The ancestral type of the R-RAS protein has oncogenic potential.

BACKGROUND: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. METHODS: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. RESULTS: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. CONCLUSIONS: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Analysis of Uncertainty and Sensitivity in Tailings Dam Breach-Runout Numerical Modelling.

Tailings dam breaches (TDBs) and subsequent flows can pose significant risk to public safety, the environment, and the economy. Numerical runout models are used to simulate potential tailings flows and understand their downstream impacts. Due to the complex nature of the breach-runout processes, the mobility and downstream impacts of these types of failures are highly uncertain. We applied the first-order second-moment (FOSM) methodology to a database of 11 back-analyzed historical tailings flows to evaluate uncertainties in TDB runout modelling and conducted a sensitivity analysis to identify key factors contributing to the variability of the HEC-RAS model output, including at different locations along the runout path. The results indicate that prioritizing resources toward advancements in estimating the values of primary contributors to the sensitivity of the selected model outputs is necessary for more reliable model results. We found that the total released volume is among the top contributors to the sensitivity of modelled inundation area and maximum flow depth, while surface roughness is among the top contributors to the sensitivity of modelled maximum flow velocity and flow front arrival time. However, the primary contributors to the sensitivity of the model outputs varied depending on the case study; therefore, the selection of appropriate rheological models and consideration of site-specific conditions are crucial for accurate predictions. The study proposes and demonstrates the FOSM methodology as an approximate probabilistic approach to model-based tailings flow runout prediction, which can help improve the accuracy of risk assessments and emergency response plans. Supplementary Information: The online version contains supplementary material available at 10.1007/s10230-024-00970-w.

Las roturas de presas de relaves (TDBs) y los flujos subsiguientes pueden suponer un riesgo significativo para la seguridad pública, el medio ambiente y la economía. Los modelos numéricos de desbordamiento se utilizan para simular posibles flujos de relaves y comprender su impacto aguas abajo. Debido a la naturaleza compleja de los procesos de rotura-desbordamiento, la movilidad y los impactos aguas abajo de este tipo de fallos tienen mucha incertidumbre. Se aplicó la metodología del segundo-momento de primer-orden (FOSM) a una base de datos de 11 flujos históricos de relaves analizados retrospectivamente para evaluar las incertidumbres en la modelización del desbordamiento de TDB y se realizó un análisis de sensibilidad para identificar los factores clave que contribuyen a la variabilidad de los resultados del modelo HEC-RAS, incluso en diferentes ubicaciones a lo largo de la trayectoria de fuga. Los resultados indican que es necesario priorizar los recursos hacia avances en la estimación de los valores de los principales contribuyentes a la sensibilidad de los resultados del modelo seleccionado para obtener resultados más fiables del modelo. El volumen total liberado se encuentra entre los principales contribuyentes a la sensibilidad del área de inundación modelizada y la profundidad máxima del flujo, mientras que la rugosidad de la superficie se encuentra entre los principales contribuyentes a la sensibilidad de la velocidad máxima del flujo modelizado y el tiempo de llegada del frente de flujo. Sin embargo, los principales factores que contribuyen a la sensibilidad de los resultados del modelo varían dependiendo del caso de estudio; por lo tanto, la selección de modelos reológicos apropiados y la consideración de las condiciones específicas del emplazamiento son cruciales para obtener predicciones precisas. El estudio propone y muestra la metodología FOSM como un enfoque probabilístico aproximado para la predicción de la extensión de flujos de relaves basada en modelos, que puede ayudar a mejorar la precisión de las evaluaciones de riesgos y los planes de respuesta a emergencias.

Genome-wide CRISPR-Cas9 screen reveals a persistent null-hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica.

Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress-induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity. Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome-wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null-hyphal phenotype-ΔRAS2, ΔRHO5, ΔSFL1, ΔSNF2, and ΔPAXIP1. Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L-1 OD-1 from a heterologous pathway with enhanced carbon flux through the mevalonate pathway. These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.

Trehalose biosynthetic pathway regulates filamentation response in Saccharomyces cerevisiae.

BACKGROUND: Diploid cells of Saccharomyces cerevisiae undergo either pseudohyphal differentiation or sporulation in response to depletion of carbon and nitrogen sources. Distinct signaling pathways regulate filamentation and sporulation in response to nutrient limitation. How these pathways are coordinated for implementing distinct cell fate decisions in response to similar nutritional cues is an enigma. Although the role of trehalose pathway in sporulation has been extensively studied, it's possible role in pseudohyphal differentiation has been unexplored. METHODS AND RESULTS: Briefly, tps1 and tps2 mutants were tested for their ability to form pseudohyphae independently as well as in the background of GPR1 and RAS2 mutations. Here, we demonstrate that disruption of TPS1 but not TPS2 inhibits pseudohyphae formation. Interestingly, deletion of GPR1 suppresses the above defect. Further genetic analysis revealed that TPS1 and TPS2 exert opposing effects in triggering filamentation. CONCLUSION: We provide new insights into the role of an otherwise well-known pathway of trehalose biosynthesis in pseudohyphal differentiation. Based on additional data we propose that downstream signaling, mediated by cAMP may be modulated by nutrient mediated differential regulation of RAS2 by TPS1 and TPS2.

Application of Unmanned Aerial Vehicle DEM in flood modeling and comparison with global DEMs: Case study of Atrak River Basin, Iran.

Digital Elevation Models (DEMs) play a significant role in hydraulic modeling and flood risk management. This study initially investigated the effect of Unmanned Aerial Vehicle (UAV) DEM resolutions, ranging from 1 m to 30 m, on flood characteristics, including the inundation area, mean flow depth, and mean flow velocity. Then, the errors of flood characteristics for global DEMs, comprising ALOS (30 m), ASTER (30 m), SRTM (30 m), and TDX (12 m) were quantified using UAV DEM measurements. For these purposes, the HEC-RAS 2D model in steady-state conditions was used to simulate the flood with return periods of 5- to 200 years along 20 km reach of Atrak River located in northeastern Iran. Results indicated when UAV DEM resolution decreased from 1 m to 30 m, inundation area and mean flow depth increased 17.0% (R2 = 0.94) and 10.2% (R2 = 0.96) respectively, while mean flow velocity decreased 16.8% (R2 = -0.94). Validation of the hydraulic modeling using the modified normalized difference water index demonstrated that the HEC-RAS 2D model in conjunction with UAV DEM simulates the flood with ⁓92% accuracy. Comparing the global DEMs with UAV DEM showed that the root mean square error (RMSE) values of the flow depth for ASTER, SRTM, ALOS, and TDX DEMs were 1.77, 1.12, 1.02, and 0.93 m, and the RMSE values of the flow velocity for the same DEMs were 0.81, 0.66, 0.55, and 0.47 m/s, respectively. Furthermore, TDX DEM with a 6.15% error in the inundation area was the nearest to UAV measurements. Overall, TDX DEM revealed a better performance in hydraulic modeling of the fluvial flood characteristics. Hence, it is recommended for environments where high-resolution topography data is scarce. The results of this study could potentially serve as a guideline for selecting global DEMs for hydraulic simulations.

Ras2 is important for growth and pathogenicity in Fusarium circinatum.

In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.

In S. cerevisiae hydroxycitric acid antagonizes chronological aging and apoptosis regardless of citrate lyase.

Caloric restriction mimetics (CRMs) are promising molecules to prevent age-related diseases as they activate pathways driven by a true caloric restriction. Hydroxycitric acid (HCA) is considered a bona fide CRM since it depletes acetyl-CoA pools by acting as a competitive inhibitor of ATP citrate lyase (ACLY), ultimately repressing protein acetylation and promoting autophagy. Importantly, it can reduce inflammation and tumour development. In order to identify phenotypically relevant new HCA targets we have investigated HCA effects in Saccharomyces cerevisiae, where ACLY is lacking. Strikingly, the drug revealed a powerful anti-aging effect, another property proposed to mark bona fide CRMs. Chronological life span (CLS) extension but also resistance to acetic acid of HCA treated cells were associated to repression of cell apoptosis and necrosis. HCA also largely prevented cell deaths caused by a severe oxidative stress. The molecule could act widely by negatively modulating cell metabolism, similarly to citrate. Indeed, it inhibited both growth reactivation and the oxygen consumption rate of yeast cells in stationary phase. Genetic analyses on yeast CLS mutants indicated that part of the HCA effects can be sensed by Sch9 and Ras2, two conserved key regulators of nutritional and stress signal pathways of primary importance. Our data together with published biochemical analyses indicate that HCA may act with multiple mechanisms together with ACLY repression and allowed us to propose an integrated mechanistic model as a basis for future investigations.

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The clinical application of immune checkpoint inhibitors (ICIs) lead to dramatic changes in the treatment strategy for patients with advanced non-small cell lung cancer (NSCLC), but the efficacy of ICIs in oncogene-driven NSCLC is controversial. Existing research shows that the efficacy of ICIs may be related to different types of driver genes, programmed cell death-ligand 1 (PD-L1) level, and tumor mutational burden (TMB). It may involved in other factors, such as clinical characteristics, and immune cell density. ICIs monotherapy or combination therapy may play a role in a subset of oncogene-driven NSCLC patients, but further studies are needed to select these patients, which may be an important direction for the future development of advanced NSCLC.

Saccharomyces cerevisiae Ras2 restores filamentation but cannot activate the first step of GPI anchor biosynthesis in Candida albicans.

Ras proteins are highly conserved small GTPases in eukaryotes. GTP-bound Ras binds to effectors to trigger signaling cascades. In order to understand how extensive is the functional homology between the highly homologous proteins, S. cerevisiae Ras2 and C. albicans Ras1, we examined whether ScRas2 could functionally complement CaRas1 in activating hyphal morphogenesis as well as GPI anchor biosynthesis. We show that ScRas2 functionally complements CaRas1 in rescuing growth as well as activating hyphal growth, a process that involves plasma membrane localized Ras activating cAMP/PKA signaling via Cyr1. However, ScRas2 is unable to activate the GPI-N-acetylglucosaminyl transferase (GPI-GnT) which catalyzes the first step of GPI biosynthesis. That CaRas1 alone activates GPI-GnT and not ScRas2 suggests that this process is cAMP independent. Interestingly, CaRas1 transcriptionally activates CaGPI2, encoding a GPI-GnT subunit that has been shown to interact with CaRas1 physically. In turn, CaGPI2 downregulates CaGPI19, encoding another GPI-GnT subunit. This has direct consequences for expression of CaERG11, encoding the target of azole antifungals. This effect too is specific to CaRas1 and ScRas2 is unable to replicate it.

Saccharomyces cerevisiae Hog1 MAP kinase pathway is activated in response to honokiol exposure.

AIM: The goal of the study was to investigate the cellular tolerance mechanism in response to honokiol exposure. METHODS AND RESULTS: The broth microdilution method was employed to test the sensitivity of different Saccharomyces cerevisiae strains to honokiol. Intracellular levels of reactive oxygen species (ROSs) were determined by DCFH-DA staining. The phosphorylation of Hog1 was evaluated by Western blot analysis. The mRNA expressions of genes involved in the Ras-cyclic AMP (cAMP) pathway were analysed by real-time reverse transcription polymerase chain reaction. We found that the sod1▵ mutant was hypersensitive to honokiol and produced more ROS compared with wild-type and sod2▵ cells. Hog1 was phosphorylated in response to honokiol exposure and deletion of HOG1 increased the sensitivity to honokiol. The expressions of genes involved in the Ras-cAMP pathway were down-regulated after honokiol exposure; exogenous cAMP significantly reduced the phosphorylation of Hog1, although the level was higher than the control level. CONCLUSIONS: In addition to SOD1, the Ras-cAMP cascade and Hog1 MAP kinase pathway is essential for protecting against honokiol-induced oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide insight into the understanding of the action mechanism of honokiol.

Rck1 promotes pseudohyphal growth via the activation of Ubp3 phosphorylation in Saccharomyces cerevisiae.

Previously, we reported that Rck1 up-regulates Ras2 and pseudohyphal growth of Saccharomyces cerevisiae. Here, we further investigate the involvement of Rck1 in the activation of pseudohyphal growth. Rck1 activated phosphorylation of the deubiquitinase Ubp3 through a direct protein interaction between Rck1 and Ubp3. The N-terminal Bre5 binding region of Ubp3 physically interacted with Rck1, and Ubp3 and Rck1 co-precipitated. Overexpression of UBP3 using a high-copy plasmid resulted in the upregulation of Ras2, and deletion of UBP3 blocked the upregulation of Ras2 by RCK1 overexpression. Treatment with the proteasome inhibitor MG132 resulted in accumulation of Ras2, indicating that Rck1 is involved in Ras2 degradation in a proteasome-dependent manner. Furthermore, deletion of UBP3 blocked the upregulation of FLO11, a flocculin required for pseudohyphal and invasive growth induced by RCK1 overexpression in S. cerevisiae. Taken together, these results demonstrate that Rck1 promotes S. cerevisiae pseudohyphal growth via the activation of Ubp3 phosphorylation.

Synchronous endometrioid adenocarcinomas in the uterine cervix and corpus.

It is frequently difficult to distinguish multiple primary carcinomas from single primary carcinoma with metastasis. Here, we report a case of synchronous endometrioid adenocarcinomas that independently occurred in the uterine cervix and corpus. A 47-year-old woman complaining of genital bleeding was preoperatively diagnosed with cervical adenocarcinoma with an endometrial lesion. On surgical treatment, two separate malignant lesions bearing endometrioid adenocarcinoma were identified in the uterine cervix and cavity. Although both lesions expressed the same type of human papillomavirus (HPV) gene, type 16, microscopic continuity was not observed. Furthermore, we detected a critical difference in PTEN mutation between the tumors and finally diagnosed this case as multiple primary cancers. This is the first report to show multiple primary endometrioid adenocarcinomas simultaneously arising in the uterine cervix and corpus. Considering the rarity of this case, the coexistence of HPV suggests its possible involvement in the carcinogenesis of the endometrioid adenocarcinomas.

Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae.

Protection against cisplatin in calorie-restricted Saccharomyces cerevisiae is mediated by the nutrient-sensor proteins Ras2, Tor1, or Sch9 through its target glutathione.

There is substantial interest in developing alternative strategies for cancer chemotherapy aiming to increase drug specificity and prevent tumor resistance. Calorie restriction (CR) has been shown to render human cancer cells more susceptible to drugs than normal cells. Indeed, deficiency of nutrient signaling proteins mimics CR, which is sufficient to improve oxidative stress response and life expectancy only in healthy cells. Thus, although CR and reduction of nutrient signaling may play an important role in cellular response to chemotherapy, the full underlying mechanisms are still not completely understood. Here, we investigate the relationship between the nutrient sensor proteins Ras2, Sch9, or Tor1 and the response of calorie-restricted Saccharomyces cerevisiae cells to cisplatin. Using wild-type and nutrient-sensing mutant strains, we show that deletion of any of these proteins mimics CR and is sufficient to increase cell protection. Moreover, we show that glutathione (GSH) is essential for proper CR protection of yeast cells under cisplatin chemotherapy. By measuring the survival rates and GSH levels, we found that cisplatin cytotoxicity leads to a decrease in GSH content reflecting in an increase of oxidative damage. Finally, investigating DNA fragmentation and apoptosis, we conclude that GSH contributes to CR-mediated cell survival.

Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae.

Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

The role of molecular analyses in the diagnosis and treatment of non-small-cell lung carcinomas.

Non-small-cell lung cancer (NSCLC) subtyping has recently been a key factor in determining patient management with novel drugs. In addition, the identification of distinct oncogenic driver mutations frequently associated with NSCLC histotype and coupled to the clinical responses to targeted therapies have revolutionized the impact of histologic type and molecular biomarkers in lung cancer. Several molecular alterations involving different genes (EGFR, KRAS, ALK, BRAF, and HER2) seem to have a remarkable predilection for adenocarcinoma and specific inhibitors of EGFR and ALK are now available for patients with adenocarcinoma harboring the relevant gene alterations. The efficacy of histology-based and molecular-targeted therapies had a deep impact in (1) re-defining classification of lung cancer (particularly adenocarcinomas) and (2) routine clinical practice of pathologists involved in optimization of handling of tissue samples in order to guarantee NSCLC subtyping with the help of immunohistochemistry and adequately preserve tumor cells for molecular analysis. In agreement with the modern multidisciplinary approach to lung cancer, we reviewed here the diagnostic and predictive value of molecular biomarkers according to the clinical, pathologic, and molecular biologist viewpoints.

Volatile fingerprints of cancer specific genetic mutations.

We report on a new concept for profiling genetic mutations of (lung) cancer cells, based on the detection of patterns of volatile organic compounds (VOCs) emitted from cell membranes, using an array of nanomaterial-based sensors. In this in-vitro pilot study we have derived a volatile fingerprint assay for representative genetic mutations in cancer cells that are known to be associated with targeted cancer therapy. Five VOCs were associated with the studied oncogenes, using complementary chemical analysis, and were discussed in terms of possible metabolic pathways. The reported approach could lead to the development of novel methods for guiding treatments, so that patients could benefit from safer, more timely and effective interventions that improve survival and quality of life while avoiding unnecessary invasive procedures. Studying clinical samples (tissue/blood/breath) will be required as next step in order to determine whether this cell-line study can be translated into a clinically useful tool. FROM THE CLINICAL EDITOR: In this novel study, a new concept for profiling genetic mutations of (lung) cancer cells is described, based on the detection of patterns of volatile organic compounds emitted from cell membranes, using an array of nano-gold based sensors.

The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

Silent information regulator 2 (Sir2) is a conserved NAD+-dependent histone deacetylase crucial for regulating cellular stress response and the aging process in Saccharomyces cerevisiae. In this study, we investigated the molecular mechanism underlying how the absence of Sir2 can lead to altered stress susceptibilities in S. cerevisiae under different environmental and physiological conditions. In a glucose-complex medium, the sir2Δ strain showed increased sensitivity to H2O2 compared to the wild-type strain during the post-diauxic phase. In contrast, it displayed increased resistance during the exponential growth phase. Transcriptome analysis of yeast cells in the post-diauxic phase indicated that the sir2Δ mutant expressed several oxidative defense genes at lower levels than the wild-type, potentially accounting for its increased susceptibility to H2O2. Interestingly, however, the sir2Δras2Δ double mutant exhibited greater resistance to H2O2 than the ras2Δ single mutant counterpart. We found that the expression regulation of the cytoplasmic catalase encoded by CTT1 was critical for the increased resistance to H2O2 in the sir2Δras2Δ strain. The expression of the CTT1 gene was influenced by the combined effect of RAS2 deletion and the transcription factor Azf1, whose level was modulated by Sir2. These findings provide insights into the importance of understanding the intricate interactions among various factors contributing to cellular stress response.

Corrigendum: The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

[This corrects the article DOI: 10.3389/fmicb.2023.1285559.].

Specific Disruption of Ras2 CAAX Proteolysis Alters Its Localization and Function.

Many CAAX proteins, such as Ras GTPase, undergo a series of posttranslational modifications at their carboxyl terminus (i.e., cysteine prenylation, endoproteolysis of AAX, and carboxylmethylation). Some CAAX proteins, however, undergo prenylation-only modification, such as Saccharomyces cerevisiae Hsp40 Ydj1. We previously observed that altering the CAAX motif of Ydj1 from prenylation-only to canonical resulted in altered Ydj1 function and localization. Here, we investigated the effects of a reciprocal change that altered the well-characterized canonical CAAX motif of S. cerevisiae Ras2 to prenylation-only. We observed that the type of CAAX motif impacted Ras2 protein levels, localization, and function. Moreover, we observed that using a prenylation-only sequence to stage hyperactive Ras2-G19V as a farnesylated and nonproteolyzed intermediate resulted in a different phenotype relative to staging by a genetic RCE1 deletion strategy that simultaneously affected many CAAX proteins. These findings suggested that a prenylation-only CAAX motif is useful for probing the specific impact of CAAX proteolysis on Ras2 under conditions where other CAAX proteins are normally modified. We propose that our strategy could be easily applied to a wide range of CAAX proteins for examining the specific impact of CAAX proteolysis on their functions. IMPORTANCE CAAX proteins are subject to multiple posttranslational modifications: cysteine prenylation, CAAX proteolysis, and carboxylmethylation. For investigations of CAAX proteolysis, this study took the novel approach of using a proteolysis-resistant CAAX sequence to stage Saccharomyces cerevisiae Ras2 GTPase in a farnesylated and nonproteolyzed state. Our approach specifically limited the effects of disrupting CAAX proteolysis to Ras2. This represented an improvement over previous methods where CAAX proteolysis was inhibited by gene knockout, small interfering RNA knockdown, or biochemical inhibition of the Rce1 CAAX protease, which can lead to pleiotropic and unclear attribution of effects due to the action of Rce1 on multiple CAAX proteins. Our approach yielded results that demonstrated specific impacts of CAAX proteolysis on the function, localization, and other properties of Ras2, highlighting the utility of this approach for investigating the impact of CAAX proteolysis in other protein contexts.