Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 59
Filtrar
1.
Colloids Surf B Biointerfaces ; 221: 113028, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36410190

RESUMEN

In this study, a nanoparticle-based sandwich-like immunoassay was designed in dispersion medium to precisely detect apoptosis over caspase antibodies in order to overcome the disadvantages of traditional apoptosis determination methods such as high cost, large sampling requirement, and appropriate laboratory and equipment conditions. For this purpose, a complementary particulate system including magnetic (MNPs) and upconversion silica (UC-SiNPs) nanoparticles while immobilizing antibodies (primary antibody to MNPs, secondary antibody to UC-SiNPs) were synthesized and characterized. Optimization and selectivity studies of the complex formed by primary antibody immobilized MNPs with standard caspase proteins were examined by the HPLC system. Within the scope of optimization studies, protein concentrations, optimal duration, and temperature parameters were evaluated. Optimal conditions were determined for pH, initial concentration, time, and temperature as 7.4, 5.6 µg/mL, 45 min, and room temperature, respectively. Furthermore, the adsorption of competitive proteins was investigated in selectivity studies as well. Moreover, the primary antibody immobilized MNPs were treated with standard caspase proteins under optimal conditions; subsequently, they were interacted with secondary antibody immobilized UC-SiNPs to demonstrate the supracomplex formation meanwhile zeta potential/size measurements and fluorescence emission spectrometry analyses were performed. As a result of these analyses, it was observed that the sandwich-like supracomplexes were successfully formed that significantly varied upconversion emission intensities of UC-SiNPs in dependence on the amounts of caspase proteins. Because this approach enabled a quantitative result, the nanoparticle-based sandwich-like immunoassay should be classified as an easy-to-handled, fast, and promising alternative to benchmark apoptosis assays.


Asunto(s)
Caspasas , Anticuerpos , Caspasas/aislamiento & purificación , Nanopartículas , Dióxido de Silicio
2.
Mol Immunol ; 132: 8-20, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33524772

RESUMEN

The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.


Asunto(s)
Apoptosis/genética , Caspasas/genética , Inmunidad Innata , Lampreas/genética , Transducción de Señal/inmunología , Animales , Apoptosis/efectos de los fármacos , Caspasa 1/química , Caspasa 1/genética , Caspasa 1/aislamiento & purificación , Caspasa 1/metabolismo , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 6/química , Caspasa 6/genética , Caspasa 6/metabolismo , Caspasa 7/química , Caspasa 7/genética , Caspasa 7/aislamiento & purificación , Caspasa 7/metabolismo , Caspasa 8/química , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/química , Caspasa 9/genética , Caspasa 9/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/química , Caspasas/aislamiento & purificación , Caspasas/metabolismo , Evolución Molecular , Duplicación de Gen , Reordenamiento Génico , Genoma , Genómica , Células HeLa , Humanos , Inmunidad Innata/genética , Lampreas/crecimiento & desarrollo , Lampreas/inmunología , Lampreas/metabolismo , Filogenia , Proteínas Recombinantes , Alineación de Secuencia , Transducción de Señal/genética , Staphylococcus aureus/efectos de los fármacos , Regulación hacia Arriba , Vibrio/efectos de los fármacos
3.
Int J Biol Macromol ; 95: 288-293, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27867055

RESUMEN

Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587µM and 4.67×103M-1min-1, respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value of doxorubicin. This is the first report of functional expression of mature phytaspase in bacterial system as well as its transfection to sensitize A549 cells at lower doxorubicin concentration.


Asunto(s)
Caspasas/genética , Caspasas/metabolismo , Nicotiana/enzimología , Células A549 , Caspasas/química , Caspasas/aislamiento & purificación , Clonación Molecular , Doxorrubicina/farmacología , Expresión Génica , Humanos , Simulación del Acoplamiento Molecular , Estructura Secundaria de Proteína , Especificidad por Sustrato , Nicotiana/genética
4.
Cell Death Dis ; 6: e1813, 2015 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-26158519

RESUMEN

The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases.


Asunto(s)
Caspasa 1/metabolismo , Caspasas/metabolismo , Inflamación/enzimología , Análisis de la Célula Individual/métodos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD , Caspasa 1/química , Caspasa 1/aislamiento & purificación , Caspasa 12/química , Caspasa 12/aislamiento & purificación , Caspasa 12/metabolismo , Caspasas/química , Caspasas/aislamiento & purificación , Caspasas Iniciadoras , Toxina del Cólera/farmacología , Proteínas del Citoesqueleto/metabolismo , Humanos , Inmunidad Innata/genética , Inflamasomas/química , Inflamasomas/metabolismo , Inflamación/patología , Macrófagos/enzimología , Imagen Molecular/métodos , Proteínas NLR , Multimerización de Proteína
5.
Methods Mol Biol ; 1280: 239-46, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25736752

RESUMEN

MALT1 (mucosa-associated lymphoid tissue protein 1) is a key regulator of antigen-induced NF-κB activation in the adaptive immune response. Activation of proteolytic activity of the MALT1 paracaspase was shown to boost the immune response. Additionally, MALT1 proteolytic activity is essential for the survival of MALT1-dependent lymphoma, such as the activated B-cell type (ABC) of diffuse large B-cell lymphoma (DLBCL) or MALT lymphoma. The functional impact of MALT1 paracaspase on T-cell activation and lymphomagenesis suggests that MALT1 is a promising therapeutic target for the treatment of autoimmune diseases and distinct lymphoma entities. To evaluate the requirement of MALT1 in further detail, direct measurement of its activity status is of great importance. We have established a fluorogenic cleavage assay which can be used to measure activity of recombinant and cellular MALT1. Here we describe the basis of the cleavage assay and include a detailed protocol for recombinant production of MALT1 and also the cellular immunoprecipitation of endogenous MALT1 to determine its proteolytic activity.


Asunto(s)
Caspasas/metabolismo , Pruebas de Enzimas , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes , Caspasas/genética , Caspasas/aislamiento & purificación , Activación Enzimática , Expresión Génica , Humanos , Inmunoprecipitación/métodos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Proteolisis
6.
Methods Mol Biol ; 1133: 3-39, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567092

RESUMEN

One of the most valuable tools that have been developed for the study of apoptosis is the availability of recombinant active caspases. The determination of caspase substrate preference, the design of sensitive substrates and potent inhibitors, the resolution of caspase structures, the elucidation of their activation mechanisms, and the identification of their substrates were made possible by the availability of sufficient amounts of enzymatically pure caspases. The current chapter describes at length the expression, purification, and basic enzymatic characterization of apoptotic caspases.


Asunto(s)
Caspasas/genética , Caspasas/aislamiento & purificación , Biología Molecular/métodos , Proteínas Recombinantes/genética , Apoptosis/genética , Inhibidores de Caspasas/química , Caspasas/química , Activación Enzimática , Humanos , Proteínas Recombinantes/química , Especificidad por Sustrato
7.
Methods Mol Biol ; 1133: 61-70, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567094

RESUMEN

Delineation of the natural substrate scope of proteases is important for understanding the functions of proteolytic pathways in physiology and disease. Herein we describe the protocol for PROTOMAP, a technique that combines SDS-PAGE with tandem mass spectrometry to globally identify shifts in protein migration indicative of proteolytic processing. When applied to cells undergoing apoptosis, this unbiased global method provides a snapshot of the topography and magnitude of proteolytic events associated with programmed cell death.


Asunto(s)
Caspasas/química , Biología Molecular/métodos , Proteómica , Apoptosis/genética , Caspasas/aislamiento & purificación , Caspasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Especificidad por Sustrato , Espectrometría de Masas en Tándem
8.
Methods Mol Biol ; 1133: 119-40, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567099

RESUMEN

The study of apoptosis and caspases has advanced greatly over recent decades. Studies conducted in the Xenopus laevis egg extract and oocyte model system have significantly contributed to these advances. Twenty years ago, Newmeyer and colleagues first showed that the X. laevis egg extract, when incubated at room temperature, reconstituted the key molecular events of cellular apoptosis including cytochrome c release, nuclear condensation, internucleosomal fragmentation, and caspase activation. The biochemical tractability of the egg extract system allows for robust study of apoptotic events and caspase activation. Its nature as a cell-free extract system allows substrates to be very simply added by pipette, and their effects on apoptosis and caspase activation and their placement in the apoptotic signaling pathway (e.g., pre- or post-mitochondrial) are subsequently very simply studied using the techniques described in this chapter. Also described in this chapter are assays that allow the study of caspase activation in intact oocytes, another valuable tool available when using the X. laevis model organism. Overall, the X. laevis egg extract/oocyte model is a robust, efficient, and biochemically tractable system that is ideal for the study of apoptosis and caspase activation.


Asunto(s)
Apoptosis/genética , Caspasas/aislamiento & purificación , Biología Molecular/métodos , Animales , Caspasas/genética , Sistema Libre de Células/enzimología , Oocitos/enzimología , Xenopus laevis
9.
Methods Mol Biol ; 1133: 141-54, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567100

RESUMEN

Members of the caspase family of proteases are evolutionarily conserved cysteine proteases that play a crucial role as the central executioners of the apoptotic pathway. Since the discovery of caspases, many methods have been developed to detect their activation and are widely used in basic and clinical studies. In a mouse tissue, caspase activation can be monitored by cleavage of caspase-specific synthetic substrates and by detecting cleaved caspase by western blot analysis of the tissue extract. In tissue sections, active caspase can be detected by immunostaining using specific antibodies to the active caspase. In addition, among the myriads of caspase-specific substrates known so far, cleaved fragments produced by caspases from the substrates such as PARP, lamin A, and cytokeratin-18 can be monitored in tissue sections by immunostaining as well as western blots of tissue extracts. In general, more than one method should be used to ascertain detection of activation of caspases in a mouse tissue.


Asunto(s)
Apoptosis/genética , Caspasas/aislamiento & purificación , Biología Molecular/métodos , Animales , Caspasas/genética , Ratones
10.
Methods Mol Biol ; 1133: 155-73, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567101

RESUMEN

The majority of caspases, cysteine-dependent aspartate-directed proteases, being in their activated state are involved in regulation of apoptosis by cleaving protein substrates harboring specific target motifs. Basically all biochemical and morphological changes in an apoptotic cell, including cell shrinkage, chromatin condensation, DNA fragmentation, and plasma membrane blebbing, are consequence of caspase-mediated proteolysis. Thus, uncovering activities of unique caspases are key determinants of the apoptotic process. This chapter describes a set of experimental protocols available for characterization, quantification and inhibition of caspase activities in mammalian cell cultures, including immunoblotting, usage of synthetic substrates, flow cytometry, and microscopic techniques.


Asunto(s)
Caspasas/aislamiento & purificación , Caspasas/metabolismo , Biología Molecular/métodos , Animales , Caspasas/genética , Línea Celular/enzimología , Activación Enzimática/genética , Citometría de Flujo , Humanos , Mamíferos/genética , Transducción de Señal
11.
Methods Mol Biol ; 1133: 177-88, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567102

RESUMEN

The paracaspase MALT1 is a Cys-dependent, Arg-specific protease that plays an essential role in the activation and proliferation of lymphocytes during the immune response. Oncogenic activation of MALT1 is associated with the development of specific forms of B-cell lymphomas. Through specific cleavage of its substrates, MALT1 controls various aspects of lymphocyte activation, including the activation of transcriptional pathways, the stabilization of mRNAs, and an increase in cellular adhesion. In lymphocytes, the activity of MALT1 is tightly controlled by its inducible monoubiquitination, which promotes the dimerization of MALT1. Here, we describe both in vitro and in vivo assays that have been developed to assess MALT1 activity.


Asunto(s)
Caspasas/genética , Caspasas/aislamiento & purificación , Biología Molecular/métodos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Caspasas/biosíntesis , Humanos , Células Jurkat , Activación de Linfocitos/genética , Linfocitos/enzimología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/metabolismo , Proteínas de Neoplasias/biosíntesis , Ubiquitinación
12.
Methods Mol Biol ; 1133: 189-202, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567103

RESUMEN

The purpose of this chapter is to give insights into metacaspase of Leishmania protozoan parasites as arginine-specific cysteine peptidase. The physiological role of metacaspase in Leishmania is still a matter of debate, whereas its peptidase enzymatic activity has been well characterized. Among the different possible expression systems, metacaspase-deficient yeast cells (Δyca1) have been instrumental in studying the activity of Leishmania major metacaspase (LmjMCA). Here, we describe techniques for purification of LmjMCA and its activity measurement, providing a platform for further identification of LmjMCA substrates.


Asunto(s)
Caspasas/aislamiento & purificación , Proteasas de Cisteína/aislamiento & purificación , Biología Molecular/métodos , Péptido Hidrolasas/aislamiento & purificación , Apoptosis/genética , Arginina/metabolismo , Caspasas/genética , Proteasas de Cisteína/genética , Leishmania major/enzimología , Péptido Hidrolasas/genética
13.
Methods Mol Biol ; 1133: 203-21, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567104

RESUMEN

Metacaspases are cysteine peptidases found in trypanosomes but absent in mammals, and despite being distantly related to the mammalian caspases they show significant disparity in their cellular and enzymatic functions. The genome of the parasitic protozoa Trypanosoma brucei (the causative agent of African sleeping sickness) encodes five metacaspases: TbMCA1-TbMCA5. Of these TbMCA2, TbMCA3, and TbMCA5 are active cysteine peptidases expressed in the bloodstream form of the parasite. To investigate the structure-function relationship of the trypanosome metacaspases and the structural basis for their divergence from the caspases, paracaspases, and other Clan CD cysteine peptidases (or vice versa), we purified and characterized TbMCA2 and determined the three-dimensional structure of an inactive mutant using X-ray crystallography. The methods presented in this chapter describe the recombinant expression of active TbMCA2 and inactive TbMCA2(C213A). The protocols produce large amounts of recombinant protein for use in structural, biochemical, and kinetic studies and include detailed information on how to produce diffraction quality crystals of TbMCA2(C213A).


Asunto(s)
Caspasas/aislamiento & purificación , Proteasas de Cisteína/aislamiento & purificación , Biología Molecular/métodos , Trypanosoma brucei brucei/enzimología , Apoptosis/genética , Caspasas/genética , Caspasas/metabolismo , Cristalografía por Rayos X , Proteasas de Cisteína/biosíntesis , Proteasas de Cisteína/genética , Humanos , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/parasitología
14.
Methods Mol Biol ; 1133: 237-53, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24567106

RESUMEN

Metacaspases are essential for cell death regulation in plants. Further understanding of biochemistry of metacaspases and their molecular function in plant biology requires a set of robust methods for detection of metacaspase activation and quantitative analysis of corresponding proteolytic activity. Here we describe methods for purification of recombinant metacaspases, measurement of enzymatic activity of recombinant and endogenous metacaspases in vitro and in cell lysates, respectively, and finally detection of metacaspase activation in vivo. Additionally, an in vitro metacaspase protein substrate cleavage assay based on the cell-free production of substrate protein followed by proteolysis with recombinant metacaspase is presented. These methods have been originally developed for type II metacaspases from Arabidopsis and Norway spruce (Picea abies), but they can be used as templates for type I metacaspases, as well as for type II metacaspases from other species.


Asunto(s)
Arabidopsis/enzimología , Caspasas/aislamiento & purificación , Biología Molecular/métodos , Caspasas/biosíntesis , Caspasas/genética , Muerte Celular/genética , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo
15.
Cytopathology ; 25(4): 250-8, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24261323

RESUMEN

OBJECTIVE: To review cytomorphological criteria and clinicopathological findings in combination with ancillary tests for the specific diagnosis of pulmonary marginal zone lymphoma (MZL) in fine needle aspiration (FNA) specimens. METHODS: Cases of pulmonary MZL diagnosed using cytological specimens from 2005 to 2012 were retrieved and reviewed by three cytopathologists. Results of immunophenotypic analysis, interphase fluorescence in situ hybridization (FISH) and molecular assays were collated, together with clinical information and imaging data. Concurrent surgical biopsies were also retrieved. RESULTS: Fifteen lung FNA specimens were identified. The smears consisted predominantly of small centrocyte-like cells. Marked plasma cell differentiation was evident in 11 cases. All cases with slides available showed tissue fragments with lymphoid tangles (TFLTs). Multinucleated giant cells were present in nine cases, two of which showed granulomas. Immunophenotyping confirmed B-cell clonality in all cases. B-cell clonality was detected by polymerase chain reaction (PCR) in two samples. FISH identified MALT1 translocation in four of 10 cases tested and trisomy 3 in three of four cases. Concurrent surgical biopsies were diagnosed independently as MZL in seven cases. CONCLUSIONS: Cytology smears from lung FNA samples consisting of small lymphoid cells with a relative abundance of plasma cells or plasmacytoid cells and large TFLTs should prompt immunophenotyping and other ancillary studies, even if multinucleated giant cells and poorly formed granulomas are also identified. Specific diagnosis of pulmonary MZL in FNA samples can be rendered on the basis of morphological features coupled with the demonstration of B-cell clonality by immunophenotyping or PCR and cytogenetic abnormalities by FISH.


Asunto(s)
Biopsia con Aguja Fina , Pulmón/patología , Linfoma de Células B de la Zona Marginal/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Caspasas/biosíntesis , Caspasas/aislamiento & purificación , Femenino , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B de la Zona Marginal/patología , Masculino , Persona de Mediana Edad , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/aislamiento & purificación
16.
Tsitologiia ; 56(6): 427-32, 2014.
Artículo en Ruso | MEDLINE | ID: mdl-25696979

RESUMEN

In this paper, we present a comparative analysis of different methods of purification of proteasomes from the culture medium in which proerithroleukemia human K562 cells were grown. The results obtained allowed us to purify proteasomes from samples of conditioned cell culture medium and control the quality of the proteasome preparations at all stages of their separation. Extracellular proteasomes purified via different approaches possess all the three types of peptidase activity described for intracellular counterparts.


Asunto(s)
Caspasas/aislamiento & purificación , Quimotripsina/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Tripsina/aislamiento & purificación , Caspasas/química , Quimotripsina/química , Medios de Cultivo Condicionados/química , Humanos , Células K562 , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Tripsina/química , Ubiquitinación
17.
Dev Neurosci ; 35(2-3): 88-101, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23445938

RESUMEN

Neuroinflammation is a complex immune response against the harmful effects of diverse stimuli within the central nervous system. Caspases are a family of intracellular cysteine proteases that mediate proteolytic events indispensable for transduction of signaling pathway-controlling biological phenomena such as apoptosis and inflammation. To date, 14 players have been identified in mammals. For many years, caspases were simply divided into 'apoptotic' and 'proinflammatory' caspases and this classification remains useful to some extent. However, increasing evidence indicates that many of these so-called apoptotic caspases also exert nonapoptotic functions. In addition, the role of certain members of the supposed inflammatory caspases in the inflammatory process per se has also been discussed. In this review, we highlight the role for 'apoptotic' and 'proinflammatory' caspases in the regulation of the inflammation response with a special focus on the central nervous system.


Asunto(s)
Apoptosis/fisiología , Caspasas/aislamiento & purificación , Sistema Nervioso Central/enzimología , Inflamación/enzimología , Animales , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Humanos , Inflamación/patología , Inflamación/fisiopatología
18.
Fungal Biol ; 117(3): 173-81, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23537874

RESUMEN

A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.


Asunto(s)
Basidiomycota/enzimología , Caspasas/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Fúngicas/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Basidiomycota/química , Basidiomycota/genética , Caspasas/genética , Caspasas/aislamiento & purificación , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/aislamiento & purificación , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Cinética , Datos de Secuencia Molecular , Alineación de Secuencia , Serina Endopeptidasas/genética , Serina Endopeptidasas/aislamiento & purificación
19.
Protein Expr Purif ; 84(2): 236-46, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22683476

RESUMEN

A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.


Asunto(s)
Caspasas/genética , Caspasas/metabolismo , Clonación Molecular/métodos , Caspasas/química , Caspasas/aislamiento & purificación , Dominio Catalítico , Cromatografía en Gel/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expresión Génica , Humanos , Cinética , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo
20.
Biochem J ; 443(1): 287-95, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22309193

RESUMEN

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB (nuclear factor κB) pathway, probably by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional-scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after arginine, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (kcat/Km values) of optimal peptidyl substrates are in the same order (10(3)-10(4) M(-1)·s(-1)) as for a putative target protein CYLD. Thus MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.


Asunto(s)
Caspasas/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Secuencia de Aminoácidos , Caspasas/química , Caspasas/aislamiento & purificación , Cromatografía de Afinidad , Citratos/química , Activación Enzimática , Activadores de Enzimas/química , Escherichia coli , Células HEK293 , Humanos , Cinética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/química , Proteínas de Neoplasias/aislamiento & purificación , Oligopéptidos/química , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Citrato de Sodio , Especificidad por Sustrato
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA