECTIVE 2 (RID2), a methyltransferase-like protein required for pre-rRNA processing. SMO2 enhanced RID2 solubility in Escherichia coli and the loss of function of SMO2 in plant cells reduced RID2 abundance, which may result in abnormal accumulation of FIBRILLARIN 1 (FIB1) and NOP56, two key nucleolar proteins, in high-molecular-weight protein complex. Taken together, our results characterized a novel plant ribosome biogenesis factor, SMO2 that maintains the abundance of RID2, thereby sustaining ribosome biogenesis during plant organ growth.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucléolo Celular/genética , Plantas/metabolismo , Ribossomos/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
We report herein a porous supramolecular framework formed by a linear mononuclear Au(I) complex (1) via the tongue-and-groove-like joinery between the pentiptycene U-cavities (grooves) and the rod-shaped π-conjugated backbone and alkyl chains (tongues) with the assistance of C-H···π and aurophilic interactions. The framework contains distorted tetrahedral Au4 units, which undergo stepwise and persistent photoinduced Au(I)-Au(I) bond shortening (excited-state aurophilicity), leading to multicolored luminescence photochromism. The one-dimensional pore channels could accommodate different solvates and guests, and the guest inclusion-induced luminescence enhancement (up to 300%) and/or vapochromism are characterized. A correlation between the aurophilic bonding and the luminescence activity is uncovered by TDDFT calculations. Isostructural derivatives 2 and 3 corroborate both the robustness of the porous supramolecular assembly and the mechanisms of the stimulation-induced luminescence properties of 1. This work demonstrates the cooperation of aurophilicity and structural porosity and adaptability in achieving novel supramolecular photochemical properties.
RESUMO
Plant lateral roots (LRs) play vital roles in anchorage and uptake of water and nutrients. Here, we reveal that degradation of lariat intronic RNAs (lariRNAs) modulated by SICKLE (SIC) is required for LR development in Arabidopsis (Arabidopsis thaliana). Loss of SIC results in hyper-accumulation of lariRNAs and restricts the outgrowth of LR primordia, thereby reducing the number of emerged LRs. Decreasing accumulation of lariRNAs by over-expressing RNA debranching enzyme 1 (DBR1), a rate-limiting enzyme of lariRNA decay, restored LR defects in SIC-deficient plants. Mechanistically, SIC interacts with DBR1 and facilitates its nuclear accumulation, which is achieved through two functionally redundant regions (SIC1-244 and SIC252-319) for nuclear localization. Of the remaining amino acids in this region, six (SIC245-251) comprise a DBR1-interacting region while two (SICM246 and SICW251) are essential for DBR1-SIC interaction. Reducing lariRNAs restored microRNA (miRNA) levels and LR development in lariRNA hyper-accumulating plants, suggesting that these well-known regulators of LR development mainly function downstream of lariRNAs. Taken together, we propose that SIC acts as an enhancer of DBR1 nuclear accumulation by driving nuclear localization through direct interaction, thereby promoting lariRNA decay to fine-tune miRNA biogenesis and modulating LR development.
Assuntos
Anemia Falciforme , Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Íntrons/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Raízes de Plantas/metabolismoRESUMO
Carbon monoxide (CO) plays an important role in signaling in cells, making its use as a therapeutic tool highly intriguing. Reduced burst emissions are important to avoid the cytotoxicity and tissue damage caused by CO. Here, we developed a stable diiron carbonyl [FeFe] hydrogenase agent that enables prolonged CO release activity (half-life of over 9 h) in cells. The integrated analysis allowed the identification of the key intermediate sites and CO accumulations with subcellular resolution. We observed that the [FeFe]A complex was enriched in neurons with S-methyl bond rupture. Furthermore, the [FeFe]A complex efficiently reduced the aggregation of tau proteins (49.3% reduction) and showed superior biocompatibility in nerve cells (â¼ 95% survival).
Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Monóxido de Carbono/química , Domínio Catalítico , Desmetilação , Hidrogenase/química , Proteínas Ferro-Enxofre/químicaRESUMO
Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Meristema/metabolismo , Peptídeos/metabolismo , Brotos de Planta/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/genéticaRESUMO
Pentatricopeptide repeat (PPR) proteins are a large family in land plants that play a role in organellular RNA processing, editing, and splicing. Here, we identify an Arabidopsis thaliana mutant, gend1-1, which exhibits a short root phenotype with reduced meristem size and cell numbers. Positional cloning of GEND1 revealed that it encodes a PPR protein, and functional analysis showed that GEND1 can bind and edit mitochondrial ccmFn-1 mRNA, causing gend1 mutants to have decreased levels of cytochrome C. GEND1 was up-regulated by high temperature conditions, to which gend1 mutants were hypersensitive. Analysis of a set of PPR mutants under high temperature showed that mutants with defects in cytochrome C had comparable temperature sensitivity to gend1. Collectively, these results suggest that cytochrome C plays an important role in root development and high temperature response in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Adaptação Fisiológica/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citocromos c/metabolismo , Temperatura Alta , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismoRESUMO
Much effort has been focused on novel nanomedicine for cancer therapy. However, tumor hypoxia limits the efficacy of various cancer therapeutics. Herein, we constructed a self-sufficient hybrid enzyme-based silk fibroin hydrogel system, consisting of Pt-decorated hollow Ag-Au trimetallic nanocages (HGN@Pt) and glucose oxidase (GOx), to supply O2 continuously and consume glucose concurrently and, thereby, synergistically enhance the anti-cancer efficacy of a combined starvation and photothermal therapy operating in a hypoxic tumor microenvironment. Thanks to the cooperative effects of the active surface atoms (resulting from the island-like features of the Pt coating), the intrinsically hollow structure, and the strain effect induced by the trimetallic composition, HGN@Pt displayed efficient catalase-like activity. The enhancement in the generation of O2 through the decomposition of H2O2 mediated by the as-designed nanozyme was greater than 400% when compared with that of hollow Ag-Pt bimetallic nanospheres or tiny Pt nanoparticles. Moreover, in the presence of HGN@Pt, significant amounts of O2 could be generated within a few minutes, even in an acidic buffer solution (pH 5.8-6.5) containing a low concentration of H2O2 (100-500 µM). Because HGN@Pt exhibited a strong surface plasmon resonance peak in the near-infrared wavelength range, it could be used as a photothermal agent for hyperthermia therapy. Furthermore, GOx was released gradually from the SF hydrogel into the tumor microenvironment to mediate the depletion of glucose, leading to glucose starvation-induced cancer cell death. Finally, the O2 supplied by HGN@Pt overcame the hypoxia of the microenvironment and, thereby, promoted the starvation therapeutic effect of the GOx-mediated glucose consumption. Meanwhile, the GOx-produced H2O2 from the oxidation of glucose could be used to regenerate O2 and, thereby, construct a complementary circulatory system. Accordingly, this study presents a self-sufficient hybrid enzyme-based system that synergistically alleviates tumor hypoxia and induces an anti-cancer effect when combined with irradiation of light from a near-infrared laser.
Assuntos
Nanopartículas/uso terapêutico , Neoplasias/terapia , Terapia Fototérmica/métodos , Hipóxia Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Antiangiogenic therapy is widely administered in many cancers, and the antiangiogenic drug sorafenib offers moderate benefits in advanced hepatocellular carcinoma (HCC). However, antiangiogenic therapy can also lead to hypoxia-driven angiogenesis and immunosuppression in the tumor microenvironment (TME) and metastasis. Here, we report the synthesis and evaluation of NanoMnSor, a tumor-targeted, nanoparticle drug carrier that efficiently codelivers oxygen-generating MnO2 and sorafenib into HCC. We found that MnO2 not only alleviates hypoxia by catalyzing the decomposition of H2O2 to oxygen but also enhances pH/redox-responsive T1-weighted magnetic resonance imaging and drug-release properties upon decomposition into Mn2+ ions in the TME. Moreover, macrophages exposed to MnO2 displayed increased mRNA associated with the immunostimulatory M1 phenotype. We further show that NanoMnSor treatment leads to sorafenib-induced decrease in tumor vascularization and significantly suppresses primary tumor growth and distal metastasis, resulting in improved overall survival in a mouse orthotopic HCC model. Furthermore, NanoMnSor reprograms the immunosuppressive TME by reducing the hypoxia-induced tumor infiltration of tumor-associated macrophages, promoting macrophage polarization toward the immunostimulatory M1 phenotype, and increasing the number of CD8+ cytotoxic T cells in tumors, thereby augmenting the efficacy of anti-PD-1 antibody and whole-cell cancer vaccine immunotherapies. Our study demonstrates the potential of oxygen-generating nanoparticles to deliver antiangiogenic agents, efficiently modulate the hypoxic TME, and overcome hypoxia-driven drug resistance, thereby providing therapeutic benefit in cancer.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Compostos de Manganês/farmacologia , Nanopartículas/química , Neovascularização Patológica/tratamento farmacológico , Óxidos/farmacologia , Inibidores da Angiogênese/química , Animais , Antineoplásicos/química , Carcinoma Hepatocelular/patologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Compostos de Manganês/química , Camundongos , Camundongos Endogâmicos C3H , Neovascularização Patológica/patologia , Óxidos/química , Tamanho da Partícula , Propriedades de Superfície , Células Tumorais Cultivadas , Evasão Tumoral/efeitos dos fármacos , Hipóxia Tumoral/efeitos dos fármacosRESUMO
A case of primary pulmonary arterial sarcoma (PPAS) treated with Endostar injection and radiotherapy and discuss the diagnosis, clinical characteristics, and pathology of PPAS. The patient complained of cough, sputum, fever, and chest pain with hemoptysis. Numerous nodules were seen in the computed tomography scan. The patient was diagnosed as pulmonary embolism (PE) by computed tomography pulmonary angiography. The pathology and immunohistochemistry results indicated soft tissue sarcomas, indicative of angiosarcoma. The nodules shrunk after 5 courses of endostatin and one course of radiotherapy, as seen by CT scan. Therefore, PPAS is clinically rare with nonspecific symptoms. Hence, it can be easily misdiagnosed as PE, biopsy for confirmation. Current treatment is limited and includes surgery. Hence, endostatin injection combined with other therapy may be an alternative treatment.
Assuntos
Endostatinas/administração & dosagem , Hemangiossarcoma/tratamento farmacológico , Hemangiossarcoma/radioterapia , Artéria Pulmonar , Proteínas Recombinantes/administração & dosagem , Neoplasias Vasculares/tratamento farmacológico , Neoplasias Vasculares/radioterapia , Adulto , Terapia Combinada , Angiografia por Tomografia Computadorizada , Diagnóstico Diferencial , Hemangiossarcoma/diagnóstico por imagem , Humanos , Injeções Intra-Arteriais , Masculino , Artéria Pulmonar/diagnóstico por imagem , Embolia Pulmonar , Resultado do Tratamento , Neoplasias Vasculares/diagnóstico por imagemRESUMO
Methoxychlor is primarily used as an insecticide and it is widely present in the environment. The objective of the present study was to investigate the direct effects of methoxychlor and its metabolite hydroxychlor (HPTE) on rat neurosteroidogenic 3α-hydroxysteroid dehydrogenase (AKR1C14) and retinol dehydrogenase 2 (RDH2) activities. Rat AKR1C14 and RDH2 were cloned and expressed in COS-1 cells, and the effects of methoxychlor and HPTE on these enzymes were measured. HPTE was more potent to inhibit AKR1C14 and RDH2 activities than methoxychlor, with IC50 values of 2.602 ± 0.057 µM and 20.473 ± 0.049 µM, respectively, while those of methoxychlor were over 100 µM. HPTE competitively inhibited AKR1C14 and RDH2 when steroid substrates were used, while it showed a mode of mixed inhibition on these enzymes when NADPH/NAD+ were used. We elucidated the binding mode of methoxychlor and HPTE to the crystal structure of AKR1C14 by molecular docking and found that HPTE had higher affinity with the enzyme than methoxychlor. In conclusion, HPTE is more potent than methoxychlor to inhibit both AKR1C14 and RDH2.
Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Inibidores de 5-alfa Redutase/farmacologia , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Metoxicloro/farmacologia , Fenóis/farmacologia , Animais , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inseticidas/farmacologia , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , RatosRESUMO
AIMS: Endotoxin induced acute lung injury (ALI) is a critical complication of some clinical illnesses. Endothelial cell dysfunction and excessive pro-inflammation cytokine release are pivotal to the injury of alveolar-capillary membrane which is the typical characteristic of endotoxic lung injury. As a potential marker of endothelial cells, endocan plays an important role in many endothelial-dependent pathophysiological diseases. We speculated that endocan have anti-inflammatory property in ALI. Here, we investigated the role of endocan in LPS-induced ALI. MATERIALS AND METHODS: Mice were randomly divided into 4 groups. LPS were used to construct ALI mice model by aerosolization for 20â¯min. Endocan was intraperitoneal injected at 30â¯min before LPS exposure. Levels of TNF-α, IFN-γ, IL-1ß, IL-6 and MPO activities were detected by indicated ELISA. Cell apoptotic rate was determined by Annexin V/PI kit, ROS level and MPTP were detected by DCFH-DA and JC-1 kit, respectively. Seahorse XF96 was applied to evaluate the alteration of OCR and ECAR. Western blot and qRT-PCR were used to detect indicated molecules. KEY FINDINGS: Endocan effectively decreased TNF-α, IFN-γ, IL-1ß, and IL-6 levels as well as relieved pulmonary epithelium cell apoptosis caused by LPS exposure. Endocan significantly reversed LPS induced UPRmt and promoted cell metabolism reprogramming which were crucial for the protective characteristic of endocan in ALI mice model. SIGNIFICANCE: The above findings suggested endocan could significantly suppress inflammatory response in ALI model through attenuating UPRmt associated apoptosis and switch cellular bioenergetics, indicating endocan could be considered as a promising compound against LPS induced ALI.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios não Esteroides/farmacologia , Lipopolissacarídeos , Proteoglicanas/farmacologia , Lesão Pulmonar Aguda/patologia , Administração por Inalação , Aerossóis , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular , Citocinas/biossíntese , Metabolismo Energético/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Pulmão/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteoglicanas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
The exocyst is a well-known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast-two-hybrid (Y2H) analysis, we found that EXO70A1 interacted with the GOLD domain of Patellin3 (PATL3). The direct EXO70A1-PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3-GFP colocalized with EXO70A1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo70a1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo70e2, PRsec6 or exo84b mutants. These data suggested that EXO70A1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicle-tethering/fusion. Both EXO70A1 and PATL3 were shown to bind PI4P and PI(4,5)P2 in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO70A1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunit-specific function, and provided data and tools for further characterization of PATL family proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a Ácido GraxoRESUMO
Taxifolin is a flavonoid in food plants. Kidney 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) is an NAD+-dependent oxidase that inactivates glucocorticoid cortisol (human) or corticosterone (rodents) into biologically inert 11 keto glucocorticoids. The present study investigated the effects of taxifolin on rat and human kidney microsomal 11ß-HSD2. Taxifolin noncompetitively inhibited rat and human 11ß-HSD2 against steroid substrates, with IC50 values of 33.08 and 13.14µM, respectively. Administration of 5 and 10mg/kg taxifolin for 30min ex vivo inhibited 11ß-HSD2 significantly and also in vivo decreased cortisol metabolism, as shown in the significant increase of area under curve (AUC). This result shows that taxifolin is a potent 11ß-HSD2 inhibitor, possibly causing side effects.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/antagonistas & inibidores , Quercetina/análogos & derivados , Animais , Corticosterona/metabolismo , Glucocorticoides/metabolismo , Humanos , Masculino , Quercetina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Arabidopsis exocyst subunit SEC3A has been reported to participate in embryo development. Here we report that SEC3A is involved during pollen germination. A T-DNA insertion in SEC3A leads to an absolute, male-specific transmission defect that can be complemented by the expression of SEC3A coding sequence from the LAT52 promoter or SEC3A genomic DNA. No obvious abnormalities in the microgametogenesis are observed in the sec3a/SEC3A mutant, however, in vitro and in vivo pollen germination are defective. Further studies reveal that the callose, pectin, and cellulose are apparently not deposited at the germination site during pollen germination. SEC3A is expressed ubiquitously, including in pollen grains and pollen tubes. Notably, SEC3A-GFP fusion proteins are specifically recruited to the future pollen germination site. This particular localization pattern is independent of phosphatidylinositol 4,5-bisphosphate (PI-4,5P2), although SEC3-HIS fusion proteins are able to bind to several phosphoinositols in vitro. These results suggest that SEC3A plays an important role in the establishment of the polar site for pollen germination.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Germinação , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Mutação/genética , Fenótipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Plantas Geneticamente Modificadas , Pólen/citologia , Transgenes , Proteínas de Transporte Vesicular/genéticaRESUMO
Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Membrana Celular/genética , Proteínas de Membrana/genética , Tubo Polínico/genética , Subunidades Proteicas/genética , Arabidopsis/crescimento & desenvolvimento , Membrana Celular/metabolismo , Parede Celular/genética , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas , Glicosilação , Complexo de Golgi/genética , Proteínas de Membrana/metabolismo , Proteínas Mutantes/genética , Pólen/genética , Pólen/crescimento & desenvolvimento , Tubo Polínico/crescimento & desenvolvimento , Transporte Proteico/genéticaRESUMO
OBJECTIVE: To observe the effects of ligustrazine hydrochloride injection(LHI) on pulmonary arterial hypertension in chronic obstructive pulmonary disease(COPD) patients and to investigate its possible mechanisms. METHODS: Twenty-two cases of patients with COPD were randomly divided into conventional treatmentgroup (group C) and ligustrazine treatment group(group L), 11 persons were randomly selected from healthy subjects without lung disease served as normal control group(group N). Group C was given bed rest, low flow oxygen inhalation, bronchial diastolic agent, glucocorticoid and antibiotics and other conventional treatment, and group L was added with ligustrazine hydrochloride injection on the above mentioned basis treatment, group N was given no treatment. After 2 weeks, lung function, blood gas analysis and pulmonary arterial pressure were compared among the three groups, and the content of H2S in plasma was tested with sensitive sulfur electrode method. RESULTS: â After two weeks treatment, in group L and group C pulmonary function, blood gas analysis, pulmonary artery pressure were obviously improved, and group L was better than group C (P<0.05); â¡ In group L the content of H2S was increased (P<0.01), group C had no significant difference (P>0.05), and there was a significant difference between the two groups (P<0.01). CONCLUSIONS: Combination with LHI can effectively improve lung function. LHI mayrelieve hypoxic hypercapnia pulmonary hypertension induced by COPD through raising the content of H2S.