RESUMEN
The spindle assembly checkpoint (SAC) is a surveillance system that preserves genome integrity by delaying anaphase onset until all chromosomes are correctly attached to spindle microtubules. Recruitment of SAC proteins to unattached kinetochores generates an inhibitory signal that prolongs mitotic duration. Chordate embryos are atypical in that spindle defects do not delay mitotic progression during early development, implying that either the SAC is inactive or the cell-cycle target machinery is unresponsive. Here, we show that in embryos of the chordate Phallusia mammillata, the SAC delays mitotic progression from the 8th cleavage divisions. Unattached kinetochores are not recognized by the SAC machinery until the 7th cell cycle, when the SAC is acquired. After acquisition, SAC strength, which manifests as the degree of mitotic lengthening induced by spindle perturbations, is specific to different cell types and is modulated by cell size, showing similarity to SAC control in early Caenorhabditis elegans embryos. We conclude that SAC acquisition is a process that is likely specific to chordate embryos, while modulation of SAC efficiency in SAC proficient stages depends on cell fate and cell size, which is similar to non-chordate embryos.
Asunto(s)
Puntos de Control de la Fase M del Ciclo Celular , Huso Acromático , Animales , Huso Acromático/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Caenorhabditis elegans/metabolismo , Tamaño de la Célula , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
Ostreopsis cf. ovata is a benthic dinoflagellate very common in tropical and temperate coastal areas, particularly in the Mediterranean Sea. This species is also found in the plankton, i.e. swimming in the water column or in aggregates floating at the sea surface. The potential links between the planktonic and benthic populations influencing their relative distribution in the water column and attached to the benthic substrate are poorly understood. To shed light on this question, a high-frequency temporal monitoring was conducted in the Villefranche bay (France) to determine the abundance of (1) epibenthic cells attached to macroalgae, (2) planktonic cells in the water column and (3) cells in aggregates floating at the sea water surface (hereafter, referred to sea surface cells) . This monitoring was realized over 3 consecutive years (2018, 2019 and 2020) and at different phases of the bloom (exponential phase - 2020, peak - 2019 and decline phase - 2018). Strong variations in benthic and planktonic O. cf. ovata abundances were observed over the 24 h sampling cycles conducted in three consecutive years. The three populations, planktonic, benthic and sea surface cells, exhibited the highest numbers during the day (light) hours and lowest values at night in 2018 and 2019. In 2020, however, benthic abundances did not differ significantly between light and dark periods. Moreover, epibenthic cells abundances peaked in the morning, followed by the peak of the cells in the plankton and in the surface aggregates during the afternoon. Monitoring of O. cf. ovata is often based on a single sampling per day without precise indications of sampling time and shows great variability in O. cf. ovata abundances. Our observations of daily variations in cell abundances along the water column clearly indicate that time and water column depth of sampling constitute a great source of variability and have to be considered when designing new monitoring strategies to reduce variability and to harmonize data acquisition and international comparisons.
Asunto(s)
Dinoflagelados , Francia , Mar Mediterráneo , Plancton , Agua de MarRESUMEN
In eukaryotic cells, a spindle assembly checkpoint (SAC) ensures accurate chromosome segregation, by monitoring proper attachment of chromosomes to spindle microtubules and delaying mitotic progression if connections are erroneous or absent. The SAC is thought to be relaxed during early embryonic development. Here, we evaluate the checkpoint response to lack of kinetochore-spindle microtubule interactions in early embryos of diverse animal species. Our analysis shows that there are two classes of embryos, either proficient or deficient for SAC activation during cleavage. Sea urchins, mussels, and jellyfish embryos show a prolonged delay in mitotic progression in the absence of spindle microtubules from the first cleavage division, while ascidian and amphioxus embryos, like those of Xenopus and zebrafish, continue mitotic cycling without delay. SAC competence during early development shows no correlation with cell size, chromosome number, or kinetochore to cell volume ratio. We show that SAC proteins Mad1, Mad2, and Mps1 lack the ability to recognize unattached kinetochores in ascidian embryos, indicating that SAC signaling is not diluted but rather actively silenced during early chordate development.
Asunto(s)
Invertebrados/embriología , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Huso Acromático/metabolismo , Animales , Puntos de Control del Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Embrión no Mamífero/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Nocodazol/farmacología , Transducción de Señal/fisiologíaRESUMEN
The mammalian zygote is a totipotent cell that generates all the cells of a new organism through embryonic development. However, if one asks about the totipotency of blastomeres after one or two zygotic divisions, opinions differ. As it is impossible to determine the individual developmental potency of early blastomeres in an intact embryo, experiments of blastomere isolation were conducted in various species, showing that two-cell blastomeres could give rise to a new organism when sister cells were separated. A mainstream interpretation was that each of the sister mammalian blastomeres was equally totipotent. However, reevaluation of those experiments raised some doubts about the real prevalence of cases in which this interpretation could truly be validated. We compiled experiments that tested the individual developmental potency of early mammalian blastomeres in a cell-autonomous way (i.e. excluding nuclear transfer and chimera production). We then confronted the developmental abilities with reported molecular differences between sister blastomeres. The reevaluated observations were at odds with the mainstream view: A viable two-cell embryo can already include one non-totipotent blastomere. We were, thus, led to propose a revised model for totipotency continuity based on the construction of the zygote as a mosaic, which accounts for differential inheritance of totipotency-relevant components between sister blastomeres. This takes place with no preordained mechanisms that would ensure a reproducible partition. This model, which is compatible with the body of data on regulative properties of mammalian early embryos, aims at tempering the rigid interpretation that discounted maternal constraints on totipotency.
Asunto(s)
Blastómeros/fisiología , Modelos Biológicos , Cigoto/fisiología , Animales , Humanos , MosaicismoRESUMEN
Early embryos develop from fertilized eggs using materials that are stored during oocyte growth and which can be defined as maternal contribution (molecules, factors, or determinants). Several heat shock proteins (HSPs) and the heat shock transcriptional factor (HSF) are part of the maternal contribution that is critical for successful embryogenesis and reproduction. A maternal role for heat shock-related genes was mainly demonstrated in genetic experimental organisms (e.g., fly, nematode, mouse). Nowadays, an increasing number of "omics" data are produced from a large panel of organisms implementing a catalog of maternal and/or embryonic HSPs and HSFs. However, for most of them, it remains to better understand their potential roles in this context. Existing and future genome-wide screens mainly set up to create loss-of-function are likely to improve this situation. This chapter will discuss available data from various experimental organisms following the developmental steps from egg production to early embryogenesis.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas de Choque Térmico/genética , Oogénesis/genética , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Cigoto/metabolismoRESUMEN
Mutations of HSPB5 (also known as CRYAB or αB-crystallin), a bona fide heat shock protein and molecular chaperone encoded by the HSPB5 (crystallin, alpha B) gene, are linked to multisystem disorders featuring variable combinations of cataracts, cardiomyopathy, and skeletal myopathy. This study aimed to investigate the pathological mechanisms involved in an early-onset myofibrillar myopathy manifesting in a child harboring a homozygous recessive mutation in HSPB5, 343delT. To study HSPB5 343delT protein dynamics, we utilize model cell culture systems including induced pluripotent stem cells derived from the 343delT patient (343delT/343delT) along with isogenic, heterozygous, gene-corrected control cells (WT KI/343delT) and BHK21 cells, a cell line lacking endogenous HSPB5 expression. 343delT/343delT and WT KI/343delT-induced pluripotent stem cell-derived skeletal myotubes and cardiomyocytes did not express detectable levels of 343delT protein, contributable to the extreme insolubility of the mutant protein. Overexpression of HSPB5 343delT resulted in insoluble mutant protein aggregates and induction of a cellular stress response. Co-expression of 343delT with WT prevented visible aggregation of 343delT and improved its solubility. Additionally, in vitro refolding of 343delT in the presence of WT rescued its solubility. We demonstrate an interaction between WT and 343delT both in vitro and within cells. These data support a loss-of-function model for the myopathy observed in the patient because the insoluble mutant would be unavailable to perform normal functions of HSPB5, although additional gain-of-function effects of the mutant protein cannot be excluded. Additionally, our data highlight the solubilization of 343delT by WT, concordant with the recessive inheritance of the disease and absence of symptoms in carrier individuals.
Asunto(s)
Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Catarata/genética , Catarata/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismo , Cardiomiopatías/etiología , Catarata/etiología , Femenino , Homocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Enfermedades Musculares/etiología , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Miocitos Cardíacos/metabolismo , Linaje , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Solubilidad , Cadena B de alfa-Cristalina/químicaRESUMEN
Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 "cardiac interactome" to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease.
Asunto(s)
Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Mitocondrias/metabolismo , Miocardio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Biología Computacional , Citosol/metabolismo , Metabolismo Energético , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/metabolismo , Corazón/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Chaperonas Moleculares/metabolismo , Desarrollo de Músculos , Oxidación-Reducción , Estrés Oxidativo , Fenotipo , Proteómica , Daño por Reperfusión , Troponina I/sangre , Técnicas del Sistema de Dos Híbridos , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Fetal alcohol spectrum disorder (FASD) is a frequent cause of mental retardation. However, the molecular mechanisms underlying brain development defects induced by maternal alcohol consumption during pregnancy are unclear. We used normal and Hsf2-deficient mice and cell systems to uncover a pivotal role for heat shock factor 2 (HSF2) in radial neuronal migration defects in the cortex, a hallmark of fetal alcohol exposure. Upon fetal alcohol exposure, HSF2 is essential for the triggering of HSF1 activation, which is accompanied by distinctive post-translational modifications, and HSF2 steers the formation of atypical alcohol-specific HSF1-HSF2 heterocomplexes. This perturbs the in vivo binding of HSF2 to heat shock elements (HSEs) in genes that control neuronal migration in normal conditions, such as p35 or the MAPs (microtubule-associated proteins, such as Dclk1 and Dcx), and alters their expression. In the absence of HSF2, migration defects as well as alterations in gene expression are reduced. Thus, HSF2, as a sensor for alcohol stress in the fetal brain, acts as a mediator of the neuronal migration defects associated with FASD.
Asunto(s)
Trastornos del Espectro Alcohólico Fetal/patología , Proteínas de Choque Térmico/metabolismo , Malformaciones del Desarrollo Cortical del Grupo II/inducido químicamente , Estrés Fisiológico , Factores de Transcripción/metabolismo , Animales , Corteza Cerebral/patología , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Proteína Doblecortina , Regulación de la Expresión Génica , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/deficiencia , Ratones , Ratones Noqueados , Unión Proteica , Factores de Transcripción/deficienciaRESUMEN
Cardiomyocytes are best known for their spontaneous beating activity, large cell size, and low regenerative capacity during adulthood. The mechanical activity of cardiomyocytes depends on a sophisticated contractile apparatus comprised of sarcomeres whose rhythmic contraction relies on Ca(2+) transients with a high level of energy consumption. Hence the proper folding and assembly of the sarcomeric and other accessory proteins involved in those diverse functions (i.e., structural, mechanical, energy exchange and production) is critical for muscle mechanics. Chaperone proteins assist other polypeptides to reach their proper conformation, activity and/or location. Consequently, chaperone-like functions are important for the healthy heart but assume greater relevance during cardiac diseases when such chaperone proteins are recruited: 1) to protect cardiac cells against adverse effects during the pathological transition, and 2) to mitigate certain pathogenic mechanisms per se. Protein misfolding is observed as a consequence of inappropriate intracellular environment with acquired conditions (e.g., ischemia/reperfusion and redox imbalance) or because of mutations, which can modify primary to quaternary protein structures. In this review, we discuss the importance of cardiac chaperones while emphasizing the genetic origin (modification of gene/protein sequence) of cardiac protein misfolding and their consequences on the cardiomyocytes leading to organ dysfunction and failure.
Asunto(s)
Cardiopatías/metabolismo , Chaperonas Moleculares/metabolismo , Deficiencias en la Proteostasis/metabolismo , Animales , Cardiopatías/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Deficiencias en la Proteostasis/genéticaRESUMEN
AIMS: The human mutation R120G in the αB-crystallin (CRYAB) causes a multisystemic disease that is characterized by hypertrophic cardiomyopathy and cytoplasmic protein aggregates. In transgenic mice, human R120GCRYAB (hR120GTg) expression in heart sequentially modifies the REDOX status, in part by the activation of the nuclear factor, erythroid derived 2, like 2 (Nrf2). Thioredoxin system (TS) components are NRF2 target genes, so it could be hypothesized that TS was affected in hR120GTg mice. RESULTS: Transgenic hearts overexpressed thioredoxin 1 (Trx1), which was identified by isotope coded affinity tag-mass spectrometry, among hundreds of peptides displaying an increased reduced/oxidized ratio. Coupled to this higher level of reduced cysteines, the activity of thioredoxin reductase 1 (TrxR1) was augmented by 2.5-fold. Combining mutiple experimental approaches, the enzymatic regulation of TrxR1 by a histone deacetylase 3 (HDAC3)-dependent level of acetylation was confirmed. In vitro and in vivo functional tests established that TrxR1 activity is required to mitigate aggregate development, and this could be mediated by Bcl-2-associated athanogene 3 (BAG3) as a potential TS substrate. INNOVATION AND CONCLUSIONS: This study uncovers the compartmentalized changes and the involvement of TS in the cardiac stress response elicited by misfolded proteins such as R120GCRYAB. Our work suggests that R120GCRYAB triggers a defensive pathway acting through the newly identified interacting partners HDAC3, TrxR1, and BAG3 to counter aggregate growth. Therefore, those interactors may function as modifier genes contributing to the variable onset and expressivity of such human diseases. Furthermore, our work underscores the potential organismal effects of pharmacological interventions targeting TS and HDAC.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Agregación Patológica de Proteínas/genética , Cadena B de alfa-Cristalina/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Marcaje Isotópico , Espectrometría de Masas , Ratones , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/metabolismo , Tiorredoxina Reductasa 1/metabolismo , Tiorredoxinas/metabolismoRESUMEN
We investigated whether a cell-penetrating peptide linked via a disulfide bond to a fluorophore-labeled cargo peptide can be used to interrogate changes in cellular redox state. A fluorescence resonance energy transfer (FRET) pair was constructed so that the cargo peptide was labeled with fluorescein amidite (FAM) and the cell-penetrating peptide was attached to a quencher. Incubation of cells in culture with the FRET construct was visualized using live-cell, time-lapse imaging, which demonstrated earlier cellular uptake of the construct when cells were treated with the reducing agent n-acetylcysteine (NAC). The FRET peptide construct was easily detected in cells cultured in 96-well plates using a plate-reader. Treatment of cells with various classes of reducing or oxidizing agents resulted in an increase or decrease in FAM fluorescence, respectively. Changes in FAM fluorescence correlated significantly with redox-sensitive green fluorescent protein ratios in cells treated with hydrogen peroxide but not NAC. Detection of relative changes in cellular redox state was enhanced by the fact that uptake of the cell-penetrating peptide occurred more quickly in relatively reduced compared with oxidized cells. We conclude that cell-penetrating peptides coupled via disulfide bonds to detectable cargo is a novel and specific approach for assessment of relative changes in cellular thiol redox state.
RESUMEN
Several mutations in αB-crystallin (CryAB), a heat shock protein with chaperone-like activities, are causally linked to skeletal and cardiac myopathies in humans. To better understand the underlying pathogenic mechanisms, we had previously generated transgenic (TG) mice expressing R120GCryAB, which recapitulated distinguishing features of the myopathic disorder (e.g., protein aggregates, hypertrophic cardiomyopathy). To determine whether induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a new experimental approach for human disease modeling, would be relevant to aggregation-prone disorders, we decided to exploit the existing transgenic mouse model to derive iPSCs from tail tip fibroblasts. Several iPSC lines were generated from TG and non-TG mice and validated for pluripotency. TG iPSC-derived cardiomyocytes contained perinuclear aggregates positive for CryAB staining, whereas CryAB protein accumulated in both detergent-soluble and insoluble fractions. iPSC-derived cardiomyocytes identified by cardiac troponin T staining were significantly larger when expressing R120GCryAB at a high level in comparison with TG low expressor or non-TG cells. Expression of fetal genes such as atrial natriuretic factor, B-type natriuretic peptide, and α-skeletal α-actin, assessed by quantitative reverse transcription-polymerase chain reaction, were increased in TG cardiomyocytes compared with non-TG, indicating the activation of the hypertrophic genetic program in vitro. Our study demonstrates for the first time that differentiation of R120G iPSCs into cardiomyocytes causes protein aggregation and cellular hypertrophy, recapitulating in vitro key pathognomonic hallmarks found in both animal models and patients. Our findings pave the way for further studies exploiting this cell model system for mechanistic and therapeutic investigations.
Asunto(s)
Cardiomiopatía Hipertrófica/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , alfa-Cristalinas/metabolismo , beta-Cristalinas/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Diferenciación Celular , Línea Celular , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Ratones , Ratones Transgénicos , Miocitos Cardíacos/patología , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/metabolismo , Multimerización de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Troponina T/metabolismo , alfa-Cristalinas/genética , beta-Cristalinas/genéticaRESUMEN
Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs). The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called α and ß, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs) deleted for both Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1α is significantly more active than the ß isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short ß isoform of HSF2 exerts a negative role on HSF1ß-dependent transactivation. To further assess the impact of HSF2ß inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relative amounts of each HSF1 and HSF2 isoforms quantitatively determine the cellular level of the proteotoxic stress response.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Factores de Transcripción/genética , Animales , Blastocisto/citología , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Western Blotting , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Leupeptinas/farmacología , Ratones , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: CryAB (HspB5) and HspB2, two small heat shock genes located adjacently in the vertebrate genome, are hypothesized to play distinct roles. Mice lacking both cryab and hspb2 (DKO) are viable and exhibit adult-onset degeneration of skeletal muscle but confounding results from independent groups were reported for cardiac responses to different stressful conditions (i.e., ischemia/reperfusion or pressure overload). To determine the specific requirements of HSPB2 in heart, we generated cardiac-specific HSPB2 deficient (HSPB2cKO) mice and examined their cardiac function under basal conditions and following cardiac pressure overload. METHODOLOGY/PRINCIPAL FINDINGS: Transverse aortic constriction (TAC) or sham surgery was performed in HSPB2cKO mice and their littermates (HSPB2wt mice). Eight weeks after TAC, we found that expression of several small HSPs (HSPB2, 5, 6) was not markedly modified in HSPB2wt mice. Both cardiac function and the hypertrophic response remained similar in HSPB2cKO and HSPB2wt hearts. In addition, mitochondrial respiration and ATP production assays demonstrated that the absence of HSPB2 did not change mitochondrial metabolism in basal conditions. However, fatty acid supported state 3 respiration rate (ADP stimulated) in TAC operated HSPB2cKO hearts was significantly reduced in compared with TAC operated HSPB2wt mice (10.5±2.2 vs. 12.8±2.5 nmol O(2)/min/mg dry fiber weight, P<0.05), and ATP production in HSPB2cKO hearts was significantly reduced in TAC compared with sham operated mice (29.8±0.2 vs. 21.1±1.8 nmol ATP/min/mg dry fiber weight, P<0.05). Although HSPB2 was not associated with mitochondria under cardiac stress, absence of HSPB2 led to changes in transcript levels of several metabolic and mitochondrial regulator genes. CONCLUSIONS/SIGNIFICANCE: The present study indicates that HSPB2 can be replaced by other members of the multigene small HSP family under basal conditions while HSPB2 is implicated in the regulation of metabolic/mitochondrial function under cardiac stress such pressure overload.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Presión Sanguínea , Cardiomegalia/metabolismo , Proteínas de Choque Térmico HSP27 , Mitocondrias Cardíacas/metabolismo , Consumo de Oxígeno , Adenosina Trifosfato/genética , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patologíaRESUMEN
A timely review series on small heat shock proteins has to appropriately examine their fundamental properties and implications in the cardiovascular system since several members of this chaperone family exhibit robust expression in the myocardium and blood vessels. Due to energetic and metabolic demands, the cardiovascular system maintains a high mitochondrial activity but irreversible oxidative damage might ensue from increased production of reactive oxygen species. How equilibrium between their production and scavenging is achieved becomes paramount for physiological maintenance. For example, heat shock protein B1 (HSPB1) is implicated in maintaining this equilibrium or redox homeostasis by upholding the level of glutathione, a major redox mediator. Studies of gain or loss of function achieved by genetic manipulations have been highly informative for understanding the roles of those proteins. For example, genetic deficiency of several small heat shock proteins such as HSPB5 and HSPB2 is well-tolerated in heart cells whereas a single missense mutation causes human pathology. Such evidence highlights both the profound genetic redundancy observed among the multigene family of small heat shock proteins while underscoring the role proteotoxicity plays in driving disease pathogenesis. We will discuss the available data on small heat shock proteins in the cardiovascular system, redox metabolism and human diseases. From the medical perspective, we envision that such emerging knowledge of the multiple roles small heat shock proteins exert in the cardiovascular system will undoubtedly open new avenues for their identification and possible therapeutic targeting in humans. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Proteínas de Choque Térmico Pequeñas/metabolismo , Estrés Oxidativo , Animales , Enfermedades Cardiovasculares/genética , Sistema Cardiovascular/embriología , Sistema Cardiovascular/crecimiento & desarrollo , Sistema Cardiovascular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Choque Térmico Pequeñas/fisiología , Humanos , Mutación , Oxidación-Reducción , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismoRESUMEN
Heat shock response (HSR) that protects cells from proteotoxic stresses is downregulated in aging, as well as upon replicative senescence of cells in culture. Here we demonstrate that HSR is suppressed in fibroblasts from the patients with segmental progerioid Werner Syndrome, which undergo premature senescence. Similar suppression of HSR was seen in normal fibroblasts, which underwent senescence in response to DNA damaging treatments. The major DNA-damage-induced signaling (DDS) pathways p53-p21 and p38-NF-kB-SASP contributed to the HSR suppression. The HSR suppression was associated with inhibition of both activity and transcription of the heat shock transcription factor Hsf1. This inhibition in large part resulted from the downregulation of SIRT1, which in turn was because of decrease in the expression of the translation regulator HuR. Importantly, we uncovered a positive feedback regulation, where suppression of Hsf1 further activates the p38-NF-κB-SASP pathway, which in turn promotes senescence. Overexpression of Hsf1 inhibited the p38-NFκB-SASP pathway and partially relieved senescence. Therefore, downregulation of Hsf1 plays an important role in the development or in the maintenance of DNA damage signaling-induced cell senescence.
Asunto(s)
Senescencia Celular/genética , Senescencia Celular/fisiología , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Línea Celular , Regulación hacia Abajo , Proteínas ELAV/metabolismo , Retroalimentación Fisiológica , Fibroblastos/metabolismo , Fibroblastos/patología , Factores de Transcripción del Choque Térmico , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Humanos , FN-kappa B/metabolismo , Transducción de Señal , Sirtuina 1/metabolismo , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Force-generating contractile cells of the myocardium must achieve and maintain their primary function as an efficient mechanical pump over the life span of the organism. Because only half of the cardiomyocytes can be replaced during the entire human life span, the maintenance strategy elicited by cardiac cells relies on uninterrupted renewal of their components, including proteins whose specialized functions constitute this complex and sophisticated contractile apparatus. Thus cardiac proteins are continuously synthesized and degraded to ensure proteome homeostasis, also termed "proteostasis." Once synthesized, proteins undergo additional folding, posttranslational modifications, and trafficking and/or become involved in protein-protein or protein-DNA interactions to exert their functions. This includes key transient interactions of cardiac proteins with molecular chaperones, which assist with quality control at multiple levels to prevent misfolding or to facilitate degradation. Importantly, cardiac proteome maintenance depends on the cellular environment and, in particular, the reduction-oxidation (REDOX) state, which is significantly different among cardiac organelles (e.g., mitochondria and endoplasmic reticulum). Taking into account the high metabolic activity for oxygen consumption and ATP production by mitochondria, it is a challenge for cardiac cells to maintain the REDOX state while preventing either excessive oxidative or reductive stress. A perturbed REDOX environment can affect protein handling and conformation (e.g., disulfide bonds), disrupt key structure-function relationships, and trigger a pathogenic cascade of protein aggregation, decreased cell survival, and increased organ dysfunction. This review covers current knowledge regarding the general domain of REDOX state and protein folding, specifically in cardiomyocytes under normal-healthy conditions and during disease states associated with morbidity and mortality in humans.
Asunto(s)
Cardiopatías/metabolismo , Proteínas Musculares/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Estrés Oxidativo , Animales , Cardiopatías/tratamiento farmacológico , Cardiopatías/fisiopatología , Homeostasis , Humanos , Proteínas Musculares/química , Contracción Miocárdica/efectos de los fármacos , Oxidación-Reducción , Conformación Proteica , Pliegue de Proteína , Relación Estructura-ActividadRESUMEN
To investigate the effects of the predominant nonprotein thiol, glutathione (GSH), on redox homeostasis, we employed complementary pharmacological and genetic strategies to determine the consequences of both loss- and gain-of-function GSH content in vitro. We monitored the redox events in the cytosol and mitochondria using reduction-oxidation sensitive green fluorescent protein (roGFP) probes and the level of reduced/oxidized thioredoxins (Trxs). Either H(2)O(2) or the Trx reductase inhibitor 1-chloro-2,4-dinitrobenzene (DNCB), in embryonic rat heart (H9c2) cells, evoked 8 or 50 mV more oxidizing glutathione redox potential, E(hc) (GSSG/2GSH), respectively. In contrast, N-acetyl-L-cysteine (NAC) treatment in H9c2 cells, or overexpression of either the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) or GCL modifier subunit (GCLM) in human embryonic kidney 293 T (HEK293T) cells, led to 3- to 4-fold increase of GSH and caused 7 or 12 mV more reducing E(hc), respectively. This condition paradoxically increased the level of mitochondrial oxidation, as demonstrated by redox shifts in mitochondrial roGFP and Trx2. Lastly, either NAC treatment (EC(50) 4 mM) or either GCLC or GCLM overexpression exhibited increased cytotoxicity and the susceptibility to the more reducing milieu was achieved at decreased levels of ROS. Taken together, our findings reveal a novel mechanism by which GSH-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.
Asunto(s)
Glutatión/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Acetilcisteína/farmacología , Animales , Citosol/metabolismo , Dinitroclorobenceno/farmacología , Depuradores de Radicales Libres/farmacología , Glutamato-Cisteína Ligasa/metabolismo , Disulfuro de Glutatión/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Corazón/efectos de los fármacos , Corazón/embriología , Homeostasis , Humanos , Peróxido de Hidrógeno/farmacología , Indicadores y Reactivos/farmacología , Miocardio/citología , Miocardio/metabolismo , Oxidantes/farmacología , Subunidades de Proteína/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/metabolismoRESUMEN
Heat shock factor 1 (HSF1), while recognized as the major regulator of the heat shock transcriptional response, also exerts important functions during mammalian embryonic development and gametogenesis. In particular, HSF1 is required for oocyte maturation, the adult phase of meiosis preceding fertilization. To identify HSF1 target genes implicated in this process, comparative transcriptomic analyses were performed with wild-type and HSF-deficient oocytes. This revealed a network of meiotic genes involved in cohesin and synaptonemal complex (SC) structures, DNA recombination, and the spindle assembly checkpoint (SAC). All of them were found to be regulated by HSF1 not only during adult but also in embryonic phases of female meiosis. Additional investigations showed that SC, recombination nodules, and DNA repair were affected in Hsf1(-/-) oocytes during prenatal meiotic prophase I. However, targeting Hsf1 deletion to postnatal oocytes (using Zp3 Cre; Hsf1(loxP/loxP)) did not fully rescue the chromosomal anomalies identified during meiotic maturation, which possibly caused a persistent SAC activation. This would explain the metaphase I arrest previously described in HSF1-deficient oocytes since SAC inhibition circumvented this block. This work provides new insights into meiotic gene regulation and points out potential links between cellular stress and the meiotic anomalies frequently observed in humans.