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1.
Cell ; 151(6): 1308-18, 2012 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-23217712

RESUMEN

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ciclinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Proliferación Celular , Ciclina D1/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSC70/metabolismo , Humanos , Fosforilación , Saccharomyces cerevisiae/citología
2.
Mol Psychiatry ; 27(9): 3842-3856, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35546635

RESUMEN

Bipolar disorder is an often-severe mental health condition characterized by alternation between extreme mood states of mania and depression. Despite strong heritability and the recent identification of 64 common variant risk loci of small effect, pathophysiological mechanisms remain unknown. Here, we analyzed genome sequences from 41 multiply-affected pedigrees and identified variants in 741 genes with nominally significant linkage or association with bipolar disorder. These 741 genes overlapped known risk genes for neurodevelopmental disorders and clustered within gene networks enriched for synaptic and nuclear functions. The top variant in this analysis - prioritized by statistical association, predicted deleteriousness, and network centrality - was a missense variant in the gene encoding D-amino acid oxidase (DAOG131V). Heterologous expression of DAOG131V in human cells resulted in decreased DAO protein abundance and enzymatic activity. In a knock-in mouse model of DAOG131, DaoG130V/+, we similarly found decreased DAO protein abundance in hindbrain regions, as well as enhanced stress susceptibility and blunted behavioral responses to pharmacological inhibition of N-methyl-D-aspartate receptors (NMDARs). RNA sequencing of cerebellar tissue revealed that DaoG130V resulted in decreased expression of two gene networks that are enriched for synaptic functions and for genes expressed, respectively, in Purkinje neurons or granule neurons. These gene networks were also down-regulated in the cerebellum of patients with bipolar disorder compared to healthy controls and were enriched for additional rare variants associated with bipolar disorder risk. These findings implicate dysregulation of NMDAR signaling and of gene expression in cerebellar neurons in bipolar disorder pathophysiology and provide insight into its genetic architecture.


Asunto(s)
Trastorno Bipolar , Receptores de N-Metil-D-Aspartato , Ratones , Animales , Humanos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Trastorno Bipolar/genética , Trastorno Bipolar/metabolismo , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/metabolismo , Redes Reguladoras de Genes/genética , Cerebelo/metabolismo
3.
BMC Genomics ; 15: 194, 2014 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-24628813

RESUMEN

BACKGROUND: Cytosolic Hsp70 is a ubiquitous molecular chaperone that is involved in responding to a variety of cellular stresses. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. To gain further insight into the functional redundancy and specialisation of the highly homologous yeast Hsp70-Ssa family we expressed each of the individual Ssa proteins as the sole source of Hsp70 in the cell and assessed phenotypic differences in prion propagation and stress resistance. Additionally we also analysed the global gene expression patterns in yeast strains expressing individual Ssa proteins, using microarray and RT-qPCR analysis. RESULTS: We confirm and extend previous studies demonstrating that cells expressing different Hsp70-Ssa isoforms vary in their ability to propagate the yeast [PSI+] prion, with Ssa3 being the most proficient. Of the four Ssa family members the heat inducible isoforms are more proficient in acquiring thermotolerance and we show a greater requirement than was previously thought, for cellular processes in addition to the traditional Hsp104 protein disaggregase machinery, in acquiring such thermotolerance. Cells expressing different Hsp70-Ssa isoforms also display differences in phenotypic response to exposure to cell wall damaging and oxidative stress agents, again with the heat inducible isoforms providing better protection than constitutive isoforms. We assessed global transcriptome profiles for cells expressing individual Hsp70-Ssa isoforms as the sole source of cytosolic Hsp70, and identified a significant difference in cellular gene expression between these strains. Differences in gene expression profiles provide a rationale for some phenotypic differences we observed in this study. We also demonstrate a high degree of correlation between microarray data and RT-qPCR analysis for a selection of genes. CONCLUSIONS: The Hsp70-Ssa family provide both redundant and variant-specific functions within the yeast cell. Yeast cells expressing individual members of the Hsp70-Ssa family as the sole source of Ssa protein display differences in global gene expression profiles. These changes in global gene expression may contribute significantly to the phenotypic differences observed between the Hsp70-Ssa family members.


Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Fenotipo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Adaptación Biológica/genética , Análisis por Conglomerados , Perfilación de la Expresión Génica , Calor , Familia de Multigenes , Unión Proteica , Replegamiento Proteico , Estrés Fisiológico/genética
4.
PLoS Comput Biol ; 9(1): e1002896, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23382668

RESUMEN

Genetic screens using Saccharomyces cerevisiae have identified an array of cytosolic Hsp70 mutants that are impaired in the ability to propagate the yeast [PSI(+)] prion. The best characterized of these mutants is the Ssa1 L483W mutant (so-called SSA1-21), which is located in the substrate-binding domain of the protein. However, biochemical analysis of some of these Hsp70 mutants has so far failed to provide major insight into the specific functional changes in Hsp70 that cause prion impairment. In order to gain a better understanding of the mechanism of Hsp70 impairment of prions we have taken an in silico approach and focused on the Escherichia coli Hsp70 ortholog DnaK. Using steered molecular dynamics simulations (SMD) we demonstrate that DnaK variant L484W (analogous to SSA1-21) is predicted to bind substrate more avidly than wild-type DnaK due to an increase in numbers of hydrogen bonds and hydrophobic interactions between chaperone and peptide. Additionally the presence of the larger tryptophan side chain is predicted to cause a conformational change in the peptide-binding domain that physically impairs substrate dissociation. The DnaK L484W variant in combination with some SSA1-21 phenotypic second-site suppressor mutations exhibits chaperone-substrate interactions that are similar to wild-type protein and this provides a rationale for the phenotypic suppression that is observed. Our computational analysis fits well with previous yeast genetics studies regarding the functionality of the Ssa1-21 protein and provides further evidence suggesting that manipulation of the Hsp70 ATPase cycle to favor the ADP/substrate-bound form impairs prion propagation. Furthermore, we demonstrate how SMD can be used as a computational tool for predicting Hsp70 peptide-binding domain mutants that impair prion propagation.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Simulación de Dinámica Molecular , Mutación , Priones/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica
5.
Nat Commun ; 15(1): 5282, 2024 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-38902255

RESUMEN

During pulmonary mucormycosis, inhaled sporangiospores adhere to, germinate, and invade airway epithelial cells to establish infection. We provide evidence that HIF1α plays dual roles in airway epithelial cells during Mucorales infection. We observed an increase in HIF1α protein accumulation and increased expression of many known HIF1α-responsive genes during in vitro infection, indicating that HIF1α signaling is activated by Mucorales infection. Inhibition of HIF1α signaling led to a substantial decrease in the ability of R. delemar to invade cultured airway epithelial cells. Transcriptome analysis revealed that R. delemar infection induces the expression of many pro-inflammatory genes whose expression was significantly reduced by HIF1α inhibition. Importantly, pharmacological inhibition of HIF1α increased survival in a mouse model of pulmonary mucormycosis without reducing fungal burden. These results suggest that HIF1α plays two opposing roles during mucormycosis: one that facilitates the ability of Mucorales to invade the host cells and one that facilitates the ability of the host to mount an innate immune response.


Asunto(s)
Células Epiteliales , Subunidad alfa del Factor 1 Inducible por Hipoxia , Mucorales , Mucormicosis , Animales , Femenino , Humanos , Ratones , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Perfilación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Pulmón/microbiología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Mucorales/metabolismo , Mucorales/genética , Mucormicosis/microbiología , Mucormicosis/metabolismo , Mucormicosis/inmunología , Transducción de Señal
6.
Sci Rep ; 9(1): 16260, 2019 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-31700027

RESUMEN

Hsp70 is a highly conserved molecular chaperone critical for the folding of new and denatured proteins. While traditional models state that cells respond to stress by upregulating inducible HSPs, this response is relatively slow and is limited by transcriptional and translational machinery. Recent studies have identified a number of post-translational modifications (PTMs) on Hsp70 that act to fine-tune its function. We utilized mass spectrometry to determine whether yeast Hsp70 (Ssa1) is differentially modified upon heat shock. We uncovered four lysine residues on Ssa1, K86, K185, K354 and K562 that are deacetylated in response to heat shock. Mutation of these sites cause a substantial remodeling of the Hsp70 interaction network of co-chaperone partners and client proteins while preserving essential chaperone function. Acetylation/deacetylation at these residues alter expression of other heat-shock induced chaperones as well as directly influencing Hsf1 activity. Taken together our data suggest that cells may have the ability to respond to heat stress quickly though Hsp70 deacetylation, followed by a slower, more traditional transcriptional response.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Levaduras/metabolismo , Acetilación , Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico/química , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Conformación Proteica , Levaduras/genética
7.
Cell Rep ; 25(5): 1135-1145.e5, 2018 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-30380406

RESUMEN

RNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth. Estimations of the number of rNMPs in DNA in these two mutants define a ribonucleotide threshold above which p53 induces apoptosis. Below the threshold, rNMPs in DNA trigger an innate immune response. Compound heterozygous cells, containing both defective enzymes, retain rNMPs above the threshold, indicative of competition for RER substrates between active and inactive enzymes, suggesting that patients with compound heterozygous mutations in RNASEH2 genes may not reflect the properties of recombinantly expressed proteins.


Asunto(s)
Desarrollo Embrionario , Mutación/genética , Ribonucleasa H/genética , Ribonucleótidos/metabolismo , Animales , ADN/metabolismo , Daño del ADN , Reparación del ADN/efectos de los fármacos , Pérdida del Embrión/patología , Embrión de Mamíferos/anomalías , Desarrollo Embrionario/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Interferones/farmacología , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Proteínas Mutantes/metabolismo , Estabilidad del ARN/efectos de los fármacos , Ribonucleasa H/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
8.
Sci Rep ; 6: 32117, 2016 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-27633137

RESUMEN

6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.


Asunto(s)
Proteínas de Choque Térmico/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Pliegue de Proteína , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Guanabenzo/farmacología , Proteínas de Choque Térmico/genética , Mutación , Factores de Terminación de Péptidos/genética , Fenantridinas/farmacología , Priones/genética , Pliegue de Proteína/efectos de los fármacos , ARN Ribosómico/metabolismo , Ribosomas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
9.
J Exp Med ; 213(3): 329-36, 2016 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-26880576

RESUMEN

The neuroinflammatory autoimmune disease Aicardi-Goutières syndrome (AGS) develops from mutations in genes encoding several nucleotide-processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species that trigger chronic type I interferon and inflammatory responses, leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2a(G37S/G37S) (G37S), to understand disease pathology. G37S homozygotes are perinatal lethal, in contrast to the early embryonic lethality previously reported for Rnaseh2b- or Rnaseh2c-null mice. Importantly, we found that the G37S mutation led to increased expression of interferon-stimulated genes dependent on the cGAS-STING signaling pathway. Ablation of STING in the G37S mice results in partial rescue of the perinatal lethality, with viable mice exhibiting white spotting on their ventral surface. We believe that the G37S knock-in mouse provides an excellent animal model for studying RNASEH2-associated autoimmune diseases.


Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Inmunidad Innata , Proteínas de la Membrana/metabolismo , Mutación/genética , Malformaciones del Sistema Nervioso/inmunología , Nucleotidiltransferasas/metabolismo , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Animales , Enfermedades Autoinmunes del Sistema Nervioso/genética , Dominio Catalítico , Células Cultivadas , Cruzamientos Genéticos , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Homocigoto , Humanos , Interferones/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Masculino , Ratones , Malformaciones del Sistema Nervioso/genética , Fenotipo , Transducción de Señal
10.
PLoS One ; 6(12): e28684, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22194885

RESUMEN

The yeast prion [PSI(+)] has been implicated in the generation of novel phenotypes by a mechanism involving a reduction in translation fidelity causing readthrough of naturally occurring stop codons. Some [PSI(+)] associated phenotypes may also be generated due to readthrough of inactivating stop codon mutations (ISCMs). Using next generation sequencing we have sequenced the genomes of two Saccharomyces cerevisiae strains that are commonly used for the study of the yeast [PSI(+)] prion. We have identified approximately 26,000 and 6,500 single nucleotide polymorphisms (SNPs) in strains 74-D694 and G600 respectively, compared to reference strain S288C. In addition to SNPs that produce non-synonymous amino acid changes we have also identified a number of SNPs that cause potential ISCMs in these strains, one of which we show is associated with a [PSI(+)]-dependent stress resistance phenotype in strain G600. We identified twenty-two potential ISCMs in strain 74-D694, present in genes involved in a variety of cellular processes including nitrogen metabolism, signal transduction and oxidative stress response. The presence of ISCMs in a subset of these genes provides possible explanations for previously identified [PSI(+)]-associated phenotypes in this strain. A comparison of ISCMs in strains G600 and 74-D694 with S. cerevisiae strains sequenced as part of the Saccharomyces Genome Resequencing Project (SGRP) shows much variation in the generation of strain-specific ISCMs and suggests this process is possible under complex genetic control. Additionally we have identified a major difference in the abilities of strains G600 and 74-D694 to grow at elevated temperatures. However, this difference appears unrelated to novel SNPs identified in strain 74-D694 present in proteins involved in the heat shock response, but may be attributed to other SNP differences in genes previously identified as playing a role in high temperature growth.


Asunto(s)
Codón de Terminación/genética , Mutación/genética , Priones/metabolismo , Saccharomyces cerevisiae/genética , Adaptación Fisiológica , Codón sin Sentido/genética , Genes Fúngicos/genética , Respuesta al Choque Térmico/genética , Sistemas de Lectura Abierta/genética , Fenotipo , Filogenia , Polimorfismo de Nucleótido Simple/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Estrés Fisiológico/genética , Temperatura
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