RESUMEN
The cytosolic isozyme of phosphoenolpyruvate carboxykinase (PCK1) was the first rate-limiting enzyme in the gluconeogenesis pathway, which exerted a critical role in maintaining the blood glucose levels. PCK1 has been established to be involved in various physiological and pathological processes, including glucose metabolism, lipid metabolism, diabetes, and tumorigenesis. Nonetheless, the association of PCK1 with aging process and the detailed underlying mechanisms of PCK1 on aging are still far to be elucidated. Hence, we herein constructed the PCK1-deficient (pck1Δ) and PCK1 overexpression (PCK1 OE) Saccharomyces cerevisiae. The results unveiled that PCK1 deficiency significantly shortened the replicative lifespan (RLS) in the S. cerevisiae, while overexpression of PCK1 prolonged the RLS. Additionally, we noted that the ROS level was significantly enhanced in PCK1-deficient strain and decreased in PCK1 OE strain. Then, a high throughput analysis by deep sequencing was performed in the pck1Δ and wild-type strains, in an attempt to shed light on the effect of PCK1 on the lifespan of aging process. The data showed that the most downregulated mRNAs were enriched in the regulatory pathways of glucose metabolism. Fascinatingly, among the differentially expressed mRNAs, PFK1 was one of the most upregulated genes, which was involved in the glycolysis process and ROS generation. Thus, we further constructed the pfk1Δpck1Δ strain by deletion of PFK1 in the PCK1-deficient strain. The results unraveled that pfk1Δpck1Δ strain significantly suppressed the ROS level and restored the RLS of pck1Δ strain. Taken together, our data suggested that PCK1 deficiency enhanced the ROS level and shortened the RLS of S. cerevisiae via PFK1.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Fosfoenolpiruvato Carboxiquinasa (ATP) , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fosfoenolpiruvato Carboxiquinasa (ATP)/deficiencia , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Beta-1,3-glucanosyltransferase (Gas1p) plays important roles in cell wall biosynthesis and morphogenesis and has been implicated in DNA damage responses and cell cycle regulation in fungi. Yeast Gas1p has also been reported to participate in endoplasmic reticulum (ER) stress responses. However, the precise roles and molecular mechanisms through which Gas1p affects these responses have yet to be elucidated. In this study, we constructed GAS1-deficient (gas1Δ) and GAS1-overexpressing (GAS1 OE) yeast strains and observed that the gas1Δ strain exhibited a decreased proliferation ability and a shorter replicative lifespan (RLS), as well as enhanced activity of the unfolded protein response (UPR) in the absence of stress. However, under the high-tunicamycin-concentration (an ER stress-inducing agent; 1.0 µg/mL) stress, the gas1Δ yeast cells exhibited an increased proliferation ability compared with the wild-type yeast strain. In addition, our findings demonstrated that IRE1 and HAC1 (two upstream modulators of the UPR) are required for the survival of gas1Δ yeast cells under the tunicamycin stress. On the other hand, we provided evidence that the GAS1 overexpression caused an obvious sensitivity to the low-tunicamycin-concentration (0.25 µg/mL). Collectively, our results indicate that Gas1p plays an important role in the ageing and ER stress responses in yeast.
Asunto(s)
Glicoproteínas de Membrana/deficiencia , Saccharomyces cerevisiae/metabolismo , Respuesta de Proteína Desplegada , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Replicación del ADN/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Viabilidad Microbiana/efectos de los fármacos , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
The Saccharomyces cerevisiae chaperone gene SSB2 belongs to the Hsp70 family. Unlike other HSP70 genes, SSB2 gene expression is reduced after heat shock. It has been reported that Ssb2p can be cross-linked to ribosome-bound nascent polypeptide chains, suggesting a potential role of SSB2 in the endoplasmic reticulum (ER) stress response. In this study, SSB2-deletion and SSB2-overexpression yeast strains were generated and applied to explore the potential mechanism by which SSB2 is involved in the tunicamycin (TM)-induced ER stress response. We demonstrate for the first time that SSB2 deficiency results in reduced resistance to TM, while overexpression of SSB2 increases resistance to TM in an IRE1-HAC1 pathway-dependent manner; these observations are related to changes in intracellular unfolded protein response activities (under the TM-stressed condition). Additionally, SSB2 deletion induces early apoptosis and it may play a causal role in the shortened replicative life span of ssb2Δ mutants observed in this study. These findings highlight the involvement of SSB2 in ER stress responses and ageing in yeast.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Estrés del Retículo Endoplásmico , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Adenosina Trifosfatasas/genética , Antifúngicos/farmacología , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Protein O-mannosyltransferase-1 (Pmt1p) deficiency extends the replicative lifespan (RLS) of Saccharomyces cerevisiae, which is related to the activation of the unfolded protein response (UPR), an important pathway for alleviating endoplasmic reticulum (ER) stress. Trafficking of Emp24p/Erv25p-dependent cargo disrupted 1 (Ted1p) has been reported as a binding partner of yeast Pmt1p. We explored the potential relationship between Pmt1p and Ted1p in the cell lifespan and ER stress responses. The TED1-deleted strain (ted1Δ) had a shorter RLS with no increase in UPR activity. However, PMT1 deficiency prolonged the short lifespan of ted1Δ in a manner dependent on Hac1p, an upstream transcription factor of the UPR pathway. In addition, PMT1 deficiency enhanced the UPR activity and alleviated the ER stress resistance of the ted1Δ strain. Thus, the enhanced UPR activity was hypothesized to explain the longevity of the pmt1Δted1Δ strain, but this long-lived pmt1Δted1Δ strain showed decreased ER stress resistance compared with the short-lived ted1Δ strain. Taken together, our results suggest a possible relationship between PMT1 and TED1 regarding lifespan regulation and the ER stress response.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Replicación del ADN , Estrés del Retículo Endoplásmico , Manosiltransferasas/genética , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Regulación Fúngica de la Expresión Génica , Manosiltransferasas/metabolismo , Pliegue de Proteína , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Respuesta de Proteína DesplegadaRESUMEN
Ubiquitin is a 76-amino acid protein that is highly conserved among higher and lower eukaryotes. The polyubiquitin gene UBI4 encodes a unique precursor protein that contains five ubiquitin repeats organized in a head-to-tail arrangement. Although the involvement of the yeast polyubiquitin gene UBI4 in the stress response was reported long ago, there are no reports regarding the underlying mechanism of this involvement. In this study, we used UBI4-deletion and UBI4-overexpressing yeast strains as models to explore the potential mechanism by which UBI4 protects yeast cells against paraquat-induced oxidative stress. Here, we show that ubi4Δ cells exhibit oxidative stress, an apoptotic phenotype, and a decreased replicative lifespan. Additionally, the reduced resistance of ubi4Δ cells to paraquat that was observed in this study was rescued by overexpression of either the catalase or the mitochondrial superoxide dismutase SOD2. We also demonstrated that only SOD2 overexpression restored the replicative lifespan of ubi4Δ cells. In contrast to the case of ubi4Δ cells, UBI4 overexpression in wild-type yeast increases the yeast's resistance to paraquat, and this overexpression is associated with large pools of expressed ubiquitin and increased levels of ubiquitinated proteins. Collectively, these findings highlight the role of the polyubiquitin gene UBI4 in apoptosis and implicate UBI4 as a modulator of the replicative lifespan.
Asunto(s)
Apoptosis/genética , Replicación del ADN/genética , Poliubiquitina/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Ubiquitina C/deficiencia , Ubiquitina C/genética , Apoptosis/efectos de los fármacos , Catalasa/metabolismo , Replicación del ADN/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mutación/genética , Paraquat/toxicidad , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Ubiquitina C/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Ksplp is a nuclear-localized Ser/Thr kinase that is not essential for the vegetative growth of yeast. A global gene function analysis in yeast suggested that Ksplp was involved in the oxidative stress response; however, the underlying mechanism remains unclear. Here, we showed that KSP1-deficient yeast cells exhibit hypersensitivity to the DNA alkylating agent methyl methanesulphonate (MMS), and treatment of the KSP1-deficient strain with MMS could trigger abnormal mitochondrial membrane potential and up-regulate reactive oxygen species (ROS) production. In addition, the mRNA expression level of the catalase gene CTT1 (which encodes cytosolic catalase) and total catalase activity were strongly down-regulated in the KSP1-deleted strain compared with those in wild-type cells. Moreover, the KSP1 deficiency also leads to a shortened replicative lifespan, which could be restored by the increased expression of CTT1. On the other hand, KSP1-overexpressed (KSP1OX) yeast cells exhibited increased resistance towards MMS, an effect that was, at least in part, CTT1 independent. Collectively, these findings highlight the involvement of Ksplp in the DNA damage response and implicate Ksplp as a modulator of the replicative lifespan.
Asunto(s)
Catalasa/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/deficiencia , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae , Catalasa/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMEN
Pmt1p is an important member of the protein O-mannosyltransferase (PMT) family of enzymes, which participates in the endoplasmic reticulum (ER) unfolded protein response (UPR), an important pathway for alleviating ER stress. ER stress and the UPR have been implicated in aging and age-related diseases in several organisms; however, a possible role for PMT1 in determining lifespan has not been previously described. In this study, we report that deletion of PMT1 increases replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae, while overexpression of PMT1 (PMT1-OX) reduces RLS. Relative to wild-type and PMT1-OX strains, the pmt1Δ strain had enhanced HAC1 mRNA splicing and elevated expression levels of UPR target genes. Furthermore, the increased RLS of the pmt1Δ strain could be completely abolished by deletion of either IRE1 or HAC1, two upstream modulators of the UPR. The double deletion strains pmt1Δhac1Δ and pmt1Δire1Δ also displayed generally reduced transcription of UPR target genes. Collectively, our results suggest that PMT1 deficiency enhances basal activity of the ER UPR and extends the RLS of yeast mother cells through a mechanism that requires both IRE1 and HAC1.