RESUMEN
Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new class of oral hypoglycemic agents for patients with type 2 diabetes mellitus and have potential antiatherosclerotic properties. Meanwhile, it is unclear how DPP-4 inhibitors have protective effects on atherosclerosis. Our aim was to determine the effects and its mechanisms of DPP-4 inhibitors on cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured in hypoxic condition. To evaluate the protective effects of DPP-4 inhibitor on HUVECs, DPP-4 inhibitor was added in the cell culture medium and the cell viability was assessed by TUNEL assay. And we examined the intracellular signaling pathways in relation to the effects of DPP-4 inhibitor. DPP-4 inhibition had beneficial effects by inhibiting the apoptosis under hypoxic conditions in HUVECs. The antiapoptotic effects of DPP-4 inhibitor were abolished by the pretreatment with a CXCR4 antagonist or a Stat3 inhibitor. DPP-4 inhibition has beneficial effects on HUVECs by inhibiting the apoptosis under hypoxic conditions. SDF-1α/CXCR4/Stat3 pathways might be involved in the mechanisms of the cytoprotective effects of DPP-4 inhibitor. These results suggested that DPP-4 inhibitor has a potential for protecting vessels.
Asunto(s)
Apoptosis/efectos de los fármacos , Hipoxia de la Célula/fisiología , Dipeptidil Peptidasa 4/biosíntesis , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL12/biosíntesis , Dipeptidil Peptidasa 4/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Oligopéptidos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
AIMS: Dipeptidyl peptidase-4 (DPP-4) inhibitors are reported to have protective effects on various cells but it is unclear how DPP-4 inhibitors have cardioprotective effects. Our aim was to study the mechanisms of cardioprotective effects by DPP-4 inhibition. METHODS AND RESULTS: C57BL/6 mice and DPP-4 knockout (DPP-4KO) mice were subjected to left coronary artery ligation to produce acute myocardial infarction (MI). C57BL/6 mice were then treated with vehicle or DPP-4 inhibitor. Left ventricular function, infarct size, the number of vessels, and myocardial ischemia were assessed at 5days after MI. The treatment with DPP-4 inhibitor significantly improved cardiac function and decreased the infarct size. DPP-4 inhibitor increased the ratio of endothelial cell numbers to a cardiomyocyte. The extent of myocardial ischemia and the number of TUNEL-positive cells in the border area were significantly decreased by DPP-4 inhibitor. Stromal cell-derived factor-1α (SDF-1α) level in myocardium was significantly increased by DPP-4 inhibitor. Those cardioprotective effects after MI were also recognized in DPP-4KO mice. DPP-4 protein was expressed on rat neonatal cardiomyocytes and DPP-4 inhibitor significantly reduced hypoxia-induced apoptosis in the cardiomyocytes. However, this effect was abolished by the pretreatment with a CXCR4 antagonist or a signal transducer and activator of transcription 3 (STAT3) inhibitor. The beneficial effects of DPP-4 inhibitor on heart failure after MI were abolished by cardiomyocyte-specific deletion of STAT3. CONCLUSIONS: DPP-4 inhibition may have direct protective effects on the post-MI heart by inducing an antiapoptotic effect and inhibiting a decrease in vessel number through the SDF-1α/CXCR4-mediated STAT3 signaling pathway.
Asunto(s)
Quimiocina CXCL12/genética , Dipeptidil Peptidasa 4/genética , Insuficiencia Cardíaca/prevención & control , Infarto del Miocardio/tratamiento farmacológico , Receptores CXCR4/genética , Factor de Transcripción STAT3/genética , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Cardiotónicos/farmacología , Quimiocina CXCL12/agonistas , Quimiocina CXCL12/metabolismo , Dipeptidil Peptidasa 4/deficiencia , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
BACKGROUND: Inflammatory responses, especially by CD4(+)T cells activated by dendritic cells, are known to be important in the pathophysiology of cardiac repair after myocardial infarction (MI). Although co-stimulatory signals through B7 (CD80/86) and CD28 are necessary for CD4(+)T cell activation and survival, the roles of these signals in cardiac repair after MI are still unclear. METHODSâANDâRESULTS: C57BL/6 (Control) mice and CD28 knockout (CD28KO) mice were subjected to left coronary artery permanent ligation. The ratio of death by cardiac rupture within 5 days after MI was significantly higher in CD28KO mice compared with Control mice. Although there were no significant differences in the infarct size between the 2 groups, left ventricular end-diastolic and end-systolic diameters were significantly increased, and fractional shortening was significantly decreased in CD28KO mice compared with Control mice. Electron microscopic observation revealed that the extent of extracellular collagen fiber was significantly decreased in CD28KO mice compared with Control mice. The number of α-smooth muscle actin-positive myofibroblasts was significantly decreased, and matrix metalloproteinase-9 activity and the mRNA expression of interleukin-1ß were significantly increased in CD28KO mice compared with Control mice. CONCLUSIONS: Deletion of CD28 co-stimulatory signals exacerbates left ventricular remodeling and increases cardiac rupture after MI through prolongation of the inflammatory period and reduction of collagen fiber in the infarct scars. (Circ J 2016; 80: 1971-1979).
Asunto(s)
Antígenos CD28/deficiencia , Eliminación de Gen , Rotura Cardíaca Posinfarto/metabolismo , Infarto del Miocardio/metabolismo , Transducción de Señal , Remodelación Ventricular , Animales , Antígenos CD28/metabolismo , Regulación de la Expresión Génica , Rotura Cardíaca Posinfarto/genética , Rotura Cardíaca Posinfarto/patología , Rotura Cardíaca Posinfarto/fisiopatología , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Masculino , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miofibroblastos/metabolismo , Miofibroblastos/ultraestructuraRESUMEN
Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of "slow-moving" Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Replicación del ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genoma Fúngico , Datos de Secuencia Molecular , Mutación , Fenotipo , Plásmidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Bi-directionality is a common feature observed for genomic replication for all three phylogenetic kingdoms: Eubacteria, Archaea, and Eukaryotes. A consequence of bi-directional replication, where the two replication forks initiated at an origin move away from each other, is that the replication termination will occur at positions away from the origin sequence(s). The replication termination processes are therefore physically and mechanistically dissociated from the replication initiation. The replication machinery is a highly processive complex that in short time copies huge numbers of bases while competing for the DNA substrate with histones, transcription factors, and other DNA-binding proteins. Importantly, the replication machinery generally wins out; meanwhile, when converging forks meet termination occurs, thus preventing over-replication and genetic instability. Very different scenarios for the replication termination processes have been described for the three phylogenetic kingdoms. In eubacterial genomes replication termination is site specific, while in archaea and eukaryotes termination is thought to occur randomly within zones where converging replication forks meet. However, a few site-specific replication barrier elements that mediate replication termination have been described in eukaryotes. This review gives an overview about what is known about replication termination, with a focus on these natural site-specific replication termination sites.
Asunto(s)
Replicación del ADN/fisiología , Archaea/genética , Archaea/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sitios de Unión , Replicación del ADN/genética , ADN de Archaea/biosíntesis , ADN de Archaea/genética , ADN Bacteriano/biosíntesis , ADN Bacteriano/genética , ADN de Hongos/biosíntesis , ADN de Hongos/genética , ADN Ribosómico/biosíntesis , ADN Ribosómico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.
Asunto(s)
Genes del Tipo Sexual de los Hongos/genética , Genes de Cambio , Recombinación Genética , Schizosaccharomyces/genética , Cromosomas Fúngicos , ADN de Hongos/genética , Fenotipo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Esporas Fúngicas/genéticaRESUMEN
Hematopoietic stem cells (HSC), the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment.
Asunto(s)
Proteína Morfogenética Ósea 4/metabolismo , Proteínas Hedgehog/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/citología , Animales , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Proteínas Smad/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Adult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they are first generated in the aorta-gonad-mesonephros region, but at later developmental stages, its role in HSCs is controversial. Here we show that HSCs in murine fetal liver and the bone marrow are of two types that can be prospectively isolated--BMP activated and non-BMP activated. Clonal transplantation demonstrates that they have distinct haematopoietic lineage outputs. Moreover, the two HSC types differ in intrinsic genetic programs, thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output. Our findings provide insight into the molecular control mechanisms that define HSC types and have important implications for reprogramming cells to HSC fate and treatments targeting distinct HSC types.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/fisiología , Transducción de Señal/fisiología , Animales , Benzofuranos , Proteínas Morfogenéticas Óseas/genética , Embrión de Mamíferos , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , QuinolinasRESUMEN
Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the "heptad" complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs.
Asunto(s)
Transdiferenciación Celular/genética , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Células CHO , Células COS , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Cricetulus , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Endoteliales/citología , Femenino , Ontología de Genes , Células Madre Hematopoyéticas/citología , Humanos , Hibridación in Situ , Ratones Endogámicos C57BL , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN/métodos , Regulación hacia ArribaRESUMEN
Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC) function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α), a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver), and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO) approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs) are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages.
Asunto(s)
Hipoxia de la Célula/fisiología , Células Madre Hematopoyéticas/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Aorta/citología , Cadherinas/metabolismo , Separación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Feto/citología , Trasplante de Células Madre Hematopoyéticas , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Placenta/citología , Embarazo , Trasplante HomólogoRESUMEN
Knowledge of the key transcription factors that drive hematopoietic stem cell (HSC) generation is of particular importance for current hematopoietic regenerative approaches and reprogramming strategies. Whereas GATA2 has long been implicated as a hematopoietic transcription factor and its dysregulated expression is associated with human immunodeficiency syndromes and vascular integrity, it is as yet unknown how GATA2 functions in the generation of HSCs. HSCs are generated from endothelial cells of the major embryonic vasculature (aorta, vitelline, and umbilical arteries) and are found in intra-aortic hematopoietic clusters. In this study, we find that GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition. Specific deletion of Gata2 in Vec (Vascular Endothelial Cadherin)-expressing endothelial cells results in a deficiency of long-term repopulating HSCs and intra-aortic cluster cells. By specific deletion of Gata2 in Vav-expressing hematopoietic cells (after HSC generation), we further show that GATA2 is essential for HSC survival. This is in contrast to the known activity of the RUNX1 transcription factor, which functions only in the generation of HSCs, and highlights the unique requirement for GATA2 function in HSCs throughout all developmental stages.
Asunto(s)
Factor de Transcripción GATA2/fisiología , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Alelos , Animales , Apoptosis , Separación Celular , Supervivencia Celular , Citometría de Flujo , Eliminación de Gen , Ratones , Ratones Noqueados , Ratones Transgénicos , Células MadreRESUMEN
We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in â¼6 d.
Asunto(s)
Aorta/citología , Embrión de Mamíferos/citología , Células Madre Hematopoyéticas/citología , Inmunohistoquímica/métodos , Microscopía Confocal/métodos , Animales , Fluorescencia , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Coloración y Etiquetado/métodos , Fijación del Tejido/métodosRESUMEN
Hematopoietic stem cells (HSCs) and an earlier wave of definitive erythroid/myeloid progenitors (EMPs) differentiate from hemogenic endothelial cells in the conceptus. EMPs can be generated in vitro from embryonic or induced pluripotent stem cells, but efforts to produce HSCs have largely failed. The formation of both EMPs and HSCs requires the transcription factor Runx1 and its non-DNA binding partner core binding factor ß (CBFß). Here we show that the requirements for CBFß in EMP and HSC formation in the conceptus are temporally and spatially distinct. Panendothelial expression of CBFß in Tek-expressing cells was sufficient for EMP formation, but was not adequate for HSC formation. Expression of CBFß in Ly6a-expressing cells, on the other hand, was sufficient for HSC, but not EMP, formation. The data indicate that EMPs and HSCs differentiate from distinct populations of hemogenic endothelial cells, with Ly6a expression specifically marking the HSC-generating hemogenic endothelium.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Células Endoteliales/fisiología , Células Eritroides/metabolismo , Células Madre Hematopoyéticas/fisiología , Células Mieloides/fisiología , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Células Endoteliales/citología , Células Eritroides/citología , Células Madre Hematopoyéticas/citología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Células Mieloides/citología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor TIE-2 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransgenesAsunto(s)
Adenosina Trifosfatasas , Transferencia de Energía , Chaperonas Moleculares , Proteínas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/fisiología , Adenosina Trifosfato/metabolismo , Hidrólisis , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiología , Conformación Proteica , Pliegue de ProteínaRESUMEN
Like other AAA proteins, Escherichia coli FtsH, a membrane-bound AAA protease, contains highly conserved aromatic and glycine residues (Phe228 and Gly230) that are predicted to lie in the central pore region of the hexamer. The functions of Phe228 and Gly230 were probed by site-directed mutagenesis. The results of both in vivo and in vitro assays indicate that these conserved pore residues are important for FtsH function and that bulkier, uncharged/apolar residues are essential at position 228. None of the point mutants, F228A, F228E, F228K, or G230A, was able to degrade sigma32, a physiological substrate. The F228A mutant was able to degrade casein, an unfolded substrate, although the other three mutants were not. Mutation of these two pore residues also affected the ATPase activity of FtsH. The F228K and G230A mutations markedly reduced ATPase activity, whereas the F228A mutation caused a more modest decrease in this activity. The F228E mutant was actually more active ATPase. The substrates, sigma32 and casein, stimulated the ATPase activity of wild type FtsH. The ATPase activity of the mutants was no longer stimulated by casein, whereas that of the three Phe228 mutants, but not the G230A mutant, remained sigma32-stimulatable. These results suggest that Phe228 and Gly230 in the predicted pore region of the FtsH hexamer have important roles in proteolysis and its coupling to ATP hydrolysis.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Western Blotting , Secuencia Conservada , Proteínas de Escherichia coli , Glutatión Transferasa/metabolismo , Glicina/química , Hidrólisis , Lisina/química , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fenilalanina/química , Plásmidos/metabolismo , Mutación Puntual , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas/metabolismo , TemperaturaRESUMEN
We have established a fluorescence polarization assay system by which degradation of sigma32, a physiological substrate, by FtsH can be monitored spectrometrically. Using the system, it was found that an FtsH hexamer degrades approximately 0.5 molecules of Cy3-sigma32 per min at 42 degrees C and hydrolyzes approximately 140 ATP molecules during the degradation of a single molecule of Cy3-sigma32. Evidence also suggests that degradation of sigma32 proceeds from the N-terminus to the C-terminus. Although FtsH does not have a robust enough unfoldase activity to unfold a tightly folded proteins such as green fluorescent protein, it can unfold proteins with lower T(m)s such as glutathione S-transferase (T(m) = 52 degrees C).