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1.
Curr Opin Chem Biol ; 59: 1-14, 2020 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-32298980

RESUMO

Exacerbation of climate change and air pollution around the world have emphasized the necessity of replacing fossil fuels with clean and sustainable energy. Metabolic engineering has provided strategies to engineer diverse organisms for the production of biofuels from renewable carbon sources. Although some of the processes are commercialized, there has been continued effort to produce advanced biofuels with higher efficiencies. In this article, metabolic engineering strategies recently exploited to enhance biofuel production and facilitate utilization of non-edible low-value carbon sources are reviewed. The strategies include engineering enzymes, exploiting new pathways, and systematically optimizing metabolism and fermentation processes, among others. In addition, metabolic and bioprocess engineering strategies to achieve competitiveness of current biofuel production systems compared with fossil fuels are discussed.

2.
Molecules ; 25(4)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32102340

RESUMO

For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32-47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

3.
Metab Eng ; 57: 13-22, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610242

RESUMO

Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.

4.
Appl Microbiol Biotechnol ; 104(3): 1001-1012, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31858190

RESUMO

Acrylamide is an important bulk chemical used for producing polyacrylamide, which is widely applied in diverse fields, such as enhanced oil recovery and water treatment. Acrylamide production with a superior biocatalyst, free-resting Rhodococcus cells containing nitrile hydratase (NHase), has been proven to be simple but effective, thereby becoming the main method adopted in industry to date. Under the harsh industrial conditions, however, NHase-containing Rhodococcus cells in a natural state are prone to deactivation. Thus, multiple genetic strategies able to evolve recombinant Rhodococcus biocatalysts at either the enzyme or cell level have been reported. While most of the methods on enzyme engineering concentrate on NHase stability enhancement by strengthening the flexible sites, Rhodococcus cell engineering with various methods can enhance both the NHase activity and stability as well. Developing some new types of reactors, especially the microreactor, is also an effective way to improve the hydration process efficiency. Compared with the conventional stirred tank reactor, the membrane dispersion microreactor can enhance the heat and mass transfer in the hydration process with Rhodococcus cells as biocatalysts, thereby significantly improving the productivity of the acrylamide bioproduction process.

5.
Biochem Biophys Res Commun ; 516(4): 1252-1257, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31301768

RESUMO

Intracerebral hemorrhage (ICH) is the most common of stroke with high mortality and severe morbidity. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a neuronprotective role in ICH. In the current study, TRIM37 mRNA expression in peripheral blood mononuclear cells (PBMCs) was found to be increased in ICH patients compared to that in healthy controls (n = 15). TRIM37 bound to PPARγ and enhanced its ubiquitination in mouse microglial BV-2 cell line. According to previous studies, thrombin is produced in the brain instantaneously after ICH and triggers the activation of microglia. Here, thrombin induced TRIM37 expression, cell apoptosis and interleukin-1ß (IL-1ß) release in BV-2 cells, while TRIM37 knockdown partially reversed the effects of thrombin on BV-2 cells. TRIM37 overexpression showed similar effects as thrombin on BV-2 cells, and PPARγ agonist rosiglitazone abolished the effects of TRIM37. In summary, TRIM37 involved in apoptosis and IL-1ß release in BV-2 microglia by regulating PPARγ ubiquitination. The present data established a potential biological role of TRIM37 in ICH-induced brain damage and may provide insight into the development of therapy strategies for ICH.

6.
N Biotechnol ; 44: 41-49, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-29689306

RESUMO

To satisfy the urgent demand for promoter engineering that can accurately regulate the metabolic circuits and expression of specific genes in the Rhodococcus microbial platform, a promoter-ribosome binding site (RBS) coupled mini-pool with fine-tuning of different activity levels was successfully established. Transcriptome analyses of R. ruber TH revealed several representative promoters with different activity levels, e.g., Pami, Pcs, Pnh, P50sl36, PcbiM, PgroE and Pniami. ß-Galactosidase (LacZ) reporter measurement demonstrated that different gene expression levels could be obtained with these natural promoters combined with an optimal RBS of ami. Further use of these promoters to overexpress the nitrile hydratase (NHase) gene with RBSami in R. ruber THdAdN produced different expression levels consistent with the transcription analyses. The -35 and -10 core elements of different promoters were further analyzed, and the conserved sequences were revealed to be TTGNNN and (T/C)GNNA(A/C)AAT. By mutating the core elements of the strong promoters, Pnh and Pami, into the above consensus sequence, two even stronger promoters, PnhM and PamiM, were obtained with 2.2-fold and 7.7-fold improvements in transcription, respectively. Integrating several strategies, including transcriptome promoter screening, -35 and -10 core element identification, core element point-mutation, RBS optimization and diverse reporter verification, a fine-tuning promoter-RBS combination mini-pool with different activity levels in Rhodococcus strains was successfully established. This development is significant for broad applications of the Rhodococcus genus as a microbial platform.


Assuntos
Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Rhodococcus , Transcrição Genética , Perfilação da Expressão Gênica , Rhodococcus/genética , Rhodococcus/metabolismo
7.
Bioprocess Biosyst Eng ; 41(7): 931-938, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29552729

RESUMO

In this work, the drastic change in the reaction rate throughout the acrylonitrile bio-hydration reaction, which was catalyzed by Rhodococcus ruber TH3 free cells in a two-liquid-phase system, was studied by changing the initial mass fraction of acrylonitrile and acrylamide. We found that the reaction rate was sensitively affected by the contact area between the acrylonitrile droplets and cells. With the acrylonitrile mass fraction of 3 wt%, the cell solution of 800 U/mL could make the superficial area of acrylonitrile droplets saturated. The sustained increase of the acrylamide concentration in the reaction process could reduce the reaction rate, and 25 wt% was the obvious inflection point. The interface adsorption of cells was visually observed with the method of fluorescence microscopy, and the uptake mechanism of substrate by direct contact was illustrated to play a main role by comparing the reaction rate of the heterogeneous system and that of the homogeneous system.


Assuntos
Acrilamida/metabolismo , Acrilonitrila/metabolismo , Biocatálise , Rhodococcus/metabolismo
8.
Appl Microbiol Biotechnol ; 101(16): 6321-6332, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28551854

RESUMO

Prevention of cell flocculation in large-scale fermentation is of great importance for most industrial microbes. Using Rhodococcus ruber TH3 as a model strain, we revealed that the undesired cell flocculation in a fermenter was associated with the colony dimorphism phenomenon, and it only occurred in the rough-type of cells (R-TH3) instead of the smooth-type of cells (S-TH3). By analyzing the transcriptome differences of R-TH3 and S-TH3, six representative genes with significantly upregulated transcription in S-TH3 were selected and overexpressed in R-TH3. The colony morphotypes of the six engineered strains changed to different extents, in which overexpressions of three lipid metabolism-related proteins LM1, LM2, and LM3 tuned the colony morphotype from rough to almost as smooth as in S-TH3. SEM observation confirmed the cell surface difference of the engineered strains from R-TH3. Their cell surface hydrophobicity also reduced, and the cell sedimentation behaviors were consequently changed as expected. Using R-TH3/LM1 as the representative of the engineered bacteria, fatty acids of the cell envelopes were measured. Fatty acid contents of S-TH3, R-TH3/LM1, and R-TH3 were 27.21, 24.10, and 22.24%, respectively. Among all the fatty acids, stearic acid binding to hydrophilic extracellular polysaccharides (EPS) in Rhodococcus showed significant differences among the cells. The EPS contents of S-TH3, R-TH3/LM1, and R-TH3 were 191, 163, and 137 mg/g cells. Hence, the hydrophilicity of the S-TH3 cells was mainly due to the EPS in the outermost layer of the cells. Increase of fatty acids especially stearic acid results in the increase of the bound EPS, finally bringing about the hydrophilicity enhancement.


Assuntos
Fermentação , Rhodococcus/genética , Rhodococcus/metabolismo , Ácidos Graxos/análise , Floculação , Perfilação da Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos/genética , Polissacarídeos/metabolismo , Rhodococcus/citologia
9.
J Mol Neurosci ; 61(3): 385-395, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27933491

RESUMO

Complement-mediated inflammation plays a vital role in intracerebral hemorrhage (ICH), implicating pro-inflammatory factor interleukin-1beta (IL-1ß) secretion. Brain samples and contralateral hemiencephalon were all collected and detected by Western blot. NLRP3 expression was located by dual immunofluorescence staining at 1, 3, and 5 days post-ICH. Brain water content was examined post-ICH. The neural deficit scores were evaluated by observers blindly. ILs were detected by ELISA. SiRNAs targeting NLRP3 (siNLRP3), siASC, and siControl were injected to inhibit NLRP3 function. To test the complement activation via Nod-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), normal rabbit complement (NRC) was injected with lipopolysaccharide (LPS) to facilitate the complement function. As a result, complement 3a (C3a) and complement 5a (C5a) were upregulated during the ICH-induced neuroinflammation, and ablation of C3 attenuates ICH-induced IL-1ß release. Though the LPS rescues the neuroinflammation in the ICH model, C3 deficiency attenuates the LPS-induced inflammatory effect. The NLRP3 inflammasome was activated after ICH and was located in the microglial cell of the mouse brain, which exhibits a time-dependent manner. However, the number of NLRP3/Iba-1 dual-labeled cells in the C3-/- group is less than that in the WT group in each time course, respectively. IL-1ß and IL-18 released in perihematoma tissue, caspase-1-p20, brain water content, and behavioral outcomes were attenuated in the siNLRP3 and siASC groups than in the siControl and ICH groups. We also found that 5% of complement supplement enhances ICH-induced IL-1ß release, while NLRP3 and ASC inhibition attenuates it. In conclusion, complement-induced ICH neuroinflammation depended on NLRP3 activation, which facilities LPS- and ICH-induced neuroinflammation, and NLRP3 is required for ICH-induced inflammation.


Assuntos
Caspase 1/metabolismo , Hemorragia Cerebral/imunologia , Inflamassomos/imunologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Encéfalo/imunologia , Complemento C3a/imunologia , Complemento C5a/imunologia , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Inflamação Neurogênica/imunologia
10.
Biotechnol Bioeng ; 114(4): 832-842, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27723092

RESUMO

Surfactin-family lipopeptides are green biosurfactants with substantial industrial potential. The major problem prohibiting surfactin use is the low titer of the wild producer, Bacillus subtilis. Using transcriptomic analysis, four strong promoters, PgroE, Pcdd, PrplK, and PsspE, were identified and cloned from the genome of B. subtilis THY-7, a novel surfactin producer that has been identified from soil with a 0.55 g/L surfactin titer. An optimal promoter, PgroE, was selected to replace the native THY-7 surfactin synthase (SrfA) promoter through single-cross homologous recombination; however, the resulting engineered strain containing the PgroE substitution did not synthesize surfactin. The sucrose-inducible promoters PsacB and PsacP were then substituted in place of PsrfA, and the resulting engineered strains produced 1.09 and 0.22 g/L surfactin, respectively. An artificial, sucrose-inducible Pg1 promoter was produced through fusion of the PgroE and PsacB ribonucleic antiterminator (RAT), and the engineered strain containing the Pg1-substitution produced a surfactin titer of 1.44 g/L. An artificial IPTG-inducible promoter, Pg2, was constructed from a PgroE-lacO fusion and then substituted for the chromosomal PsrfA locus, and the surfactin titer of the resulting THY-7/Pg2-srfA increased to 5.98 g/L. The driving capacity of Pg2 was further improved by the inclusion of two point mutations in the -35 and -10 regions to produce the novel promoter Pg3. Pg3 exhibited super-strong activity as measured by lacZ reporter gene overexpression (approximately 3000 U). The Pg3-substitution strain THY-7/Pg3-srfA produced up to 9.74 g/L surfactin in a 5 L fermentor. The maximum productivity was 0.30 g/L/h, and the greatest yield reached 0.14 g surfactin/g sucrose. Biotechnol. Bioeng. 2017;114: 832-842. © 2016 Wiley Periodicals, Inc.


Assuntos
Bacillus subtilis/genética , Lipopeptídeos/metabolismo , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/genética , Bacillus subtilis/metabolismo , Fermentação , Perfilação da Expressão Gênica , Lipopeptídeos/genética , Tensoativos , Transcriptoma
11.
eNeuro ; 3(4)2016.
Artigo em Inglês | MEDLINE | ID: mdl-27517085

RESUMO

Accumulation of amyloid-ß (Aß) peptide in the brain is a central hallmark of Alzheimer's disease (AD) and is thought to be the cause of the observed neurodegeneration. Many animal models have been generated that overproduce Aß yet do not exhibit clear neuronal loss, questioning this Aß hypothesis. We previously developed an in vivo mouse model that expresses a humanized amyloid precursor protein (hAPP) in olfactory sensory neurons (OSNs) showing robust apoptosis and olfactory dysfunction by 3 weeks of age, which is consistent with early OSN loss and smell deficits, as observed in AD patients. Here we show, by deleting the ß-site APP cleaving enzyme 1 (BACE1) in two distinct transgenic mouse models, that hAPP-induced apoptosis of OSNs is Aß independent and remains cell autonomous. In addition, we reveal that the intrinsic apoptosis pathway is responsible for hAPP-induced OSN death, as marked by mitochondrial damage and caspase-9 activation. Given that hAPP expression causes OSN apoptosis despite the absence of BACE1, we propose that Aß is not the sole cause of hAPP-induced neurodegeneration and that the early loss of olfactory function in AD may be based on a cell-autonomous mechanism, which could mark an early phase of AD, prior to Aß accumulation. Thus, the olfactory system could serve as an important new platform to study the development of AD, providing unique insight for both early diagnosis and intervention.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Apoptose/fisiologia , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Caspase 9/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Potencial da Membrana Mitocondrial/fisiologia , Camundongos Transgênicos , Microscopia Eletrônica , Neurônios/patologia
12.
Cereb Cortex ; 26(2): 576-85, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25246509

RESUMO

Gamma-frequency oscillatory activity plays an important role in information integration across brain areas. Disruption in gamma oscillations is implicated in cognitive impairments in psychiatric disorders, and 5-HT3 receptors (5-HT3Rs) are suggested as therapeutic targets for cognitive dysfunction in psychiatric disorders. Using a 5-HT3aR-EGFP transgenic mouse line and inducing gamma oscillations by carbachol in hippocampal slices, we show that activation of 5-HT3aRs, which are exclusively expressed in cholecystokinin (CCK)-containing interneurons, selectively suppressed and desynchronized firings in these interneurons by enhancing spike-frequency accommodation in a small conductance potassium (SK)-channel-dependent manner. Parvalbumin-positive interneurons therefore received diminished inhibitory input leading to increased but desynchronized firings of PV cells. As a consequence, the firing of pyramidal neurons was desynchronized and gamma oscillations were impaired. These effects were independent of 5-HT3aR-mediated CCK release. Our results therefore revealed an important role of 5-HT3aRs in gamma oscillations and identified a novel crosstalk among different types of interneurons for regulation of network oscillations. The functional link between 5-HT3aR and gamma oscillations may have implications for understanding the cognitive impairments in psychiatric disorders.


Assuntos
Ritmo Gama/fisiologia , Hipocampo/citologia , Interneurônios/fisiologia , Parvalbuminas/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Animais , Apamina/farmacologia , Benzodiazepinas/farmacologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Potenciais Pós-Sinápticos Excitadores/genética , Antagonistas de Receptores de GABA-A/farmacologia , Ritmo Gama/genética , Antagonistas de Hormônios/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Picrotoxina/análogos & derivados , Picrotoxina/farmacologia , Receptores 5-HT3 de Serotonina/genética , Serotoninérgicos/farmacologia , Análise Espectral
13.
Neuropsychiatr Dis Treat ; 11: 1609-14, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26170673

RESUMO

BACKGROUND: The purpose of this study was to investigate the epidemiology of sporadic ruptured cerebral aneurysm in the Chinese population. METHODS: We retrospectively analyzed the medical records of 264 consecutive Chinese patients admitted to the Affiliated Hospital of Zunyi Medical University of Guizhou Province in Southwest China between December 2012 and March 2015 for spontaneous subarachnoid hemorrhage due to a ruptured cerebral artery aneurysm. RESULTS: The study population comprised 171 females and 93 males with a median age of 50 (range 5-76) years. The female to male ratio was 1.84:1. For both males and females, aneurysm rupture was most common in the 40-49-year age group (34.5%). Most of the ruptured aneurysms were in the size range of 2-5 mm (47.2%), followed by 5-10 mm (43.8%). Ruptured aneurysms occurred most often in the posterior communicating artery (36.6%) or the anterior communicating artery (25.7%). There were more cases of anterior communicating artery aneurysm on the left side (53 [Left side]/16 [Right side]=3.31, P>0.001) and slightly more cases of posterior communicating artery aneurysm on the right side (54 [Right side]/44[Left side]=1.23, P<0.05). CONCLUSION: This study provides valuable information on the epidemiology of ruptured cerebral aneurysm in the Chinese population.

14.
IUBMB Life ; 66(6): 379-86, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24979663

RESUMO

Prostate cancer (PCa) is the second most commonly occurring malignant tumor in Europe and America. Normal and neoplastic growth of prostate gland are dependent on androgen receptor (AR) expression and function. PCa is driven by androgen and its receptor, and they continue to be the key drivers of castration-resistant prostate cancer (CRPC). CRPC is the terminal stage of PCa and seriously jeopardizes the patient's quality of life and lifespan. miRNAs are small noncoding RNAs, 18-25 nt in length that destabilize mRNA or repress protein synthesis by interacting with the 3'-untranslated regions (3'-UTR) of target mRNAs. miRNAs can regulate AR or be regulated by AR and then affect various signaling pathways related to cellular functions and tumor processes. In this review, we focus on the relationship between miRNAs and AR in PCa and elucidate their roles in the induction of malignant changes in PCa.


Assuntos
Regulação da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Receptores Androgênicos/metabolismo , Progressão da Doença , Regulação da Expressão Gênica/genética , Humanos , Masculino , Neoplasias da Próstata/genética
15.
Mol Cell Proteomics ; 12(12): 3719-31, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24023391

RESUMO

The cysteine protease caspase-3, best known as an executioner of cell death in apoptosis, also plays a non-apoptotic role in N-methyl-d-aspartate receptor-dependent long-term depression of synaptic transmission (NMDAR-LTD) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor endocytosis in neurons. The mechanism by which caspase-3 regulates LTD and AMPA receptor endocytosis, however, remains unclear. Here, we addressed this question by using an enzymatic N-terminal peptide enrichment method and mass spectrometry to identify caspase-3 substrates in neurons. Of the many candidates revealed by this proteomic study, we have confirmed BASP1, Dbn1, and Gap43 as true caspase-3 substrates. Moreover, in hippocampal neurons, Gap43 mutants deficient in caspase-3 cleavage inhibit AMPA receptor endocytosis and LTD. We further demonstrated that Gap43, a protein well-known for its functions in axons, is also localized at postsynaptic sites. Our study has identified Gap43 as a key caspase-3 substrate involved in LTD and AMPA receptor endocytosis, uncovered a novel postsynaptic function for Gap43 and provided new insights into how long-term synaptic depression is induced.


Assuntos
Caspase 3/genética , Proteína GAP-43/genética , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/genética , Neurônios/metabolismo , Receptores de AMPA/genética , Transmissão Sináptica/genética , Animais , Caspase 3/metabolismo , Embrião de Mamíferos , Endocitose , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Plasticidade Neuronal/genética , Neurônios/citologia , Técnicas de Patch-Clamp , Cultura Primária de Células , Ligação Proteica , Mapeamento de Interação de Proteínas , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/genética , Sinapses/metabolismo , Técnicas de Cultura de Tecidos
16.
J Neurochem ; 120(4): 502-14, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22118516

RESUMO

Cholesterol is a major component of membrane lipid rafts. It is more abundant in the brain than in other tissues and plays a critical role in maintaining brain function. We report here that a significant enhancement in apoptosis in rat cerebellar granule neurons (CGNs) was observed upon incubation with 5mM K(+) /serum free (LK-S) medium. Cholesterol enrichment further potentiated CGN apoptosis incubated under LK-S medium. On the contrary, cholesterol depletion using methyl-beta-cyclodextrin protected the CGNs from apoptosis induced by LK-S treatment. Cholesterol enrichment, however, did not induce apoptosis in CGNs that have been incubated with 25mM K(+) /serum medium. Mechanistically, increased I(K) currents and DNA fragmentation were found in CGNs incubated in LK-S, which was further potentiated in the presence of cholesterol. Cholesterol-treated CGNs also exhibited increased cAMP levels and up-regulation of Kv2.1 expression. Increased levels of activated form of PKA and phospho-CREB further supported activation of the cAMP/PKA pathway upon treatment of CGNs with cholesterol-containing LK-S medium. Conversely, inhibition of PKA or small G protein Gs abolished the increase in I(K) current and the potentiation of Kv2.1 expression, leading to reduced susceptibility of CGNs to LK-S and cholesterol-induced apoptosis. Our results demonstrate that the elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1. Our data provide new evidence for the role of cholesterol in eliciting neuronal cell death.


Assuntos
Apoptose/genética , Proteína de Ligação a CREB/metabolismo , Colesterol/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Neurônios/metabolismo , Canais de Potássio Shab/biossíntese , Regulação para Cima/fisiologia , Animais , Células Cultivadas , AMP Cíclico/fisiologia , Feminino , Predisposição Genética para Doença , Ratos , Ratos Sprague-Dawley , Canais de Potássio Shab/genética , Transdução de Sinais/genética
17.
Neuron ; 70(4): 758-72, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21609830

RESUMO

It has recently been found that caspases not only function in apoptosis, but are also crucial for nonapoptotic processes such as NMDA receptor-dependent long-term depression (LTD) of synaptic transmission. It remains unknown, however, how caspases are activated and how neurons escape death in LTD. Here we show that caspase-3 is activated by the BAD-BAX cascade for LTD induction. This cascade is required specifically for NMDA receptor-dependent LTD but not for mGluR-LTD, and its activation is sufficient to induce synaptic depression. In contrast to apoptosis, however, BAD is activated only moderately and transiently and BAX is not translocated to mitochondria, resulting in only modest caspase-3 activation. We further demonstrate that the intensity and duration of caspase-3 activation determine whether it leads to cell death or LTD, thus fine-tuning of caspase-3 activation is critical in distinguishing between these two pathways.


Assuntos
Apoptose/fisiologia , Caspase 3/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Transmissão Sináptica/fisiologia , Proteína X Associada a bcl-2/fisiologia , Proteína de Morte Celular Associada a bcl/fisiologia , Animais , Células Cultivadas , Ativação Enzimática/fisiologia , Hipocampo/enzimologia , Hipocampo/fisiologia , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley
18.
Cell ; 141(5): 859-71, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20510932

RESUMO

NMDA receptor-dependent synaptic modifications, such as long-term potentiation (LTP) and long-term depression (LTD), are essential for brain development and function. LTD occurs mainly by the removal of AMPA receptors from the postsynaptic membrane, but the underlying molecular mechanisms remain unclear. Here, we show that activation of caspase-3 via mitochondria is required for LTD and AMPA receptor internalization in hippocampal neurons. LTD and AMPA receptor internalization are blocked by peptide inhibitors of caspase-3 and -9. In hippocampal slices from caspase-3 knockout mice, LTD is abolished whereas LTP remains normal. LTD is also prevented by overexpression of the anti-apoptotic proteins XIAP or Bcl-xL, and by a mutant Akt1 protein that is resistant to caspase-3 proteolysis. NMDA receptor stimulation that induces LTD transiently activates caspase-3 in dendrites, without causing cell death. These data indicate an unexpected causal link between the molecular mechanisms of apoptosis and LTD.


Assuntos
Apoptose , Caspase 3/metabolismo , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo , Receptores de AMPA/metabolismo , Animais , Células Cultivadas , Citocromos c/metabolismo , Hipocampo/citologia , Potenciação de Longa Duração , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína bcl-X/metabolismo
19.
J Neurochem ; 100(4): 979-91, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17156132

RESUMO

Previously, we have reported that apoptosis of cerebellar granular neurons induced by incubation in 5 mm K(+) and serum-free medium (LK-S) was associated with an increase in the delayed rectifier K(+) current (I(K)). Here, we show that I(K) associated with apoptotic neurons is mainly encoded by a Kv2.1 subunit. Silencing Kv2.1 expression by small interfering RNA reduces I(K) and increases neuron viability. Forskolin is able to decrease the I(K) amplitude recording from neurons of both the LK-S and control group, and prevents apoptosis of granule cells that are induced by LK-S. Dibutyryl cAMP mimicks the effect of forskolin on the modulation of I(K) and, accordingly, the inhibitor of protein kinase A, H-89, aborts the neuron-protective effect induced by forskolin. Whereas the expression of Kv2.1 was silenced by Kv2.1 small interfering RNA, the inhibition of forskolin on the current amplitude was significantly reduced. Quantitative RT-PCR and whole-cell recording revealed that the expression of Kv2.1 was elevated in the apoptotic neurons, and forskolin significantly depressed the expression of Kv2.1. We conclude that the protection against apoptosis via the protein kinase A pathway is associated with a double modulation on I(K) channel properties and its expression of alpha-subunit that is mainly encoded by the Kv2.1 gene.


Assuntos
Apoptose/fisiologia , Cerebelo/citologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/fisiologia , Neurônios/fisiologia , Canais de Potássio Shab/fisiologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Cloreto de Potássio/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Canais de Potássio Shab/genética , Transfecção/métodos
20.
J Pineal Res ; 36(2): 109-16, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14962062

RESUMO

Activation of K(+) current plays a critical role in the control of programmed cell death. In the present study, whole-cell patch-clamp recording, a caspase-3 activity assay, and flow cytometric analysis were used to examine the effects of the MT2 melatonin receptor agonist 2-iodomelatonin on the delayed-rectifier K(+) current (IK) and the prevention of apoptosis. It was found that apoptosis of cerebellar granular neurons induced by low-K(+) (5 mm) incubation was associated with an increase in IK amplitude and caspase-3 activity. After 6 hr of low-K(+) treatment, IK was increased by 45% (n = 86). Flow cytometry showed that the apoptosis rate increased by 333% compared with the control neurons. In addition, exposure of cultured granule cells to low K(+) also resulted in a significant activation of caspase-3, by 466%. 2-Iodomelatonin (10 microm in injection pipette) inhibited the IK amplitude recorded from control cells and from cells undergoing apoptosis. However, 2-iodomelatonin only modified the IK-channel activation kinetics of cells under both conditions. Furthermore, 2-iodomelatonin reduced the rate of apoptosis and caspase-3 activation, by 66 and 64%, respectively. The melatonin receptor antagonist, 4P-PDOT, abrogated the effect of 2-iodomelatonin on the IK augmentation, caspase-3 activity, and apoptosis. These results suggest that the neuroprotective effects of melatonin are not only because of its function as a powerful antioxidant, but also to its interactions with specific receptors. The effect of 2-iodomelatonin against apoptosis may be mediated by activating a melatonin receptor, which modulates IK channels and reduces K(+) efflux.


Assuntos
Apoptose/efeitos dos fármacos , Melatonina/análogos & derivados , Melatonina/farmacologia , Neurônios/efeitos dos fármacos , Receptores de Melatonina/agonistas , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Ratos
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