Yeast Tor complex 1 phosphorylates eIF4E-binding protein, Caf20.

Tor complex 1 (TORC1), a master regulator of cell growth, is an evolutionarily conserved protein kinase within eukaryotic organisms. To control cell growth, TORC1 governs translational processes by phosphorylating its substrate proteins in response to cellular nutritional cues. Mammalian TORC1 (mTORC1) assumes the responsibility of phosphorylating the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) to regulate its interaction with eIF4E. The budding yeast Saccharomyces cerevisiae possesses a pair of 4E-BP genes, CAF20 and EAP1. However, the extent to which the TORC1-4E-BP axis regulates translational initiation in yeast remains uncertain. In this study, we demonstrated the influence of TORC1 on the phosphorylation status of Caf20 in vivo, as well as the direct phosphorylation of Caf20 by TORC1 in vitro. Furthermore, we found the TORC1-dependent recruitment of Caf20 to the 80S ribosome. Consequently, our study proposes a plausible involvement of yeast's 4E-BP in the efficacy of translation initiation, an aspect under the control of TORC1.

Serine metabolism contributes to cell survival by regulating extracellular pH and providing an energy source in Saccharomyces cerevisiae.

Changes in extracellular pH affect the homeostasis and survival of unicellular organisms. Supplementation of culture media with amino acids can extend the lifespan of budding yeast, Saccharomyces cerevisiae, by alleviating the decrease in pH. However, the optimal amino acids to use to achieve this end, and the underlying mechanisms involved, remain unclear. Here, we describe the specific role of serine metabolism in the regulation of pH in a medium. The addition of serine to synthetic minimal medium suppressed acidification, and at higher doses increased the pH. CHA1, which encodes a catabolic serine hydratase that degrades serine into ammonium and pyruvate, is essential for serine-mediated alleviation of acidification. Moreover, serine metabolism supports extra growth after glucose depletion. Therefore, medium supplementation with serine can play a prominent role in the batch culture of budding yeast, controlling extracellular pH through catabolism into ammonium and acting as an energy source after glucose exhaustion.

Genomic profile of radiation-induced early-onset mouse B-cell lymphoma recapitulates features of Philadelphia chromosome-like acute lymphoblastic leukemia in humans.

Epidemiological studies have revealed a radiation-related increase in the risk of developing acute lymphoblastic leukemia (ALL). Our recent study revealed early induction and increased risk of precursor B-cell (pB) lymphomas in mice after radiation exposure. However, the genomic landscape of radiation-induced B-cell lymphomas remains unclear. To identify the relevant genetic alterations in mice, whole-exome sequencing was performed on both early-onset and late-onset B-cell lymphomas that developed spontaneously or after gamma-irradiation. In addition to multiple driver mutations, the data revealed that interstitial deletion of chromosome 4, including Pax5, and missense mutations in Jak3 are unique genomic alterations in radiation-induced, early-onset B-cell lymphomas. RNA sequencing revealed a pB-cell-type gene-expression profile with no involvement of known fusion genes for human ALLs in the early-onset B-cell lymphomas. Activation of Jak3/Stat5 signaling in early-onset B-cell lymphomas was validated using western capillary electrophoresis. Those features were similar to those of Philadelphia chromosome-like ALL. Our data suggest a critical role for Pax5 loss-of-function mutations in initiating B-cell leukemogenesis coupled with activation of Jak3/Stat5 signaling as a basis for the rapid development of radiation-induced pB-ALL. These molecular signatures for radiation-induced cancers will inform both risk assessment and potential targeted therapies for pB-ALL.

Labeling and measuring stressed mitochondria using a PINK1-based ratiometric fluorescent sensor.

Mitochondria are essential organelles that carry out a number of pivotal metabolic processes and maintain cellular homeostasis. Mitochondrial dysfunction caused by various stresses is associated with many diseases such as type 2 diabetes, obesity, cancer, heart failure, neurodegenerative disorders, and aging. Therefore, it is important to understand the stimuli that induce mitochondrial stress. However, broad analysis of mitochondrial stress has not been carried out to date. Here, we present a set of fluorescent tools, called mito-Pain (mitochondrial PINK1 accumulation index), which enable the labeling of stressed mitochondria. Mito-Pain uses PTEN-induced putative kinase 1 (PINK1) stabilization on mitochondria and quantifies mitochondrial stress levels by comparison with PINK1-GFP, which is stabilized under mitochondrial stress, and RFP-Omp25, which is constitutively localized on mitochondria. To identify compounds that induce mitochondrial stress, we screened a library of 3374 compounds using mito-Pain and identified 57 compounds as mitochondrial stress inducers. Furthermore, we classified each compound into several categories based on mitochondrial response: depolarization, mitochondrial morphology, or Parkin recruitment. Parkin recruitment to mitochondria was often associated with mitochondrial depolarization and aggregation, suggesting that Parkin is recruited to heavily damaged mitochondria. In addition, many of the compounds led to various mitochondrial morphological changes, including fragmentation, aggregation, elongation, and swelling, with or without Parkin recruitment or mitochondrial depolarization. We also found that several compounds induced an ectopic response of Parkin, leading to the formation of cytosolic puncta dependent on PINK1. Thus, mito-Pain enables the detection of stressed mitochondria under a wide variety of conditions and provides insights into mitochondrial quality control systems.

Forced lipophagy reveals that lipid droplets are required for early embryonic development in mouse.

Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.

Impact of the Antithrombotic Effects of Prasugrel on Mid-Term Vascular Healing in Acute Coronary Syndrome vs. Stable Coronary Artery Disease.

BACKGROUND: The impact of antiplatelet drug effects on mid-term local arterial responses following percutaneous coronary intervention (PCI) remains uncertain. We evaluated the impact of the platelet reactivity of prasugrel on mid-term vascular healing between acute coronary syndrome (ACS) and stable coronary artery disease (CAD).Methods and Results:We conducted a prospective, 12-center study in 125 patients with ACS and 126 patients with stable CAD who underwent PCI with an everolimus-eluting stent (EES) and received dual antiplatelet therapy (DAPT) with prasugrel and aspirin. Serial optical coherence tomography (OCT) was performed immediately after PCI and at the 9-month follow-up to assess the association of P2Y12reaction units (PRU) with the frequency of malapposed or uncovered struts and intrastent thrombi (IST). The incidence of abnormal mid-term OCT findings did not different between the ACS and CAD arms, regardless of clinical presentation, except that uncovered struts were more frequent in the ACS than CAD arm. PRU at PCI was significantly associated with the frequency of IST at follow-up, but not with uncovered and malapposed struts. PRU at PCI was the only independent predictor of IST detected at follow-up (odds ratio 1.009). CONCLUSIONS: In patients undergoing EES implantation and receiving prasugrel, achieving an adequate antiplatelet effect at the time of stent implantation may regulate thrombus formation throughout the follow-up period.

Reversible DNA damage checkpoint activation at the presenescent stage in telomerase-deficient cells of Saccharomyces cerevisiae.

The telomere protects the ends of eukaryotic linear chromosomes, and its shortening or erosion is recognized as DNA damage, leading to loss of proliferation activity and, thus, cellular senescence at the population level. Here, using a GFP-based DNA damage checkpoint marker suited for single-cell observation of Saccharomyces cerevisiae cells, we correlated the checkpoint status of telomere-shortened cells with their behavior. We show that some cells possessing short telomeres retain proliferation capacity even after the DNA damage checkpoint is activated. At the presenescent stage, the activation of the checkpoint causes cell cycle delay, but does not induce permanent cell cycle arrest, eventually leading to the expansion of cell size that is characteristic of cellular senescence. Moreover, the proliferation capacity of checkpoint-activated cells is not dependent on homologous recombination or the checkpoint adaptation pathway. The retention of proliferation capacity is specific to the telomere-derived DNA damage response, suggesting that damaged telomeres differ functionally from other types of DNA damage. Our data establish the role of the presenescent stage in telomere shortening-induced senescence, which proceeds gradually and is associated with a variety of changes, including altered cell morphology and metabolism.

Phosphatase-dependent fluctuations in DNA-damage checkpoint activation at partially defective telomeres.

Telomeres protect the ends of eukaryotic chromosomes, and telomere shortening causes irreversible cell-cycle arrest through activation of the DNA-damage checkpoint. In this study, we found that deletion of PPH3, encoding a 2A-like protein phosphatase, accelerated telomere-shortening-mediated senescence without affecting normal telomere length or the telomere erosion rate in Saccharomyces cerevisiae. Moreover, the loss of PPH3 increased sensitivity to telomere dysfunction. The detection of telomere abnormalities by DNA-damage sensors was not an all-or-none response, implying that Pph3 helps determine the border between normal and dysfunctional telomeres by suppressing premature activation of the DNA-damage checkpoint.

Impact of dual antiplatelet therapy with adjusted-dose prasugrel on mid-term vascular response in patients undergoing elective percutaneous coronary intervention with everolimus-eluting stents.

The impact of dual antiplatelet therapy (DAPT) with adjusted-dose (3.75 mg/day) prasugrel for Japanese patients has not been fully investigated in terms of local arterial healing following the elective percutaneous coronary intervention (PCI). The ROUTE-01 elective study was a prospective, 12-center and single-arm registry that enrolled 123 patients who underwent elective PCI with everolimus-eluting stents (EESs) under DAPT with a combination of adjusted-dose prasugrel and aspirin. Serial optical coherence tomography (OCT) was performed at the index PCI and 9-month follow-up to assess the relationship between in-stent thorombus (IST) and residual platelet reactivity measuring platelet reactivity unit (PRU). The patients were classified as extensive, intermediate, and poor metabolizers by cytochrome P450 2C19 (CYP2C19) loss-of-function polymorphisms. The prevalence of IST was 9.0% by 9-month OCT, with no difference amongst the three groups (p = 0.886). The incidences of malapposed and uncovered struts were not different among the groups. PRU was not statistically different among the groups. In multivariate logistic regression analysis, the independent predictor for IST on 9-month OCT was irregular protrusion (odds ratio = 8.952, p = 0.037) on post-PCI OCT, not CYP2C19 loss-of-function polymorphisms. An adequate anti-thrombotic effect with an acceptable incidence of IST was observed irrespective of CYP2C19 loss-of-function polymorphisms. Our data suggests that adjusted-dose prasugrel and aspirin is a feasible treatment option in Japanese patients treated with EESs in elective PCI.

Use of CK-548 and CK-869 as Arp2/3 complex inhibitors directly suppresses microtubule assembly both in vitro and in vivo.

Two types of Arp2/3 complex inhibitors, CK-666/636 and CK-548/869, are commonly used to study Arp2/3 complex-dependent actin assembly both in vitro and in vivo. However, we found that CK-548 and CK-869 directly suppress microtubule (MT) assembly independent of the actin cytoskeleton. Treatment of cultured mammalian cells with 50 µM CK-869 dramatically decreased MT networks and, instead, accumulated tubulin at the cell periphery, as did nocodazole that inhibits MT assembly. An in vitro MT-sedimentation assay revealed that CK-548 and CK-869 significantly suppressed MT polymerization. In budding yeast, although CK-548 and CK-869 are reported to lack binding abilities in the yeast Arp3, CK-548 treatment decreased cytoplasmic MT at several tens of micromolar concentrations. In addition, we found that the effects of CK-548 and CK-869 on MT assembly varied according to species. We propose that CK-548 and CK-869 are not suitable for studying the cytoskeleton in living cells.

The role of autophagy in genome stability through suppression of abnormal mitosis under starvation.

The coordination of subcellular processes during adaptation to environmental change is a key feature of biological systems. Starvation of essential nutrients slows cell cycling and ultimately causes G1 arrest, and nitrogen starvation delays G2/M progression. Here, we show that budding yeast cells can be efficiently returned to the G1 phase under starvation conditions in an autophagy-dependent manner. Starvation attenuates TORC1 activity, causing a G2/M delay in a Swe1-dependent checkpoint mechanism, and starvation-induced autophagy assists in the recovery from a G2/M delay by supplying amino acids required for cell growth. Persistent delay of the cell cycle by a deficiency in autophagy causes aberrant nuclear division without sufficient cell growth, leading to an increased frequency in aneuploidy after refeeding the nitrogen source. Our data establish the role of autophagy in genome stability through modulation of cell division under conditions that repress cell growth.

Newly isolated Penicillium oxalicum A592-4B secretes enzymes that degrade milled rice straw with high efficiency.

An enzyme producing micro-organism, which can directly saccharify rice straw that has only been crushed without undergoing the current acid or alkaline pretreatment, was found. From the homology with the ITS, 28S rDNA sequence, the strain named A592-4B was identified as Penicillium oxalicum. Activities of the A592-4B enzymes and commercial enzyme preparations were compared by Novozymes Cellic CTec2 and Genencore GC220. In the present experimental condition, activity of A592-4B enzymes was 2.6 times higher than that of CTec2 for degrading milled rice straw. Furthermore, even when a quarter amount of A592-4B enzyme was applied to the rice straw, the conversion rate was still higher than that by CTec2. By utilizing A592-4B enzymes, improved lignocellulose degradation yields can be achieved without pre-treatment of the substrates; thus, contributing to cost reduction as well as reducing environmental burden.

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair.

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.

A Steroidal Saponin from Ophiopogon japonicus Extends the Lifespan of Yeast via the Pathway Involved in SOD and UTH1.

Nolinospiroside F is a steroidal saponin isolated from Ophiopogon japonicus (O. japonicus). In this study, we found that nolinospiroside F significantly extends the replicative lifespan of K6001 yeast at doses of 1, 3 and 10 µM, indicating that it has an anti-aging effect. This may be attributed to its anti-oxidative effect, as nolinospiroside F could increase yeast survival under oxidative stress conditions and decrease the level of malondialdehyde (MDA), an oxidative stress biomarker. It could also increase anti-oxidative stress genes, SOD1 and SOD2, expression, and the activity of superoxide dismutase (SOD). It increase the activity of SIRT1, an upstream inducer of SOD2 expression. In sod1 and sod2 mutant yeast strains, nolinospiroside F failed to extend their replicative lifespan. These results indicate that SOD participates in the anti-aging effect of nolinospiroside F. Furthermore, nolinospiroside F inhibited the expression of UTH1, a yeast-aging gene that is involved in the oxidative stress of yeast, and failed to extend the replicative lifespan of uth1 or skn7 mutant yeast cells. SKN7 is the transcriptional activator of UTH1. We also demonstrate that SOD and UTH1 regulate each other's expression. Together, these results suggest that SOD and UTH1 genes are required for and play interactive roles in nolinospiroside F-mediated yeast lifespan extension.

Artificial control of mating type and repeated mating to produce polyploid cells in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is an excellent model for examining the effects of ploidy. Here, we provide a protocol for producing polyploid cells by creating a basic unit (matΔ) and polyploidizing it via repeated mating. We describe steps for basic unit construction by one-step transformation, increased ploidy via repeated mating, and ploidy confirmation using flow cytometry. This protocol can be broadly applied to evaluate the physiology of polyploid cells. For complete details on the use and execution of this protocol, please refer to Oya and Matsuura (2022).1.

Alpha 2-macroglobulin acts as a clearance factor in the lysosomal degradation of extracellular misfolded proteins.

Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many age-related diseases. Within the cell, molecular chaperones facilitate the refolding of misfolded proteins into their bioactive forms, thus preventing undesirable interactions and aggregation. Although the mechanisms of intracellular protein degradation pathways for intracellular misfolded proteins have been extensively studied, the protein degradation pathway for extracellular proteins remain poorly understood. In this study, we identified several misfolded proteins that are substrates for alpha 2-macroglobulin (α2M), an extracellular chaperone. We also established a lysosomal internalization assay for α2M, which revealed that α2M mediates the lysosomal degradation of extracellular misfolded proteins. Comparative analyses of α2M and clusterin, another extracellular chaperone, indicated that α2M preferentially targets aggregation-prone proteins. Thus, we present the degradation pathway of α2M, which interacts with aggregation-prone proteins for lysosomal degradation via selective internalization.

Isoquercitrin from Apocynum venetum L. Exerts Antiaging Effects on Yeasts via Stress Resistance Improvement and Mitophagy Induction through the Sch9/Rim15/Msn Signaling Pathway.

BACKGROUND: With the development of an aging sociality, aging-related diseases, such as Alzheimer's disease, cardiovascular disease, and diabetes, are dramatically increasing. To find small molecules from natural products that can prevent the aging of human beings and the occurrence of these diseases, we used the lifespan assay of yeast as a bioassay system to screen an antiaging substance. Isoquercitrin (IQ), an antiaging substance, was isolated from Apocynum venetum L., an herbal tea commonly consumed in Xinjiang, China. AIM OF THE STUDY: In the present study, we utilized molecular-biology technology to clarify the mechanism of action of IQ. METHODS: The replicative lifespans of K6001 yeasts and the chronological lifespans of YOM36 yeasts were used to screen and confirm the antiaging effect of IQ. Furthermore, the reactive oxygen species (ROS) and malondialdehyde (MDA) assay, the survival assay of yeast under stresses, real-time polymerase chain reaction (RT-PCR) and Western blotting analyses, the replicative-lifespan assay of mutants, such as Δsod1, Δsod2, Δgpx, Δcat, Δskn7, Δuth1, Δatg32, Δatg2, and Δrim15 of K6001, autophagy flux analysis, and a lifespan assay of K6001 yeast after giving a mitophagy inhibitor and activator were performed. RESULTS: IQ extended the replicative lifespans of the K6001 yeasts and the chronological lifespans of the YOM36 yeasts. Furthermore, the reactive nitrogen species (RNS) showed no change during the growth phase but significantly decreased in the stationary phase after treatment with IQ. The survival rates of the yeasts under oxidative- and thermal-stress conditions improved upon IQ treatment, and thermal stress was alleviated by the increasing superoxide dismutase (Sod) activity. Additionally, IQ decreased the ROS and MDA of the yeast while increasing the activity of antioxidant enzymes. However, it could not prolong the replicative lifespans of Δsod1, Δsod2, Δgpx, Δcat, Δskn7, and Δuth1 of K6001. IQ significantly increased autophagy and mitophagy induction, the presence of free green fluorescent protein (GFP) in the cytoplasm, and ubiquitination in the mitochondria of the YOM38 yeasts at the protein level. IQ did not prolong the replicative lifespans of Δatg2 and Δatg32 of K6001. Moreover, IQ treatment led to a decrease in Sch9 at the protein level and an increase in the nuclear translocation of Rim15 and Msn2. CONCLUSIONS: These results indicated that the Sch9/Rim15/Msn signaling pathway, as well as antioxidative stress, anti-thermal stress, and autophagy, were involved in the antiaging effects of IQ in the yeasts.

CCPG1 recognizes endoplasmic reticulum luminal proteins for selective ER-phagy.

The endoplasmic reticulum (ER) is a major cell compartment where protein synthesis, folding, and posttranslational modifications occur with assistance from a wide variety of chaperones and enzymes. Quality control systems selectively eliminate abnormal proteins that accumulate inside the ER due to cellular stresses. ER-phagy, that is, selective autophagy of the ER, is a mechanism that maintains or reestablishes cellular and ER-specific homeostasis through removal of abnormal proteins. However, how ER luminal proteins are recognized by the ER-phagy machinery remains unclear. Here, we applied the aggregation-prone protein, six-repeated islet amyloid polypeptide (6xIAPP), as a model ER-phagy substrate and found that cell cycle progression 1 (CCPG1), which is an ER-phagy receptor, efficiently mediates its degradation via ER-phagy. We also identified prolyl 3-hydroxylase family member 4 (P3H4) as an endogenous cargo of CCPG1-dependent ER-phagy. The ER luminal region of CCPG1 contains several highly conserved regions that we refer to as cargo-interacting regions (CIRs); these interact directly with specific luminal cargos for ER-phagy. Notably, 6xIAPP and P3H4 interact directly with different CIRs. These findings indicate that CCPG1 is a bispecific ER-phagy receptor for ER luminal proteins and the autophagosomal membrane that contributes to the efficient removal of aberrant ER-resident proteins through ER-phagy.

Anti-aging effects of hesperidin on Saccharomyces cerevisiae via inhibition of reactive oxygen species and UTH1 gene expression.

This study used a replicative lifespan assay of K6001 yeast to screen anti-aging food factors in commercial flavonoids. Hesperidin derived from the Citrus genus extended the lifespan of yeast at doses of 5 and 10 µM as compared with the control group (p<0.01, p<0.01). Reactive oxygen species (ROS), real-time PCR (RT-PCR), and lifespan assays of uth1 and skn7 mutants with the K6001 background were used to study the anti-aging mechanisms in yeast. The results indicate that hesperidin significantly inhibits the ROS of yeast, and UTH1 gene expression, and that SKN7 gene are involved in hesperidin-mediated lifespan extension. Further, increases in the Sir2 homolog, SIRT1 activity, and SOD gene expression were confirmed at doses of 5 (p<0.01) and 10 µM (p<0.05). This suggests that Sir2, UTH1 genes, and ROS inhibition after administration of hesperidin have important roles in the anti-aging effects of yeast. However, the aglycon hesperetin did not exhibit anti-aging effects in yeast.

Effects of a proton pump inhibitor on the physiological accumulation of fluoro-2-deoxy-D-glucose (FDG) in FDG-positron emission tomography.

PURPOSES: The physiological accumulation of fluoro-2-deoxy-D-glucose (FDG) is common in medical examinations of the digestive tract conducted using FDG-positron emission tomography (PET). The aim of this study was to determine the effects of a proton pump inhibitor (PPI) on the physiological FDG accumulation in the digestive tract. METHODS: A total of 130 patients examined from July 2007 to October 2008 were included in the final analysis. A PPI was administered orally prior to FDG-PET in 65 patients. The remaining 65 patients underwent FDG-PET without administration of the PPI. The assessments used visual and quantitative evaluations. RESULTS: Visual evaluation showed that physiological FDG accumulation in the stomach was significantly reduced (p = 0.037) in the PPI group compared with the control group. The quantitative evaluation also revealed a significant reduction in the maximum standardized uptake values (SUV(max)) in the stomach in the PPI group compared with the control group (p < 0.0001). Physiological FDG accumulation in the colon showed a decreasing trend on visual evaluation in the PPI group compared with the control group, and the quantitative evaluation found a significant reduction in the physiological FDG accumulation in the colon in the PPI group (p = 0.045). CONCLUSIONS: The oral administration of a PPI was effective for reducing the physiological accumulation of the FDG in the alimentary tract. However, based on the error associated with SUV(max) measurement, a quantitative evaluation should therefore be combined with the visual evaluation.