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1.
Braz J Microbiol ; 54(2): 739-752, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37157054

RESUMO

Bacterial proteases have extensive applications in various fields of industrial microbiology. In this study, protease-producing organisms were screened on skimmed milk agar media using serial dilution. Through microbial biomass production, biochemical tests, protease-specific activity, and 16 s RNA gene sequencing, the isolates were identified as Bacillus subtilis and submitted to NCBI. The strain accession numbers were designated as A1 (MT903972), A2 (MT903996), A4 (MT904091), and A5 (MT904796). The strain A4 Bacillus subtilis showed highest protease-specific activity as 76,153.84 U/mg. A4 Bacillus subtilis was unaffected by Ca2+, Cu2+, Fe2+, Hg2+, Mg2+, Na, Fe2+, and Zn2+ but was inhibited by 80% by Mn2+ (5 mM). The protease activity was inhibited by up to 30% by iodoacetamide (5 mM). These findings confirm the enzyme to be a cysteine protease which was further confirmed by MALDI-TOF. The identified protease showed 71% sequence similarity with Bacillus subtilis cysteine protease. The crude cysteine protease significantly aided in fabric stain removal when added to a generic detergent. It also aided in the recovery of silver from used X-ray films and de-hairing of goat skin hides and showed decent application in meat tenderization. Thus, the isolated cysteine protease has high potential for industrial applications.


Assuntos
Bacillus subtilis , Cisteína Proteases , Peptídeo Hidrolases/metabolismo , Proteólise , Microbiologia Industrial , Proteínas de Bactérias/metabolismo
2.
Curr Microbiol ; 79(5): 142, 2022 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-35322302

RESUMO

Metal-binding proteins occur in the cytosol of most eubacteria. The hypothetical metal responsive protein MreA (PP-2969 gene; NreA) seems responsible for zinc, chromium, cadmium accumulation, and metal ion homeostasis. However, there is a lack of definitive evidence regarding the specific metal-binding sites of MreA protein. The present study aimed to identify putative metal-binding regions for MreA. In silico analysis revealed that amino acids C40, H65, and C69 (CHC region) seem critical for metal-protein interactions. We created site-directed mutants (SDM's) of MreA for interacted amino acids to validate in silico results. The differential scanning fluorimetry (DSF) and atomic absorption spectroscopy (AAS) showed that SDM strains of MreA protein curtailed metal accumulation compared to the wild types indicating C40, H65, and C69 amino acids are critical for metal binding. Thus, we report potential implications for MreA-bioengineered strains of Pseudomonas putida KT2440 for metal ion homeostasis by alleviating metal toxicity in the biological environment.


Assuntos
Pseudomonas putida , Sítios de Ligação , Cádmio/metabolismo , Metais/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Zinco/metabolismo
3.
Brain Res Bull ; 182: 1-11, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35143926

RESUMO

Stroke leads to disturbance in the physiology of the ER (Endoplasmic Reticulum) that triggers UPR (Unfolded Protein Response) pathways aimed to compensate neuronal cell damage. However, sustained UPR causes stressful conditions in the ER lumen forming abnormal protein aggregates. Stroke-induced oxidative stress also amalgamates with UPR to safeguard and ensure the proper functioning of brain cells. Thus we tested the effect of apocynin (a potent antioxidant) post-treatment in experimental stroke on the outcome of ER stress and UPR branch pathways. We administered a low dose of apocynin at 1 mg/kg (intraperitoneal) to adult Sprague-Dawley rats subjected to Middle Cerebral Artery Occlusion (MCAO) for two-time points. The first dose immediately after re-establishing the blood flow and another at 6 h of reperfusion. Apocynin post-treatment significantly reduced ROS (Reactive Oxygen Species) generation at an early reperfusion time point of 4 h. It preserved neuronal morphology, dendritic spine density, reduced protein aggregation, and brain damage after 24 h of reperfusion. Apocynin post-treatment regulates the two UPR branch pathways in our experimental paradigm. 1) Down-regulation of eIF2α (Eukaryotic Initiation Factor 2α) phosphorylation, and CHOP (C/EBP homologous protein) 2) by reducing the XBP-1 (X-Box binding Protein-1) mRNA splicing downstream to PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α (Inositol Requiring Enzyme 1alpha) UPR pathways, respectively. Bioinformatics prediction showed that apocynin has binding sites for PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α proteins. The amino acid residues interacting with apocynin were Cys891 and Gln889 (for PERK), and the amino acids Ser726, Arg722, and Ala719 (for IRE1α) lying within their activation loop. Overall, these studies indicate that apocynin post-treatment might regulate ER stress/UPR pathways and minimize stroke brain damage, thus having implications for developing newer strategies for stroke treatment.


Assuntos
Lesões Encefálicas , Acidente Vascular Cerebral , Acetofenonas , Animais , Endorribonucleases , Fator de Iniciação 2 em Eucariotos , Proteínas Serina-Treonina Quinases , RNA , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/tratamento farmacológico
4.
Biochem Biophys Rep ; 28: 101161, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34765745

RESUMO

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of ∼8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of ∼31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and kcat of 1356 nmol/min/mg and 0.69sec-1 respectively. Steady state kinetic analysis of NcFADS exhibited a Km of NcFADS for FMN is 2.7 µM and for MgATP-2 is 88.7 µM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent Kd of 20.8 µM for FMN and 16.6 µM for MgATP-2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the Tm is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.

5.
Biochim Biophys Acta Mol Cell Res ; 1867(1): 118573, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678591

RESUMO

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction. Mutation of these lysine residues abrogates the apoptosome formation causing inhibition of caspase activation. Using in-silico molecular docking, we have identified Cyt c-binding interface on Apaf-1. Although mutant Cyt c shows higher affinity for Apaf-1, the presence of Cyt c-WT restores the apoptosome activity. ATP addition modulates only mutant Cyt c binding to Apaf-1 but not WT Cyt c binding to Apaf-1. Using TCGA and cBioPortal, we identified multiple mutations in both Apaf-1 and Cyt c that are predicted to interfere with apoptosome assembly. We also demonstrate that transcript levels of various enzymes involved with dATP or ATP synthesis are increased in various cancers. Silencing of nucleotide metabolizing enzymes such as ribonucleotide reductase subunit M1 (RRM1) and ATP-producing glycolytic enzymes PKM2 attenuated ATP production and enhanced caspase activation. These findings suggest important role for lysine residues of Cyt c and nucleotides in the regulation of apoptosome-dependent apoptotic cell death as well as demonstrate how these mutations and nucleotides may have a pivotal role in human diseases such as cancer.


Assuntos
Apoptossomas/fisiologia , Citocromos c/química , Simulação de Acoplamento Molecular , Neoplasias/patologia , Nucleotídeos/química , Alanina/química , Alanina/genética , Substituição de Aminoácidos , Apoptossomas/química , Fator Apoptótico 1 Ativador de Proteases/química , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Estudos de Casos e Controles , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Citocromos c/genética , Citocromos c/metabolismo , Feminino , Humanos , Lisina/química , Lisina/genética , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Neoplasias/genética , Neoplasias/metabolismo , Nucleotídeos/metabolismo , Células PC-3 , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Multimerização Proteica/genética , Transdução de Sinais/genética
6.
Free Radic Biol Med ; 90: 261-71, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26627937

RESUMO

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins.


Assuntos
Apoptose/efeitos dos fármacos , Azadirachta/química , Limoninas/farmacologia , Neoplasias/patologia , Fosforilação Oxidativa , Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , DNA Mitocondrial/análise , Dinaminas , Complexo I de Transporte de Elétrons/fisiologia , GTP Fosfo-Hidrolases/análise , Células HCT116 , Humanos , Proteínas Associadas aos Microtúbulos/análise , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Mitocondriais/análise , Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/fisiologia
7.
Appl Microbiol Biotechnol ; 99(21): 9203-13, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26112211

RESUMO

The aim of the present work was to engineer bacteria for the removal of Co in contaminated effluents. Radioactive cobalt ((60)Co) is known as a major contributor for person-sievert budgetary because of its long half-life and high γ-energy values. Some bacterial Ni/Co transporter (NiCoT) genes were described to have preferential uptake for cobalt. In this study, the NiCoT genes nxiA and nvoA from Rhodopseudomonas palustris CGA009 (RP) and Novosphingobium aromaticivorans F-199 (NA), respectively, were cloned under the control of the groESL promoter. These genes were expressed in Deinococcus radiodurans in reason of its high resistance to radiation as compared to other bacterial strains. Using qualitative real time-PCR, we showed that the expression of NiCoT-RP and NiCoT-NA is induced by cobalt and nickel. The functional expression of these genes in bioengineered D. radiodurans R1 strains resulted in >60 % removal of (60)Co (≥5.1 nM) within 90 min from simulated spent decontamination solution containing 8.5 nM of Co, even in the presence of >10 mM of Fe, Cr, and Ni. D. radiodurans R1 (DR-RP and DR-NA) showed superior survival to recombinant E. coli (ARY023) expressing NiCoT-RP and NA and efficiency in Co remediation up to 6.4 kGy. Thus, the present study reports a remarkable reduction in biomass requirements (2 kg) compared to previous studies using wild-type bacteria (50 kg) or ion-exchanger resins (8000 kg) for treatment of ~10(5)-l spent decontamination solutions (SDS).


Assuntos
Radioisótopos de Cobalto/metabolismo , Deinococcus/metabolismo , Poluentes Ambientais/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Engenharia Metabólica , Centrais Nucleares , Soluções/metabolismo , Deinococcus/genética , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Viabilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopseudomonas/enzimologia , Rodopseudomonas/genética , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética , Ativação Transcricional/efeitos dos fármacos
8.
J Biol Chem ; 288(1): 368-81, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23152504

RESUMO

Proapoptotic Bcl-2 homology 3-only protein Bim plays an important role in Bax/Bak-mediated cytochrome c release and apoptosis. Here, we provide evidence for a novel prosurvival function of Bim in cancer cells. Bim was constitutively overexpressed in multiple prostate and breast cancer cells as well as in primary tumor cells. Quantitative real time PCR analysis showed that Bim was transcriptionally up-regulated. We have identified eight endogenous E2F1-binding sites on the Bim promoter using in silico analysis. Luciferase assay demonstrated that Bim expression was E2F1-dependent as mutation of the E2F1-binding sites on the Bim promoter inhibited luciferase activities. In support, E2F1 silencing led to the loss of Bim expression in cancer cells. Bim primarily localized to mitochondrial and cytoskeleton-associated fractions. Bim silencing or microinjection of anti-Bim antibodies into the cell cytoplasm resulted in cell rounding, detachment, and subsequent apoptosis. We observed up-regulation of prosurvival proteins Bcl-xL and Mcl-1, which sequester Bim in cancer cells. In addition, a phosphorylated form of Bim was also elevated in cancer cells. These findings suggest that the constitutively overexpressed Bim may function as a prosurvival molecule in epithelial cancer cells, and phosphorylation and association with Bcl-xL/Mcl-1 block its proapoptotic functions.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Fator de Transcrição E2F1/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Sobrevivência Celular , Inativação Gênica , Humanos , Imuno-Histoquímica/métodos , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Transdução de Sinais , Regulação para Cima
9.
Mitochondrion ; 13(5): 493-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23088850

RESUMO

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.


Assuntos
Antineoplásicos Fitogênicos/metabolismo , Apoptose , Autofagia , Caspases/metabolismo , DNA Mitocondrial/metabolismo , Mitocôndrias/efeitos dos fármacos , Estilbenos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Resveratrol
10.
Carcinogenesis ; 33(11): 2199-207, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22915764

RESUMO

Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Glicerídeos/química , Limoninas/farmacologia , Terpenos/química , Proteína Supressora de Tumor p53/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Fator de Indução de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Western Blotting , Caspases/química , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Imunofluorescência , Humanos , Inseticidas/farmacologia , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo
11.
Cell Cycle ; 10(23): 4128-37, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22101335

RESUMO

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.


Assuntos
Apoptose , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Curcumina/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 3/genética , Caspase 3/metabolismo , Inibidores de Caspase , Inibidor de Quinase Dependente de Ciclina p21/genética , Citocromos c/genética , Citocromos c/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Células Jurkat , Lentivirus/genética , Lentivirus/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
12.
J Biol Chem ; 286(33): 28749-28760, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21712378

RESUMO

Resveratrol, a naturally occurring phytoalexin, is known to induce apoptosis in multiple cancer cell types, but the underlying molecular mechanisms remain unclear. Here, we show that resveratrol induced p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated translocation of Bax to mitochondria where it underwent oligomerization to initiate apoptosis. Resveratrol treatment promoted interaction between Bax and XIAP in the cytosol and on mitochondria, suggesting that XIAP plays a critical role in the activation and translocation of Bax to mitochondria. This process did not involve p53 but required accumulation of Bim and t-Bid on mitochondria. Bax primarily underwent homo-oligomerization on mitochondria and played a major role in release of cytochrome c to the cytosol. Bak, another key protein that regulates the mitochondrial membrane permeabilization, did not interact with p53 but continued to associate with Bcl-xL. Thus, the proapoptotic function of Bak remained suppressed during resveratrol-induced apoptosis. Caspase-9 silencing inhibited resveratrol-induced caspase activation, whereas caspase-8 knockdown did not affect caspase activity, suggesting that resveratrol induces caspase-9-dependent apoptosis. Together, our findings characterize the molecular mechanisms of resveratrol-induced caspase activation and subsequent apoptosis in cancer cells.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Caspase 8/metabolismo , Caspase 9/metabolismo , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estilbenos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 8/genética , Caspase 9/genética , Citocromos c/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Humanos , Células Jurkat , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Multimerização Proteica/genética , Resveratrol , Proteína Supressora de Tumor p53/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteína X Associada a bcl-2/genética
13.
PLoS One ; 6(1): e16379, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21297999

RESUMO

In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.


Assuntos
Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 9/metabolismo , Nucleotídeos/deficiência , Trifosfato de Adenosina/deficiência , Caspases/metabolismo , Citocromos c/metabolismo , Citosol , Fibroblastos , Humanos , Ligação Proteica
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