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1.
PLoS Biol ; 17(9): e3000451, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31525189

RESUMEN

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1-/- mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity.


Asunto(s)
Encefalomielitis Autoinmune Experimental/etiología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Adulto , Animales , Astrocitos/fisiología , Estudios de Casos y Controles , Sistema Nervioso Central/patología , Codón sin Sentido , Enfermedades Desmielinizantes , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Inmunidad Innata , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Mutación Missense , Adulto Joven
2.
Biochem J ; 478(13): 2681-2696, 2021 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-34156061

RESUMEN

Apoptosis is a regulated form of cell death essential to the removal of unwanted cells. At its core, a family of cysteine peptidases named caspases cleave key proteins allowing cell death to occur. To do so, each caspase catalytic pocket recognizes preferred amino acid sequences resulting in proteolysis, but some also use exosites to select and cleave important proteins efficaciously. Such exosites have been found in a few caspases, notably caspase-7 that has a lysine patch (K38KKK) that binds RNA, which acts as a bridge to RNA-binding proteins favoring proximity between the peptidase and its substrates resulting in swifter cleavage. Although caspase-7 interaction with RNA has been identified, in-depth characterization of this interaction is lacking. In this study, using in vitro cleavage assays, we determine that RNA concentration and length affect the cleavage of RNA-binding proteins. Additionally, using binding assays and RNA sequencing, we found that caspase-7 binds RNA molecules regardless of their type, sequence, or structure. Moreover, we demonstrate that the N-terminal peptide of caspase-7 reduces the affinity of the peptidase for RNA, which translates into slower cleavages of RNA-binding proteins. Finally, employing engineered heterodimers, we show that a caspase-7 dimer can use both exosites simultaneously to increase its affinity to RNA because a heterodimer with only one exosite has reduced affinity for RNA and cleavage efficacy. These findings shed light on a mechanism that furthers substrate recognition by caspases and provides potential insight into its regulation during apoptosis.


Asunto(s)
Apoptosis , Caspasa 7/metabolismo , Lisina/metabolismo , ARN/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/química , Caspasa 7/genética , Células HCT116 , Células HEK293 , Humanos , Immunoblotting , Lisina/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Unión Proteica , Multimerización de Proteína , Proteolisis , ARN/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato
3.
Proc Natl Acad Sci U S A ; 116(43): 21521-21528, 2019 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-31586028

RESUMEN

To achieve swift cell demise during apoptosis, caspases cleave essential proteins for cell survival and removal. In addition to the binding of preferred amino acid sequences to its substrate-binding pocket, caspase-7 also uses exosites to select specific substrates. 4 lysine residues (K38KKK) located in the N-terminal domain of caspase-7 form such an exosite and promote the rapid proteolysis of the poly(ADP-ribose) polymerase 1 (PARP-1), but the mechanism of recognition remains mostly unknown. In this study, we show that the overall positive charge of the exosite is the critical feature of this evolutionarily conserved binding site. Additionally, interaction with the caspase-7 exosite involves both the Zn3 and BRCT domains of PARP-1 and is mediated by RNA. Indeed, PARP-1 proteolysis efficacy is sensitive to RNase A and promoted by added RNA. Moreover, using affinity chromatography and gel shift assays, we demonstrate that caspase-7, but not caspase-3 or a caspase-7 with a mutated exosite, binds nucleic acids. Finally, we show that caspase-7 prefers RNA-binding proteins (RNA-BPs) as substrates compared to caspase-3 and that RNA enhances proteolysis by caspase-7 of many of these RNA-BPs. Thus, we have uncovered an unusual way by which caspase-7 selects and cleaves specific substrates.


Asunto(s)
Caspasa 7/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencias de Aminoácidos , Animales , Caspasa 7/química , Caspasa 7/genética , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasa-1/química , Poli(ADP-Ribosa) Polimerasa-1/genética , Unión Proteica , Dominios Proteicos , Proteolisis , ARN/genética , Proteínas de Unión al ARN/genética , Especificidad por Sustrato
4.
Biochemistry ; 56(38): 5099-5111, 2017 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-28863261

RESUMEN

Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, caspase-7 is more proficient at cleaving poly(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure-activity relationship analyses using mutagenesis of the caspase-7 NTD and kinetics show that residues 36-45 of caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD142↓G), which is far from the optimal sequence, we tested all residues at that position and found notable differences in the preference of caspase-7 and magnitude of differences between residues compared to the results of studies that have used small peptidic substrate libraries. Finally, bioinformatics shows that the regions we identified in caspase-7 and p23 are intrinsically disordered regions that contain molecular recognition features that permit a transient interaction between these two proteins. In summary, we characterized the binding mode for a caspase that is tailored to the specific recognition and cleavage of a substrate, highlighting the importance of studying the peptidase-substrate pair to understand the modalities of substrate recognition by caspases.


Asunto(s)
Caspasa 7/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Caspasa 7/genética , Dicroismo Circular , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Cinética , Chaperonas Moleculares/genética , Mutación , Fosfoproteínas/genética , Prostaglandina-E Sintasas , Dominios Proteicos , Relación Estructura-Actividad
5.
Pharmacol Res ; 104: 176-85, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26707030

RESUMEN

Advanced glycation end-products (AGE) and the receptor for AGE (RAGE) have been linked to numerous diabetic vascular complications. RAGE activation promotes a self-sustaining state of chronic inflammation and has been shown to induce apoptosis in various cell types. Although previous studies in vascular smooth muscle cells (VSMC) showed that RAGE activation increases vascular calcification and interferes with their contractile phenotype, little is known on the potential of RAGE to induce apoptosis in VSMC. Using a combination of apoptotic assays, we showed that RAGE stimulation with its ligand CML-HSA promotes apoptosis of VSMC. The formation of stress granules and the increase in the level of the associated protein HuR point toward RAGE-dependent endoplasmic reticulum (ER) stress, which is proposed as a key contributor of RAGE-induced apoptosis in VSMC as it has been shown to promote cell death via numerous mechanisms, including up-regulation of caspase-9. Chronic NF-κB activation and modulation of Bcl-2 homologs are also suspected to contribute to RAGE-dependent apoptosis in VSMC. With the goal of reducing RAGE signaling and its detrimental impact on VSMC, we designed a RAGE antagonist (iRAGE) derived from the primary amino acid sequence of HSA. The resulting CML peptide was selected for the high glycation frequency of the primary sequence in the native protein in vivo. Pretreatment with iRAGE blocked 69.6% of the increase in NF-κB signaling caused by RAGE activation with CML-HSA after 48h. Preincubation with iRAGE was successful in reducing RAGE-induced apoptosis, as seen through enhanced cell survival by SPR and reduced PARP cleavage. Activation of executioner caspases was 63.5% lower in cells treated with iRAGE before stimulation with CML-HSA. To our knowledge, iRAGE is the first antagonist shown to block AGE-RAGE interaction and we propose the molecule as an initial candidate for drug discovery.


Asunto(s)
Lisina/análogos & derivados , Péptidos/farmacología , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Albúmina Sérica/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Productos Finales de Glicación Avanzada/metabolismo , Lisina/química , Lisina/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Albúmina Sérica/química
6.
Biogerontology ; 17(5-6): 817-828, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27465500

RESUMEN

Death-associated protein 6 (DAXX) is a ubiquitous protein implicated in various cellular processes such as apoptosis, tumorigenesis, development and transcription. The role of DAXX is however ambiguous and many contradictory results regarding its function in apoptosis upon various cellular stresses are described in the literature. In order to have a better understanding of the role of DAXX throughout the entire organism under physiological stress conditions, we have characterized the mRNA levels, protein expression and the proteolytic processing of DAXX in the normal aging process in peripheral organs and brain regions in C57BL/6 male mice. Overall, Daxx mRNA expression decreases with aging in the liver, kidney, heart, cortex and cerebellum. In contrast, an increase is observed in the striatum. The protein expression of DAXX and of its proteolytic fragments increases with aging in the kidney, heart and cortex. In liver and spleen, no changes are observed while in the striatum and cerebellum, certain forms increase and others decrease with age, suggesting that the functions of DAXX may be cell type dependent. This study provides important details regarding the expression and post-translational modifications of DAXX in aging in the entire organism and provides reference data for the deregulation observed in age-associated diseases.


Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Vísceras/metabolismo , Animales , Proteínas Co-Represoras , Masculino , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares , Especificidad de Órganos/fisiología
7.
Proc Natl Acad Sci U S A ; 109(15): 5669-74, 2012 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-22451931

RESUMEN

During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K(38)KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7's N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis.


Asunto(s)
Caspasa 7/química , Caspasa 7/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteolisis , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Apoptosis , Caspasa 3/metabolismo , Línea Celular , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Prostaglandina-E Sintasas , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato
8.
Biosci Rep ; 44(6)2024 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-38836326

RESUMEN

Cumulative research findings support the idea that endocytic trafficking is crucial in regulating receptor signaling and associated diseases. Specifically, strong evidence points to the involvement of sorting nexins (SNXs), particularly SNX1 and SNX2, in the signaling and trafficking of the receptor tyrosine kinase (RTK) MET in colorectal cancer (CRC). Activation of hepatocyte growth factor (HGF) receptor MET is a key driver of CRC progression. In the present study, we utilized human HCT116 CRC cells with SNX1 and SNX2 genes knocked out to demonstrate that their absence leads to a delay in MET entering early endosomes. This delay results in increased phosphorylation of both MET and AKT upon HGF stimulation, while ERK1/2 (extracellular signal-regulated kinases 1 and 2) phosphorylation remains unaffected. Despite these changes, HGF-induced cell proliferation, scattering, and migration remain similar between the parental and the SNX1/2 knockout cells. However, in the absence of SNX1 and SNX2, these cells exhibit increased resistance to TRAIL-induced apoptosis. This research underscores the intricate relationship between intracellular trafficking, receptor signaling, and cellular responses and demonstrates for the first time that the modulation of MET trafficking by SNX1 and SNX2 is critical for receptor signaling that may exacerbate the disease.


Asunto(s)
Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales , Factor de Crecimiento de Hepatocito , Proteínas Proto-Oncogénicas c-met , Nexinas de Clasificación , Humanos , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Células HCT116 , Factor de Crecimiento de Hepatocito/metabolismo , Transducción de Señal , Fosforilación , Endosomas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transporte de Proteínas
9.
Sci Transl Med ; 16(739): eabn8529, 2024 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-38507466

RESUMEN

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2-related MD, and collagen VI-related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13-amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.


Asunto(s)
Distrofia Muscular de Duchenne , Nicho de Células Madre , Ratones , Animales , Apelina/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Transducción de Señal
10.
Biol Chem ; 399(12): 1351, 2018 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-30352023
11.
J Cell Biochem ; 113(8): 2775-84, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22473799

RESUMEN

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.


Asunto(s)
Caspasas/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Calcio/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular , Endopeptidasas/metabolismo , Células HeLa , Humanos , Ratas
12.
Apoptosis ; 17(8): 916-25, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22644572

RESUMEN

Apoptosis can be routinely characterized using biomolecular markers such as in the TUNEL and the annexin V assays or by using fluorescent caspase substrates. Apoptosis can also be semi-quantitatively characterized using microscopy, which targets morphological features such as cell rounding, nuclear condensation and fragmentation as well as cell membrane blebbing. This label-free approach provides a limited resolution for the evolution of these events in time and relies heavily on subjective identification of the morphological features. Here we propose a label-free assay based on surface plasmon resonance (SPR) detection of minute morphology changes occurring as a result of apoptosis induction in an endothelial cell model (EA.hy926). At first, annexin V assays confirmed that our cellular model was responsive to TRAIL over a 12-hour period. Then, we show that SPR allows accurate monitoring of apoptosis by measuring (1) the duration of the latency period during which the apoptotic signal is integrated by the initiator caspases and transmitted to the executioner caspases, (2) the rate of the execution phase in which death substrates are cleaved and morphological changes occur, and (3) the total extent of apoptosis. Using these parameters, we characterized the responses obtained with TRAIL (EA.hy926, HeLa, AD-293) and the anti-Fas antibody (HeLa) for the extrinsic pathways and UV exposure (HeLa) for the intrinsic pathways. By comparing the SPR time-course of apoptosis with phase contrast micrographs, we demonstrate that the cell morphological hallmarks of apoptosis are the major contributors to the SPR signal. Altogether, our results validate the use of SPR as an accurate label-free assay for the real-time monitoring of apoptosis-triggered cell morphological changes.


Asunto(s)
Apoptosis , Forma de la Célula , Resonancia por Plasmón de Superficie , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Células Cultivadas , Humanos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología
13.
J Biol Chem ; 285(22): 16632-42, 2010 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-20308068

RESUMEN

Caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.


Asunto(s)
Caspasa 8/metabolismo , Apoptosis , Sitios de Unión , Caspasas/metabolismo , Dominio Catalítico , Línea Celular , Dimerización , Endopeptidasas/metabolismo , Activación Enzimática , Células HeLa , Humanos , Células Jurkat , Modelos Biológicos , Mutación , Estructura Terciaria de Proteína
14.
Cell Physiol Biochem ; 26(4-5): 629-40, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-21063100

RESUMEN

BACKGROUND/AIMS: The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). Three isoforms of IP(3)R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP(3)R signalling is involved in the activation of the Ca(2+)-sensitive transcription factors NFAT and CREB. METHODS: Each IP(3)R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca(2+) was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. RESULTS: IP(3)R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca(2+) response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP(3)R-2-knocked down HEK 293A cells. Overexpression of IP(3)R-1 could fully compensate for IP(3)R-2 knock down to mobilize Ca(2+) and to activate NFAT. CONCLUSION: Our results show that the knock down of IP(3)R-2 significantly reduced the intracellular Ca(2+) response of HEK 293 cells. This reduced Ca(2+) response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca(2+) signals required for the activation of NFAT are stronger than those required for the activation of CREB.


Asunto(s)
Calcio/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Factores de Transcripción NFATC/metabolismo , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Interferencia de ARN , ARN Interferente Pequeño
15.
Methods Mol Biol ; 414: 191-220, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18175821

RESUMEN

Caspases are central to the execution of apoptosis. Their proteolytic activity is responsible for the demise of cells in many physiological and pathological states. Great advances in understanding caspases have been made using recombinant caspase expression and enzymatic characterization. Assays to measure caspase activity in apoptotic cell extracts and the development of a reconstituted cell-free assay were also critical in establishing the hierarchy in the caspase activation cascade and comprehend how caspase-9 is activated by the apoptosome. More recently, new tools such as activity-based probes allowed us to detect caspase activation in their working environment providing readout of the system with minimal interference. This chapter describes some of the methods used by our group to study the activation mechanisms of caspases and their activity.


Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Extractos Celulares/análisis , Activación Enzimática , Humanos , Immunoblotting , Proteínas Recombinantes/metabolismo , Coloración y Etiquetado , Estreptavidina/análisis
16.
Biochem J ; 405(1): 11-9, 2007 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-17437405

RESUMEN

During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp(330), and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp(330) removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.


Asunto(s)
Caspasa 3/metabolismo , Caspasa 9/metabolismo , Inhibidores de Caspasas , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Secuencia de Aminoácidos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Ácido Aspártico/metabolismo , Caspasa 9/genética , Línea Celular , Inhibidores de Cisteína Proteinasa/metabolismo , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica
17.
FEBS J ; 284(10): 1394-1420, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28371378

RESUMEN

During apoptosis, caspases feast on several hundreds of cellular proteins to orchestrate rapid cellular demise. Indeed, caspases are known to get a taste of every cellular process in one way or another, activating some, but most often shutting them down. Thus, it is not surprising that caspases proteolyze proteins involved in intracellular trafficking with particularly devastating consequences for this important process. This review article focuses on how caspases target the machinery responsible for smuggling goods within and outside the cell.


Asunto(s)
Caspasas/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Transporte Biológico/fisiología , Caspasas/genética , Humanos , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteolisis
18.
Cell Death Discov ; 3: 16100, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28179995

RESUMEN

The unfolding of apoptosis involves the cleavage of hundreds of proteins by the caspase family of cysteinyl peptidases. Among those substrates are proteins involved in intracellular vesicle trafficking with a net outcome of shutting down the crucial processes governing protein transport to organelles and to the plasma membrane. However, because of the intertwining of receptor trafficking and signaling, cleavage of specific proteins may lead to unintended consequences. Here we show that in apoptosis, sorting nexin 1 and 2 (SNX1 and SNX2), two proteins involved in endosomal sorting, are cleaved by initiator caspases and also by executioner caspase-6 in the case of SNX2. Moreover, SNX1 is cleaved at multiple sites, including following glutamate residues. Cleavage of SNX2 results in a loss of association with the endosome-to-trans-Golgi network transport protein Vps35 and in a delocalization from endosomes of its associated partner Vps26. We also demonstrate that SNX2 depletion causes an increase in hepatocyte growth factor receptor tyrosine phosphorylation and Erk1/2 signaling in cells. Finally, we show that SNX2 mRNA and protein levels are decreased in colorectal carcinoma and that lower SNX2 gene expression correlates with an increase in cancer patient mortality. Our study reveals the importance to characterize the cleavage fragments produced by caspases of specific death substrates given their potential implication in the mechanism of regulation of physiological (signaling/trafficking) pathways or in the dysfunction leading to pathogenesis.

19.
Neurobiol Aging ; 47: 50-62, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27552481

RESUMEN

Caspases and their substrates are key mediators of apoptosis and strongly implicated in various physiological processes. As the serine/threonine kinase family is involved in apoptosis and serine/threonine kinase 3 (STK3) is a recently identified caspase-6 substrate, we assessed the expression and cleavage of STK3 in murine peripheral organs and brain regions during the aging process. We also assessed caspase-3, -6, -7, and -8 expression and activity in order to delineate potential mechanism(s) underlying the generation of the STK3 fragments observed and their relation to the apoptotic pathway. We demonstrate for the first time the cleavage of STK3 by caspase-7 and show that STK3 protein levels globally increase throughout the organism with age. In contrast, caspase-3, -6, -7, and -8 expression and activity vary significantly among the different organs analyzed suggesting differential effects of aging on the apoptotic mechanism and/or nonapoptotic functions of caspases throughout the organism. These results further our understanding of the role of caspases and their substrates in the normal aging process and highlight a potential role for STK3 in neurodegeneration.


Asunto(s)
Envejecimiento/genética , Envejecimiento/metabolismo , Apoptosis/genética , Caspasas/genética , Caspasas/metabolismo , Expresión Génica/genética , Especificidad de Órganos/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Animales , Encéfalo/metabolismo , Caspasas/fisiología , Masculino , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/genética , Serina-Treonina Quinasa 3
20.
Biochem J ; 382(Pt 2): 651-7, 2004 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-15209560

RESUMEN

The first step in caspase activation is transition of the latent zymogen to an active form. For the initiator caspases, this occurs through dimerization of monomeric zymogens at an activating complex. Recent studies have suggested that FLIP(L) [FLICE-like inhibitory protein, long form; FLICE is FADD (Fas-associated death domain protein)-like interleukin-1beta-converting enzyme], previously thought to act solely as an inhibitor of caspase-8 activation, can under certain circumstances function to enhance caspase activation. Using an in vitro induced-proximity assay, we demonstrate that activation of caspases-8 and -10 occurs independently of cleavage of either the caspase or FLIP(L). FLIP(L) activates caspase-8 by forming heterodimeric enzyme molecules with substrate specificity and catalytic activity indistinguishable from those of caspase-8 homodimers. Significantly, the barrier for heterodimer formation is lower than that for homodimer formation, suggesting that FLIP(L) is a more potent activator of caspase-8 than is caspase-8 itself.


Asunto(s)
Caspasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Caspasa 10 , Caspasa 8 , Línea Celular Tumoral , Dimerización , Activación Enzimática/fisiología , Precursores Enzimáticos/metabolismo , Humanos , Hidrólisis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat/enzimología
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