RESUMEN
Induction of apoptosis is often necessary for successful cancer therapy, and the non-invasive monitoring of apoptosis post-therapy could assist in clinical decision making. Isatins are a class of compounds that target activated caspase-3 during apoptosis. Here we report the synthesis of the 5-iodo-1,2,3-triazole (FITI) analog of the PET tracer [18F]ICMT11 as a candidate tracer for imaging of apoptosis with SPECT, as well as PET. Labelling with radioiodine (123,125I) was achieved in 55 ± 12% radiochemical yield through a chelator-accelerated one-pot cycloaddition reaction mediated by copper(I) catalysis. The caspase-3 binding affinity and selectivity of FITI compares favourably to that of [18F]ICMT11 (Ki = 6.1 ± 0.9 nM and 12.4 ± 4.7 nM, respectively). In biodistribution studies, etoposide-induced cell death in a SW1222 xenograft model resulted in a 2-fold increase in tumour uptake of the tracer. However, the tumour uptake was too low to allow in vivo imaging of apoptosis with SPECT.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/aislamiento & purificación , Radioisótopos de Yodo/farmacología , Neoplasias/diagnóstico por imagen , Animales , Apoptosis/genética , Caspasa 3/química , Caspasa 3/genética , Línea Celular Tumoral , Cobre/química , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacología , Xenoinjertos , Humanos , Radioisótopos de Yodo/química , Isatina/síntesis química , Isatina/farmacología , Ratones , Neoplasias/patología , Neoplasias/terapia , Radiofármacos/síntesis química , Radiofármacos/farmacología , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodos , Triazoles/síntesis química , Triazoles/farmacologíaRESUMEN
Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.
Asunto(s)
Caspasa 10/aislamiento & purificación , Caspasa 8/aislamiento & purificación , Caspasa 9/aislamiento & purificación , Péptidos/química , Apoptosis/genética , Caspasa 10/química , Caspasa 10/genética , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/aislamiento & purificación , Caspasa 8/química , Caspasa 8/genética , Caspasa 9/química , Caspasa 9/genética , Inhibidores de Caspasas/química , Inhibidores de Caspasas/farmacología , Humanos , Células Jurkat , Cinética , Transducción de Señal , Especificidad por SustratoRESUMEN
Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population. Here we describe a flow cytometric assay fully realising that potential by the use of anti-RIP-3 (Receptor-interacting serine/threonine-protein kinase 3) and anti-active caspase-3 fluorescently tagged antibodies and a fixable live dead fluorescent dye. This allows the determination of the degree of necroptosis, apoptosis and RIP1-dependent apoptosis within live and dead populations. Necroptosis was identified by the up-regulation of RIP3, while RIP1-dependent apoptosis was described by double positive for RIP3/active Caspase-3 events in live and dead populations. Apoptotic cells were defined by an active-Caspase-3+ve/RIP3-ve phenotype. Pan-caspase blocker zVAD and RIP1 inhibitors GSK'481 or necrostatin-1 revealed interesting modulations of such sub-populations of Jurkat cells. This novel flow cytometric assay employing two antibodies and a fixable viability probe provides the researcher with in-depth analysis of various forms of regulated forms of cell death beyond what is currently available and is a major methodological advancement in this field.
Asunto(s)
Apoptosis/genética , Citometría de Flujo/métodos , Inmunofenotipificación/métodos , Caspasa 3/genética , Caspasa 3/aislamiento & purificación , Línea Celular Tumoral , Humanos , Necrosis/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/aislamiento & purificación , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
Since human Caspase-3, a member of the cysteine protease family, plays important roles not only in the apoptosis pathway as an executioner protein, but also in neurological disorders as a critical factor, biomedical researchers have been interested in the development of modulators of caspase-3 activity. Such studies require large quantities of purified active caspase-3. So far, purification of soluble caspase-3 from full-length human caspase-3 in Escherichia coli (E. coli) yields only several mg from a liter of culture media. Therefore, a number of alternative strategies to purify active caspase-3 have been described in the literature, including refolding and protein engineering. In this study, we systematically study the effects of host E. coli strains and growth conditions on purifications of active caspase-3 from full-length human caspase-3. Using a combination of conditions that include use of the C41(DE3) strain, low-temperature expression, and auto-induction that induces caspase-3 expression depending on metabolic state of the individual host cell, we are able to obtain 14-17 mg caspase-3 per liter of culture, an amount that is about 7 times larger than published results. This optimized expression and purification method for caspase-3 can be easily scaled up to facilitate the demand for active enzyme.
Asunto(s)
Caspasa 3 , Expresión Génica , Caspasa 3/biosíntesis , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
This paper reports a novel approach for the simple assays of cell apoptosis using electrochemical technique. In this study, caspase-3 activity, which was detected with differential plus voltammetry (DPV) as an alternative to conventional spectrometry approach, was employed as an indicator of cell apoptosis and, while an acetylated peptide Ac-GGHDEVDHGGGC was used as the blocked substrate. In the presence of casepase-3, the hydrolysis of blocked peptide might release active amine groups, which could covalently conjugate with graphene oxide. Therefore, electroactive methylene blue molecules could be further attached to the electrode surface through π-π stacking and electrostatic interactions. Using this proposed new method, a very sensitive detection of caspase-3 could be achieved with a low detection limit of 0.06 pg/mL, and a new method for sensitive detection of cell apoptosis was developed. Moreover, we have successfully used this new method to detect cell apoptosis with human pulmonary carcinoma A549 cell after apoptosis inducing.
Asunto(s)
Apoptosis , Técnicas Biosensibles , Caspasa 3/aislamiento & purificación , Grafito/química , Caspasa 3/química , Oro , Humanos , Límite de Detección , Óxidos/químicaRESUMEN
As apoptosis occurs via a complex signaling cascade that is tightly regulated at multiple cell points, different methods exist to evaluate the activity of the proteins involved in the intracellular apoptotic pathways and the phenotype of apoptotic neurons. Detention of the activity of the enzyme caspase-3, the key executioner caspase in programmed cell death, by laser scanning confocal fluorescence microscopy and the fluorescence resonance energy transfer technology is an alternative approach to classical standard techniques, such as Western blotting, activity assays, or histological techniques, and allows working with both fixed and living cells. This technique combined with the organotypic culture approach ex vivo represents a valid tool for the study of the mechanisms of neuronal survival /death and neuroprotection.
Asunto(s)
Apoptosis/genética , Caspasa 3/aislamiento & purificación , Neuronas/ultraestructura , Caspasa 3/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos , Microscopía Confocal , Biología Molecular/métodos , Neuronas/metabolismoRESUMEN
Caspase-3 is an apoptotic cysteine protease and its aberrancy is highly implicated to numerous diseases thereby rendering caspase-3 activity as an important disease marker. Most caspase-3 sensors are caspase-3 substrates of which the fluorescence signals are turned on upon catalytic cleavage by active caspase-3. However, once the signal is generated, the fluorescence does not disappear albeit caspase-3 activity is abolished. Recently, we and other groups have developed the intrinsic Förster resonance energy transfer (iFRET) technique, which utilizes tryptophan residues of the target proteins and target-specific probes, as FRET donors and acceptors, respectively. Due to this principle, iFRET does not require the labeling of target proteins. In this work, we report the development of caspase-3 specific iFRET probes by structure-based design and synthesis, and the successful detection of caspase-3 in cell lysates as well as in its purified form. The limit-of-detection (LOD) of the probes in case of purified caspase-3 was found to be 1.4-1.5 nM. The designed probes did not bind to either procaspase-3 or C163S caspase-3, which are catalytic inactive, confirming that the observed iFRET signal correlates to the catalytic activity of caspase-3. Furthermore, in competition experiments with Ac-DEVD-CHO, a known competitive inhibitor of caspase-3, the iFRET signal was inhibited.
Asunto(s)
Técnicas Biosensibles , Caspasa 3/aislamiento & purificación , Transferencia Resonante de Energía de Fluorescencia , Caspasa 3/química , Catálisis , Colorantes Fluorescentes/química , Humanos , Límite de Detección , Oligopéptidos/química , Especificidad por SustratoRESUMEN
The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.
Asunto(s)
Antiinflamatorios/uso terapéutico , Ácido Glutámico/toxicidad , Ácido Glicirrínico/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Células PC12/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/aislamiento & purificación , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Citocromos c/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Morfolinas/farmacología , Células PC12/clasificación , Células PC12/citología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/aislamiento & purificación , Ratas , Proteína X Asociada a bcl-2/aislamiento & purificaciónRESUMEN
Caenorhabditis elegans genome has four genes (ced-3, csp-1, csp-2, and csp-3) encoding caspase-like proteins. Among these four proteins, CED-3 is the most well-known cell-killing caspase. Elucidation of the role of CED-3 as a central component of the apoptotic pathway in C. elegans has contributed to the understanding of the more complex apoptosis network in mammals and in other metazoa. In the highly conserved pathway of programmed cell death in C. elegans, CED-3 functions at the terminal step of this cell-killing pathway. Identification of CED-3 caspase substrates is essential for bridging the gaps between CED-3 activation and various downstream cell death execution events. If a protein is cleaved by CED-3 in vitro, this protein could be a potential CED-3 substrate in vivo. Here, we describe the method for purification of active CED-3 caspase. We will also describe in vitro assays for determining CED-3 proteolytic activity, CED-3 substrates, and CED-3 cleavage sites in the substrates.
Asunto(s)
Caspasa 3/genética , Caspasa 3/aislamiento & purificación , Biología Molecular/métodos , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Caenorhabditis elegans/enzimología , Caspasa 3/biosíntesisRESUMEN
Caspases are a highly specialized class of cell death proteases. Since they are synthesized as inactive full-length zymogens, activation--at least of effector caspases and to some extent also of initiator caspases-requires a proteolytic cleavage event, generating a large and a small subunit, two of each forming the active caspase. The proteolytic cleavage event generates neo-epitopes at both the C-terminus of the large subunit and the N-terminus of the small subunit. The cleaved Caspase-3 (CC3) antibody was raised against the neo-epitope of the large subunit and thus detects only cleaved, but not full-length, Caspase-3. Although raised against human cleaved Caspase-3, the CC3 antibody cross-reacts in other species and detects cleaved caspases, most notably DrICE and Dcp-1, in Drosophila. This protocol describes the procedure for use of the CC3 antibody to detect caspase activity in larval imaginal discs in Drosophila.
Asunto(s)
Apoptosis/genética , Caspasa 3/aislamiento & purificación , Biología Molecular/métodos , Animales , Caspasa 3/genética , Drosophila melanogaster/enzimología , Humanos , Discos Imaginales/enzimología , Larva/enzimologíaRESUMEN
We describe here a simple fluorometric assay for the highly sensitive detection of caspase-3 activities on the basis of the inner-filter effect of gold nanoparticles (AuNPs) on CdTe quantum dots (QDs). The method takes advantage of the high molar absorptivity of the plasmon band of gold nanoparticles as well as the large absorption band shift from 520 to 680 nm upon nanoparticle aggregation. When labeled with a peptide possessing the caspase-3 cleavage sequence (DEVD), the monodispersed Au-Ps (peptide-modified AuNPs) exhibited a tendency to aggregate when exposed to caspase-3, which induced the absorption band transition from 520 to 680 nm and turned on the fluorescence of the CdTe QDs for caspase-3 sensing. Under optimum conditions, a high sensitivity towards caspase-3 was achieved with a detection limit as low as 18 pM, which was much lower than the corresponding assays based on absorbance or other approaches. Overall, we demonstrated a facile and sensitive approach for caspase-3 detection, and we expected that this method could be potentially generalized to design more fluorescent assays for sensing other bioactive entities.
Asunto(s)
Técnicas Biosensibles , Caspasa 3/aislamiento & purificación , Oro/química , Nanopartículas del Metal/química , Absorción , Compuestos de Cadmio/química , Caspasa 3/química , Humanos , Puntos Cuánticos/química , Telurio/químicaRESUMEN
Caspases are required for essential biological functions, most notably apoptosis and pyroptosis, but also cytokine production, cell proliferation, and differentiation. One of the most well studied members of this cysteine protease family includes executioner caspase-3, which plays a central role in cell apoptosis and differentiation. Unfortunately, there exists a dearth of chemical tools to selectively monitor caspase-3 activity under complex cellular and in vivo conditions due to its close homology with executioner caspase-7. Commercially available activity-based probes and substrates rely on the canonical DEVD tetrapeptide sequence, which both caspases-3 and -7 recognize with similar affinity, and thus the individual contributions of caspase-3 and/or -7 toward important cellular processes are irresolvable. Here, we analyzed a variety of permutations of the DEVD peptide sequence in order to discover peptides with biased activity and recognition of caspase-3 versus caspases-6, -7, -8, and -9. Through this study, we identify fluorescent and biotinylated probes capable of selective detection of caspase-3 using key unnatural amino acids. Likewise, we determined the X-ray crystal structures of caspases-3, -7, and -8 in complex with our lead peptide inhibitor to elucidate the binding mechanism and active site interactions that promote the selective recognition of caspase-3 over other highly homologous caspase family members.
Asunto(s)
Caspasa 3/química , Caspasa 7/química , Inhibidores Enzimáticos/química , Sondas Moleculares/química , Secuencia de Aminoácidos/genética , Sustitución de Aminoácidos , Biotinilación , Caspasa 3/genética , Caspasa 3/aislamiento & purificación , Caspasa 7/genética , Caspasa 7/aislamiento & purificación , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Células HL-60 , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Péptidos/química , Péptidos/genética , Especificidad por SustratoRESUMEN
Early response prediction is considered an essential tool to obtain a more customized anticancer treatment because it allows for the identification of patients who will benefit most from a particular therapy and prevents the exposure of those patients to toxic, non-effective regimens. Recent discoveries of novel markers in functional imaging have created exciting opportunities for in vivo visualization and quantification of cell death. This review will focus on in vivo apoptosis imaging with various radiotracers as predictive tools for tumor response after anticancer therapy. Particular focus will be on annexin V imaging, a technique with the largest clinical experience to date. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".
Asunto(s)
Anexina A5 , Apoptosis/efectos de los fármacos , Imagen Molecular/métodos , Neoplasias/tratamiento farmacológico , Anexina A5/análisis , Anexina A5/química , Antineoplásicos/administración & dosificación , Caspasa 3/aislamiento & purificación , Humanos , Neoplasias/fisiopatología , Nitrilos/análisis , Nitrilos/química , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven ß subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin ß-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.
Asunto(s)
Péptido Hidrolasas/aislamiento & purificación , Populus/enzimología , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Xilema/enzimología , Apoptosis , Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Caspasa 3/aislamiento & purificación , Caspasa 3/metabolismo , Diferenciación Celular , Pared Celular/metabolismo , Lactonas/farmacología , Oligopéptidos/farmacología , Péptido Hidrolasas/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Tallos de la Planta/citología , Tallos de la Planta/efectos de los fármacos , Tallos de la Planta/enzimología , Tallos de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Populus/citología , Populus/efectos de los fármacos , Populus/crecimiento & desarrollo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Plantones/citología , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/crecimiento & desarrollo , Xilema/citología , Xilema/crecimiento & desarrolloRESUMEN
Caspase 3c (MrCasp3c) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrCasp3c consisted of 2080 bp nucleotide encoded 521 polypeptide with an estimated molecular mass of 59 kDa. MrCasp3c sequence contains caspase family p20 domain profile and caspase family p10 domain profile at 236-367 and 378-468 respectively. The quantitative real time PCR analysis revealed a broad expression of MrCasp3c with the highest expression in haemocyte and the lowest in stomach. The expression of MrCasp3c after challenge with the infectious hypodermal and haematopoietic necrosis virus (IHHNV) was tested in haemocyte. In addition, MrCasp3c was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The enzyme activity of MrCasp3c was also found to be up-regulated by IHHNV in haemocyte and hepatopancreas tissues. This study suggested that MrCasp3c may be an effector caspase associated with the induction of apoptosis which is potentially involved in the immune defence of M. rosenbergii.
Asunto(s)
Caspasa 3/genética , Caspasa 3/metabolismo , Densovirinae/fisiología , Regulación Enzimológica de la Expresión Génica , Palaemonidae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caspasa 3/inmunología , Caspasa 3/aislamiento & purificación , Densovirinae/inmunología , Perfilación de la Expresión Génica , Hepatopáncreas/enzimología , Datos de Secuencia Molecular , Palaemonidae/clasificación , Palaemonidae/enzimología , Palaemonidae/genética , Palaemonidae/virología , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentación , Apoptosis , Bioensayo/métodos , Caspasa 3/química , Caspasa 3/aislamiento & purificación , Descubrimiento de Drogas , Electrodos , Diseño de Equipo , Células HeLa , Humanos , Dispositivos Laboratorio en un ChipRESUMEN
During stress-induced apoptosis, the initiator caspase-9 is activated by the Apaf-1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase-9 is paradoxically observed outside the complex. We show herein that apoptosome-mediated cleavage of procaspase-9 occurs exclusively through a CARD-displacement mechanism, so that unlike the effector procaspase-3, procaspase-9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase-9 possessed higher affinity for the apoptosome and could displace the processed caspase-9 from the complex, thereby facilitating a continuous cycle of procaspase-9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase-9 per se contributed little to the activation of procaspase-3. Thus, the Apaf-1 apoptosome functions as a proteolytic-based 'molecular timer', wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which the processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer 'ticks' over.
Asunto(s)
Apoptosomas/metabolismo , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Caspasa 9/metabolismo , Animales , Apoptosis , Apoptosomas/genética , Apoptosomas/aislamiento & purificación , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/aislamiento & purificación , Caspasa 3/genética , Caspasa 3/aislamiento & purificación , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/aislamiento & purificación , Línea Celular , Clonación Molecular , Activación Enzimática , Humanos , Ratones , MutaciónRESUMEN
Apoptosis, or programmed cell death, is an essential process affecting homeostasis of cell growth, development, and the elimination of damaged or dangerous cells. Inappropriate cell death caused by oxidative stress has been implicated in the development of neurodegenerative diseases such as Alzheimer's, Parkinson's, and stroke. On the other hand, a defect in the cell death process leads to the development of cancer. For example, the main player of apoptosis, p53, is defective in many of the human cancers. Apoptosis is regulated by the interplay of pro-apoptotic and anti-apoptotic proteins from the Bcl-2 family and caspases. In particular, specific modulators of the activity of Caspase 3 could be very important for the development of therapies for diseases such as neurodegeneration and cancer. In this study, two V(H)Hs specific to Caspase 3 (VhhCasp31 and VhhCasp32) were isolated from a heavy chain antibody variable domain (V(H)H) phage display library and tested for their apoptosis-modulating effects. While VhhCasp31 was found to be antagonistic towards Caspase 3, VhhCasp32 was agonistic. Furthermore, when expressed as intrabodies in SHSY-5Y neuroblastoma cells, VhhCasp31 rendered cells resistant to oxidative-stress-induced apoptosis, whereas VhhCasp32 resulted in apoptosis. These V(H)H antagonist and agonist of apoptosis could have potential for the development of therapeutics for neurodegenerative diseases and cancer, respectively.