RESUMEN
In budding yeast, the nucleolus serves as the site to sequester Cdc14, a phosphatase essential for mitotic exit. Nucleolar proteins Tof2, Net1, and Fob1 are required for this sequestration. Although it is known that these nucleolar proteins are SUMOylated, how SUMOylation regulates their activity remains unknown. Here, we show that Tof2 exhibits cell-cycle-regulated nucleolar delocalization and turnover. Depletion of the nuclear small ubiquitin-like modifier (SUMO) protease Ulp2 not only causes Tof2 polySUMOylation, nucleolar delocalization, and degradation but also leads to Cdc14 nucleolar release and activation. This outcome depends on polySUMOylation and the activity of downstream enzymes, including SUMO-targeted ubiquitin ligase and Cdc48/p97 segregase. We further developed a system to tether SUMO machinery to Tof2 and generated a SUMO-deficient tof2 mutant, and the results indicate that Tof2 polySUMOylation is necessary and sufficient for its nucleolar delocalization and degradation. Together, our work reveals a polySUMO-dependent mechanism that delocalizes Tof2 from the nucleolus to facilitate mitotic exit.
Asunto(s)
Nucléolo Celular , Mitosis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sumoilación , Nucléolo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/metabolismo , Endopeptidasas/metabolismo , Proteína que Contiene Valosina/metabolismoRESUMEN
Actinidia arguta, the most widely distributed Actinidia species and the second cultivated species in the genus, can be distinguished from the currently cultivated Actinidia chinensis on the basis of its small and smooth fruit, rapid softening, and excellent cold tolerance. Adaptive evolution of tetraploid Actinidia species and the genetic basis of their important agronomic traits are still unclear. Here, we generated a chromosome-scale genome assembly of an autotetraploid male A. arguta accession. The genome assembly was 2.77 Gb in length with a contig N50 of 9.97 Mb and was anchored onto 116 pseudo-chromosomes. Resequencing and clustering of 101 geographically representative accessions showed that they could be divided into two geographic groups, Southern and Northern, which first diverged 12.9 million years ago. A. arguta underwent two prominent expansions and one demographic bottleneck from the mid-Pleistocene climate transition to the late Pleistocene. Population genomics studies using paleoclimate data enabled us to discern the evolution of the species' adaptation to different historical environments. Three genes (AaCEL1, AaPME1, and AaDOF1) related to flesh softening were identified by multi-omics analysis, and their ability to accelerate flesh softening was verified through transient expression assays. A set of genes that characteristically regulate sexual dimorphism located on the sex chromosome (Chr3) or autosomal chromosomes showed biased expression during stamen or carpel development. This chromosome-level assembly of the autotetraploid A. arguta genome and the genes related to important agronomic traits will facilitate future functional genomics research and improvement of A. arguta.
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Actinidia , Genoma de Planta , Tetraploidía , Actinidia/genética , Evolución Molecular , Adaptación Fisiológica/genética , Evolución BiológicaRESUMEN
Developing electrochemical high-energy storage systems is of crucial importance toward a green and sustainable energy supply. A promising candidate is fluoride-ion batteries (FIBs), which can deliver a much higher volumetric energy density than lithium-ion batteries. However, typical metal fluoride cathodes with conversion-type reactions cause a low-rate capability. Recently, layered perovskite oxides and oxyfluorides, such as LaSrMnO4 and Sr3Fe2O5F2, have been reported to exhibit relatively high rate performance and cycle stability compared to typical metal fluoride cathodes with conversion-type reactions, but their discharge capacities (â¼118 mA h/g) are lower than those of typical cathodes used in lithium-ion batteries. Here, we show that double-layered perovskite oxyfluoride La1.2Sr1.8Mn2O7-δF2 exhibits (de) intercalation of two fluoride ions to rock-salt slabs and further (de) intercalation of excess fluoride ions to the perovskite layer, leading to a reversible capacity of 200 mA h/g. The additional fluoride-ion intercalation leads to the formation of O-O bond in the structure for charge compensation (i.e., anion redox). These results highlight the layered perovskite oxyfluorides as a new class of active materials for the construction of high-performance FIBs.
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Light and temperature are key factors influencing the accumulation of anthocyanin in fruit crops. To assess the effects of fruit bagging during development and high post-ripening temperature on 'Hongyang' kiwifruit, we compared the pigmentation phenotypes and expression levels of anthocyanin-related genes between bagged and unbagged treatments, and between 25 °C and 37 °C postharvest storage temperatures. Both the bagging and 25 °C treatments showed better pigmentation phenotypes with higher anthocyanin concentrations. The results of the qRT-PCR analysis revealed that the gene expression levels of LDOX (leucoanthocyanidin dioxygenase), F3GT (UDP-flavonoid 3-O-glycosyltransferase ), AcMYB10, and AcbHLH42 were strongly correlated and upregulated by both the bagging treatment and 25 °C storage. The results of bimolecular fluorescence complementation and luciferase complementation imaging assays indicated an interaction between AcMYB10 and AcbHLH42 in plant cells, whereas the results of a yeast one-hybrid assay further demonstrated that AcMYB10 activated the promoters of AcLODX and AcF3GT. These results strongly suggest that enhanced anthocyanin synthesis is caused by the promoted expression of AcLODX and AcF3GT, regulated by the complex formed by AcMYB10-AcbHLH42.
Asunto(s)
Actinidia , Antocianinas , Frutas/genética , Temperatura , Flavonoides , Actinidia/genética , Saccharomyces cerevisiaeRESUMEN
Actinidia ('Mihoutao' in Chinese) includes species with complex ploidy, among which diploid Actinidia chinensis and hexaploid Actinidia deliciosa are economically and nutritionally important fruit crops. Actinidia deliciosa has been proposed to be an autohexaploid (2n = 174) with diploid A. chinensis (2n = 58) as the putative parent. A CCS-based assembly anchored to a high-resolution linkage map provided a chromosome-resolved genome for hexaploid A. deliciosa yielded a 3.91-Gb assembly of 174 pseudochromosomes comprising 29 homologous groups with 6 members each, which contain 39 854 genes with an average of 4.57 alleles per gene. Here we provide evidence that much of the hexaploid genome matches diploid A. chinensis; 95.5% of homologous gene pairs exhibited >90% similarity. However, intragenome and intergenome comparisons of synteny indicate chromosomal changes. Our data, therefore, indicate that if A. deliciosa is an autoploid, chromosomal rearrangement occurred following autohexaploidy. A highly diversified pattern of gene expression and a history of rapid population expansion after polyploidisation likely facilitated the adaptation and niche differentiation of A. deliciosa in nature. The allele-defined hexaploid genome of A. deliciosa provides new genomic resources to accelerate crop improvement and to understand polyploid genome evolution.
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Actinidia , Actinidia/genética , Mapeo Cromosómico , Genoma de Planta/genética , Ploidias , Cromosomas , Frutas/genéticaRESUMEN
Protein misfolding, aggregation, and accumulation cause neurodegenerative disorders. One such disorder, Huntington's disease, is caused by an increased number of glutamine-encoding trinucleotide repeats CAG in the first exon of the huntingtin (HTT) gene. Mutant proteins of Htt exon 1 with polyglutamine expansion are prone to aggregation and form pathological inclusion bodies in neurons. Extensive studies have shown that misfolded proteins are cleared by the ubiquitin-proteasome system or autophagy to alleviate their cytotoxicity. Misfolded proteins can form small soluble aggregates or large insoluble inclusion bodies. Previous works have elucidated the role of autophagy in the clearance of misfolded protein aggregates, but autophagic clearance of inclusion bodies remains poorly characterized. Here we use mutant Htt exon 1 with 103 polyglutamine (Htt103QP) as a model substrate to study the autophagic clearance of inclusion bodies in budding yeast. We found that the core autophagy-related proteins were required for Htt103QP inclusion body autophagy. Moreover, our evidence indicates that the autophagy of Htt103QP inclusion bodies is selective. Interestingly, Cue5/Tollip, a known autophagy receptor for aggrephagy, is dispensable for this inclusion body autophagy. From the known selective autophagy receptors in budding yeast, we identified three that are essential for inclusion body autophagy. Amyloid beta peptide (Aß42) is a major component of amyloid plaques found in Alzheimer's disease brains. Interestingly, a similar selective autophagy pathway contributes to the clearance of Aß42 inclusion bodies in budding yeast. Therefore, our results reveal a novel autophagic pathway specific for inclusion bodies associated with neurodegenerative diseases, which we have termed IBophagy.
RESUMEN
The conserved chromosomal passenger complex (CPC) consists of Ipl1Aurora-B, Sli15INCENP, Bir1Survivin, and Nbl1Borealin, and localizes at the kinetochore/centromere to correct kinetochore attachment errors and to prevent checkpoint silencing. After anaphase entry, the CPC moves from the kinetochore/centromere to the spindle. In budding yeast, CPC subunit Sli15 is phosphorylated by both cyclin-dependent kinase (CDK) and Ipl1 kinase. Following anaphase onset, activated Cdc14 phosphatase reverses Sli15 phosphorylation imposed by CDK to promote CPC translocation. Although abolished Sli15 phosphorylation imposed by Ipl1 also causes CPC translocation, the regulation of Ipl1-imposed Sli15 phosphorylation remains unclear. In addition to Sli15, Cdc14 also dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. Here, we present evidence supporting the notion that kinetochore-localized Fin1-PP1 likely reverses Ipl1-imposed Sli15 phosphorylation to promote CPC translocation from the kinetochore/centromere to the spindle. Importantly, premature Fin1 kinetochore localization or phospho-deficient sli15 mutation causes checkpoint defects in response to tensionless attachments, resulting in chromosome missegregation. In addition, our data indicate that reversion of CDK- and Ipl1-imposed Sli15 phosphorylation shows an additive effect on CPC translocation. Together, these results reveal a previously unidentified pathway to regulate CPC translocation, which is important for accurate chromosome segregation.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Saccharomycetales/metabolismo , Centrómero/metabolismo , Cinetocoros/metabolismo , Huso Acromático/metabolismo , FosforilaciónRESUMEN
Background: Pin1 is a member of the evolutionarily conserved peptidyl-prolyl isomerase (PPIase) family of proteins. Following phosphorylation, Pin1-catalyzed prolyl-isomerization induces conformational changes, which serve to regulate the function of many phosphorylated proteins that play important roles during oncogenesis. Thus, the inhibition of Pin1 provides a unique means of disrupting oncogenic pathways and therefore represents an appealing target for novel anticancer therapies. Methods: As Pin1 is conserved between yeast and humans, we employed budding yeast to establish a high-throughput screening method for the primary screening of Pin1 inhibitors. This effort culminated in the identification of the compounds HWH8-33 and HWH8-36. Multifaceted approaches were taken to determine the inhibition profiles of these compounds against Pin1 activity in vitro and in vivo, including an isomerization assay, surface plasmon resonance (SPR) technology, virtual docking, MTT proliferation assay, western blotting, cell cycle analysis, apoptosis analysis, immunofluorescence analysis, wound healing, migration assay, and nude mouse assay. Results: In vitro, HWH8-33 and HWH8-36 could bind to purified Pin1 and inhibited its enzyme activity; showed inhibitory effects on cancer cell proliferation; led to G2/M phase arrest, dysregulated downstream protein expression, and apoptosis; and suppressed cancer cell migration. In vivo, HWH8-33 suppressed tumor growth in the xenograft mice after oral administration for 4 weeks, with no noticeable toxicity. Together, these results show the anticancer activity of HWH8-33 and HWH8-36 against Pin1 for the first time. Conclusion: In summary, we identified two hit compounds HWH8-33 and HWH8-36, which after further structure optimization have the potential to be developed as antitumor drugs.
RESUMEN
Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting virus protease. For complete details on the use and execution of this protocol, please refer to Li et al. (2022), Yan et al. (2021), and Yan et al. (2022c).
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Ensayos Analíticos de Alto Rendimiento , Humanos , SARS-CoV-2 , Proteínas no Estructurales Virales , Cisteína Endopeptidasas , Inhibidores de Proteasas/farmacología , Antivirales/farmacología , Polarización de FluorescenciaRESUMEN
Accurate chromosome segregation depends on bipolar chromosome-microtubule attachment and tension generation on chromosomes. Incorrect chromosome attachment results in chromosome missegregation, which contributes to genome instability. The kinetochore is a protein complex that localizes at the centromere region of a chromosome and mediates chromosome-microtubule interaction. Incorrect chromosome attachment leads to checkpoint activation to prevent anaphase onset. Kinetochore detachment activates the spindle assembly checkpoint (SAC), while tensionless kinetochore attachment relies on both the SAC and tension checkpoint. In budding yeast Saccharomyces cerevisiae, kinesin-5 motor proteins Cin8 and Kip1 are needed to separate spindle pole bodies for spindle assembly, and deletion of CIN8 causes lethality in the absence of SAC. To study the function of Cin8 and Kip1 in chromosome segregation, we constructed an auxin-inducible degron (AID) mutant, cin8-AID. With this conditional mutant, we first confirmed that cin8-AID kip1∆ double mutants were lethal when Cin8 is depleted in the presence of auxin. These cells arrested in metaphase with unseparated spindle pole bodies and kinetochores. We further showed that the absence of either the SAC or tension checkpoint was sufficient to abolish the cell-cycle delay in cin8-AID mutants, causing chromosome missegregation and viability loss. The tension checkpoint-dependent phenotype in cells with depleted Cin8 suggests the presence of tensionless chromosome attachment. We speculate that the failed spindle pole body separation in cin8 mutants could increase the chance of tensionless syntelic chromosome attachments, which depends on functional tension checkpoint for survival.
Asunto(s)
Segregación Cromosómica/genética , Cinesinas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Dineínas/genética , Ácidos Indolacéticos/metabolismo , Cinesinas/genética , Cinetocoros/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC50 value of 24.26 ± 0.4 µM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening.
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COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Proteasas Similares a la Papaína de Coronavirus , Polarización de Fluorescencia , Humanos , Papaína , Péptido Hidrolasas , Estudios ProspectivosRESUMEN
Protein misfolding and aggregation are implicated in many neurodegenerative diseases. One of these diseases is Huntington's, which is caused by increased glutamine-encoding trinucleotide repeats within the Huntingtin gene. Like other misfolded proteins, mutated Huntingtin proteins with polyglutamine expansions are prone to aggregation. Misfolded proteins exist as soluble monomers, small aggregates, or as large insoluble inclusion bodies. Misfolded protein aggregates are believed to be cytotoxic by stressing the protein degradation machinery, disrupting membrane structure, or sequestering other proteins. We recently showed that expression of misfolded proteins lowers cellular free ubiquitin levels, which compromises the protein degradation machinery. Therefore, the efficient degradation of misfolded proteins is critical to preserve cell health. Cells employ two major mechanisms to degrade misfolded proteins. The first is the ubiquitin-proteasome system (UPS), which ubiquitinates and degrades misfolded proteins with the assistance of segregase Cdc48/p97. The UPS pathway is mainly responsible for the clearance of misfolded proteins present as monomers or smaller aggregates. The second pathway is macroautophagy/autophagy, in which protein aggregates or inclusion bodies are recruited into an autophagosome before transport to the vacuole/lysosome for degradation. This review is focused on the current understanding of the cytotoxicity of misfolded proteins as well as their clearance pathways, with a particular emphasis on mutant Huntingtin.
Asunto(s)
Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Pliegue de Proteína , Animales , Autofagia , Muerte Celular , Humanos , Modelos Biológicos , Agregado de ProteínasRESUMEN
DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.
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Anafase/genética , Replicación del ADN/fisiología , Proteína Fosfatasa 2/metabolismo , Saccharomyces cerevisiae/genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Hidroxiurea/farmacología , Microorganismos Modificados Genéticamente , Mutación , Fosforilación , Proteína Fosfatasa 2/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Fase S/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Securina/genética , Securina/metabolismoRESUMEN
The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation.
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Anafase , Aurora Quinasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Segregación Cromosómica , Cinetocoros/química , Cinetocoros/efectos de los fármacos , Viabilidad Microbiana/genética , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fosforilación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático/efectos de los fármacos , Factores de TiempoRESUMEN
The demand for novel antibiotics is imperative for drug-resistant Gram-negative bacteria which causes diverse intractable infection disease in clinic. Here, a comprehensive screening was implemented to identify potential agents that disrupt the assembly of ß-barrel outer-membrane proteins (OMPs) in the outer membrane (OM) of Gram-negative bacteria. The assembly of OMPs requires ubiquitous ß-barrel assembly machinery (BAM). Among the five protein subunits in BAM, the interaction between BamA and BamD is essential for the function of this complex. We first established a yeast two-hybrid (Y2H) system to confirm the interaction between BamA and BamD, and then screened agents that specifically disrupt this interaction. From this screen, we identified a compound IMB-H4 that specially blocks BamA-BamD interaction and selectively inhibits the growth of Escherichia coli and other Gram-negative bacteria. Moreover, our results suggest that IMB-H4 disrupts BamA-BamD interaction by binding to BamA. Strikingly, E. coli cells having been treated with IMB-H4 showed impaired OM integrity and decreased the abundance of OMPs. Therefore, an antibacterial agent was identified successfully using Y2H system, and this compound likely blocks the assembly of OMPs by targeting BamA-BamD interaction in Gram-negative bacteria.
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The accumulation of misfolded proteins is associated with multiple neurodegenerative disorders, but it remains poorly defined how this accumulation causes cytotoxicity. Here, we demonstrate that the Cdc48/p97 segregase machinery drives the clearance of ubiquitinated model misfolded protein Huntingtin (Htt103QP) and limits its aggregation. Nuclear ubiquitin ligase San1 acts upstream of Cdc48 to ubiquitinate Htt103QP. Unexpectedly, deletion of SAN1 and/or its cytosolic counterpart UBR1 rescues the toxicity associated with Cdc48 deficiency, suggesting that ubiquitin depletion, rather than compromised proteolysis of misfolded proteins, causes the growth defect in cells with Cdc48 deficiency. Indeed, Cdc48 deficiency leads to elevated protein ubiquitination levels and decreased free ubiquitin, which depends on San1/Ubr1. Furthermore, enhancing free ubiquitin levels rescues the toxicity in various Cdc48 pathway mutants and restores normal turnover of a known Cdc48-independent substrate. Our work highlights a previously unappreciated function for Cdc48 in ensuring the regeneration of monoubiquitin that is critical for normal cellular function.
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Homeostasis , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Proteína que Contiene Valosina/metabolismo , Muerte Celular , Proteína Huntingtina/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Temperatura , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación , Proteína que Contiene Valosina/genéticaRESUMEN
Accurate chromosome segregation during cell division is essential to maintain genome integrity in all eukaryotic cells, and chromosome missegregation leads to aneuploidy and therefore represents a hallmark of many cancers. Accurate segregation requires sister kinetochores to attach to microtubules emanating from opposite spindle poles, known as bipolar attachment or biorientation. Recent studies have uncovered several mechanisms critical to chromosome bipolar attachment. First, a mechanism exists to ensure that the conformation of sister centromeres is biased toward bipolar attachment. Second, the phosphorylation of some kinetochore proteins destabilizes kinetochore attachment to facilitate error correction, but a protein phosphatase reverses this phosphorylation. Moreover, the activity of the spindle assembly checkpoint is regulated by kinases and phosphatases at the kinetochore, and this checkpoint prevents anaphase entry in response to faulty kinetochore attachment. The fine-tuned kinase/phosphatase balance at kinetochores is crucial for faithful chromosome segregation during both mitosis and meiosis. Here, we discuss the function and regulation of protein phosphatases in the establishment of chromosome bipolar attachment with a focus on the model organism budding yeast.
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Segregación Cromosómica , Meiosis , Mitosis , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cinetocoros , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The R2R3 MYB genes associated with the flavonoid/anthocyanidin pathway feature two repeats, and represent the most abundant classes of MYB genes in plants; however, the physiological role and regulatory function of most R2R3 MYBs remain poorly understood in kiwifruit (Actinidia). Here, genome-wide analysis identified 155 R2R3-MYBs in the 'Red 5' version of the Actinidia chinensis genome. Out of 36 anthocyanin-related AccR2R3-MYBs, AcMYB10 was the most highly expressed in inner pericarp of red-fleshed kiwifruit. The expression of AcMYB10 was highly correlated with anthocyanin accumulation in natural pigmentation during fruit ripening and light-/temperature-induced pigmentation in the callus. AcMYB10 is localized in the nuclei and has transcriptional activation activity. Overexpression of AcMYB10 elevates anthocyanin accumulation in transgenic A. chinensis. In comparison, A. chinensis fruit infiltrated with virus-induced gene silencing showed delayed red coloration, lower anthocyanin content, and lower expression of AcMYB10. The transient expression experiment in Nicotiana tabacum leaves and Actinidia arguta fruit indicated the interaction of AcMYB10 with AcbHLH42 might strongly activate anthocyanin biosynthesis by activating the transcription of AcLDOX and AcF3GT. In conclusion, this study provides novel molecular information about R2R3-MYBs in kiwifruit, advances our understanding of light- and temperature-induced anthocyanin accumulation, and demonstrates the important function of AcMYB10 in the biosynthesis of anthocyanin in kiwifruit.
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Actinidia/metabolismo , Antocianinas/biosíntesis , Respuesta al Choque Térmico , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Actinidia/genética , Antocianinas/genética , Frutas/genética , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Luz Solar , Factores de Transcripción/genéticaRESUMEN
Fungal infection is a leading cause of mortality in immunocompromised population; thus, it is urgent to develop new and safe antifungal agents. Different from human cells, fungi have a cell wall, which is composed mainly of polysaccharide glucan and chitin. The unique cell wall structure is an ideal target for antifungal drugs. In this research, a chemical-genetic method was used to isolate antifungal agents that target chitin synthesis in yeast cells. From a compound library, we isolated two benzothiazole compounds that showed greater toxicity to yeast mutants lacking glucan synthase Fks1 compared to wild-type yeast cells and mutants lacking chitin synthase Chs3. Both of them inhibited the activity of chitin synthase in vitro and reduced chitin level in yeast cells. Besides, these compounds showed clear synergistic antifungal effect with a glucan synthase inhibitors caspofungin. Furthermore, these compounds inhibited the growth of Saccharomyces cerevisiae and opportunistic pathogen Candida albicans. Surprisingly, the genome-wide mass-spectrometry analysis showed decreased protein level of chitin synthases in cells treated with one of these drugs, and this decrease was not a result of downregulation of gene transcription. Therefore, we successfully identified two new antifungal agents that inhibit chitin synthesis using a chemical-genetic method.