The mechanism of multistep progression of the transcriptional cascade in activated microglia as approached by a proteome approach.

The ocular cytokine network plays pivotal roles in terms of the initiation and progression of retinal degeneration. Several types of immunocompetent cells such as microglia participate in inflammation, and a temporal transition in the molecular events of inflammation has been hypothesized. We previously found that the Csf2 gene was induced in the early phase of retinal degeneration. CSF2 participates in the transcriptional activation of several cytokines expressed by microglia; however, whether CSF2 is essential in this context is not known. In this work, we approach this question by using anti-CSF2 neutralizing bntibody and the protein synthesis inhibitor cycloheximide (CHX). We first revealed that CSF2 positively regulated the cytokine induction cascade using a CSF2-neutralizing antibody (anti-CSF2) to treat the microglial cell line that were activated by lipopolysaccharide (LPS). LPS or Lipid A stimulation in the presence of the protein synthesis inhibitor cycloheximide (CHX) led to cytokine superinduction, but suppression of the expression of a few cytokines was also noted in MG5 cells. To examine transitions of the molecular events within LPS-activated microglia, we next performed proteome analysis of MG5 cells stimulated with LPS for 0, 4, and 9 h. The Database for Annotation, Visualization, and Integrated Discovery analysis of differentially expressed proteins showed that various mRNA-modifying molecules were induced after LPS stimulation, in addition to molecules involved in inflammation. However, the numbers of common proteins founded in the comparison between the induced proteins of 4 and 9 h were only one-third and one-half of induced proteins at 4 and 9 h, respectively, suggesting dynamic transition of the induced proteins. LPS-induced mRNA-modifying proteins were almost completely suppressed by CHX, as expected, suggesting that transient induction of transcription-editing proteins plays an important role in terms of the phenotype of inflammation that develops in microglia after LPS stimulation.

Loss of the yeast transporter Agp2 upregulates the pleiotropic drug-resistant pump Pdr5 and confers resistance to the protein synthesis inhibitor cycloheximide.

The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.

FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity.

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

Retrograde amnesia for the stress-induced impairment of extinction: time-dependent and not so forgotten.

We investigated whether retrograde amnesia for the stress-induced impairment of extinction retrieval shares similar characteristics with original acquisition memories. The first experiment demonstrated that cycloheximide administered shortly after a single restraint stress session alleviated the impairment of extinction retrieval but not when administered following a longer delay (i.e., the amnesia for stress is time-dependent). A second experiment showed that the retrograde amnesia for stress could be alleviated by a second brief exposure to the stressor. These results demonstrating that amnesia for stress shares characteristics similar to original memories are explained using a retrieval-based memory integration model of retrograde amnesia.

Context and Time Regulate Fear Memory Consolidation and Reconsolidation in the Basolateral Amygdala Complex.

It is widely accepted that fear memories are consolidated through protein synthesis-dependent changes in the basolateral amygdala complex (BLA). However, recent studies show that protein synthesis is not required to consolidate the memory of a new dangerous experience when it is similar to a prior experience. Here, we examined whether the protein synthesis requirement for consolidating the new experience varies with its spatial and temporal distance from the prior experience. Female and male rats were conditioned to fear a stimulus (S1, e.g., light) paired with shock in stage 1 and a second stimulus (S2, e.g., tone) that preceded additional S1-shock pairings (S2-S1-shock) in stage 2. The latter stage was followed by a BLA infusion of a protein synthesis inhibitor, cycloheximide, or vehicle. Subsequent testing with S2 revealed that protein synthesis in the BLA was not required to consolidate fear to S2 when the training stages occurred 48 h apart in the same context; was required when they were separated by 14 d or occurred in different contexts; but was again not required if S1 was re-presented after the delay or in the different context. Similarly, protein synthesis in the BLA was not required to reconsolidate fear to S2 when the training stages occurred 48 h apart but was required when they occurred 14 d apart. Thus, the protein synthesis requirement for consolidating/reconsolidating fear memories in the BLA is determined by similarity between present and past experiences, the time and place in which they occur, and reminders of the past experiences.

Changes in the conflicting nongenomic effects of progesterone in rat myometrium during pregnancy.

AIMS: Although the functions of progesterone in the myometrium are well-established, the nongenomic effects of progesterone in pregnant myometrial contractions are still unclear. Therefore, this study aimed to investigate changes in the nongenomic effects of progesterone during pregnancy. MAIN METHODS: Myometrial strips were obtained from non-pregnant, pregnant, and postpartum rats, and the nongenomic effects of progesterone in the myometrium during pregnancy were examined. Additionally, the influence of actinomycin D and cycloheximide and the effects of Org OD-02-0 (a specific membrane progesterone receptor (mPR) agonist) in the myometrium were investigated. Moreover, DNA microarray and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to identify genes involved in progesterone-induced effects in the myometrium. KEY FINDINGS: Progesterone did not cause rhythmic contractions in non-pregnant myometrium but induced rhythmic contractions in pregnant myometrium, with the effects peaking at 20 d + 8 h of pregnancy. However, myometrial contractions decreased after delivery and were restored to non-pregnant levels at 7 d postpartum. Additionally, progesterone stably inhibited high KCl-induced myometrial contractions during pregnancy. Moreover, the nongenomic effects of progesterone were unaffected by actinomycin D or cycloheximide, and Org OD-02-0 effectively mimicked these effects. DNA microarray analysis and qRT-PCR revealed a significant increase in mPRß gene expression during pregnancy. However, mPRα, mPRγ, mPRδ, and mPRε expression levels remained unchanged. SIGNIFICANCE: The stimulatory nongenomic effect of progesterone, which was inducible and mPRß-dependent during pregnancy, may be involved in parturition. The inhibitory effect, which was constitutive and depended on other mPRs, may be involved in pregnancy maintenance.

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Short-Term Inhibition of Translation by Cycloheximide Concurrently Affects Mitochondrial Function and Insulin Secretion in Islets from Female Mice.

Since glucose stimulates protein biosynthesis in beta cells concomitantly with the stimulation of insulin release, the possible interaction of both processes was explored. The protein biosynthesis was inhibited by 10 µM cycloheximide (CHX) 60 min prior to the stimulation of perifused, freshly isolated or 22 h-cultured NMRI mouse islets. CHX reduced the insulinotropic effect of 25 mM glucose or 500 µM tolbutamide in fresh but not in cultured islets. In cultured islets the second phase of glucose stimulation was even enhanced. In fresh and in cultured islets CHX strongly reduced the content of proinsulin, but not of insulin, and moderately diminished the [Ca2+]i increase during stimulation. The oxygen consumption rate (OCR) of fresh islets was about 50% higher than that of cultured islets at basal glucose and was significantly increased by glucose but not tolbutamide. In fresh, but not in cultured, islets CHX diminished the glucose-induced OCR increase and changes in the NAD(P)H- and FAD-autofluorescence. It is concluded that short-term CHX exposure interferes with the signal function of the mitochondria, which have different working conditions in fresh and in cultured islets. The interference may not be an off-target effect but may result from the inhibited cytosolic synthesis of mitochondrial proteins.

Redefining pleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells.

TP53 (p53) and MYC are amongst the most frequently altered genes in cancer. Both are thus attractive targets for new anticancer therapies. Historically, however, both genes have proved challenging to target and currently there is no approved therapy against either. The aim of this study was to investigate the effect of the mutant p53 reactivating drug, COTI-2 on MYC. Total MYC, pSer62 MYC and pThr58 MYC were detected using Western blotting. Proteasome-mediated degradation was determined using the proteasome, inhibitor MG-132, while MYC half-life was measured using pulse chase experiments in the presence of cycloheximide. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Treatment of 5 mutant p53 breast cancer cell lines with COTI-2 resulted in dose-dependent MYC degradation. Addition of the proteasome inhibitor, MG132, rescued the degradation, suggesting that this proteolytic system was at least partly responsible for the inactivation of MYC. Using cycloheximide in pulse chase experiments, COTI-2 was found to reduce the half-life of MYC in 2 different mutant p53 breast cancer cell lines, i.e., from 34.8 to 18.6 min in MDA-MB-232 cells and from 29.6 to 20.3 min in MDA-MB-468 cells. Co-treatment with COTI-2 and the MYC inhibitor, MYCi975 resulted in synergistic growth inhibition in all 4 mutant p53 cell lines investigated. The dual ability of COTI-2 to reactivate mutant p53 and degrade MYC should enable this compound to have broad application as an anticancer drug.

Efficient production of recombinant proteins in suspension CHO cells culture using the Tol2 transposon system coupled with cycloheximide resistance selection.

DNA recombination techniques in mammalian cells has been applied to the production of therapeutic proteins for several decades. To be used for commercial production, established cell lines should stably express target proteins with high productivity and acceptable quality for human use. In the conventional transfection method, the screening process is laborious and time-consuming since superior cell lines had to be selected from an enormous number of transfected cell pools and clonal cell lines with a wide variety of transgene insertion locations. In this study, we demonstrated that the combination of a Tol2 transposon system and cell selection by cycloheximide resistance is an efficient method to express therapeutic proteins, such as human antibody in suspension culture of Chinese hamster ovary cells. The resulting stable cell lines showed constant productivity and cell growth over a long enough cultivation periods for recombinant protein production. We anticipate that this approach will prove widely applicable to protein production in research and development of pharmaceutical products.

The Yeast Permease Agp2 Senses Cycloheximide and Undergoes Degradation That Requires the Small Protein Brp1-Cellular Fate of Agp2 in Response to Cycloheximide.

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.

Evaluation of Broad Anti-Coronavirus Activity of Autophagy-Related Compounds Using Human Airway Organoids.

To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.

Activation of the ß­TrCP/IκBα/inflammation axis limits the sensitivity of liver cancer cells to neddylation inhibition.

Upregulation of protein neddylation occurs in numerous types of human cancer, including liver cancer. MLN4924, a potent neddylation­inhibiting pharmacological agent, demonstrates anticancer ability in numerous cancers. However, the sensitivity of MLN4924 in liver cancer remains unsatisfactory due to factors causing resistance. RT­qPCR and western blotting were utilized to assess the mRNA and protein levels of genes, respectively. Cell Counting Kit­8 assay and colony formation assays were employed to assess cell viability and proliferation. The pathway of protein degradation and stability were determined by western blotting after treatment with MG132 and cycloheximide. An immunoprecipitation assay was utilized to detect the ubiquitination of protein. An in vitro ubiquitination assay was used to determine the ubiquitin linkage. To the best of our knowledge, the present study was the first to demonstrate that NF­κB inhibitor α (IκBα) downregulation and subsequent inflammation in response to MLN4924 limited the antitumor potential of MLN4924. Ectopic expression of IκBα enhanced the antitumor potential of MLN4924 in liver cancer cells. Moreover, the results of the present study demonstrated that MLN4924 decreased IκBα via promoting the K48 linkage of ubiquitin to IκBα. Mechanistic studies demonstrated that MLN4924 enhanced the protein stability of ß­transducin repeat­containing protein (ß­TrCP), promoting the ubiquitination of IκBα, which led to the ubiquitin­mediated degradation of IκBα. In addition, the results of the present study also demonstrated that ß­TrCP knockdown markedly inhibited MLN4924 from suppressing the growth of liver cancer cells, via attenuating MLN4924­mediated IκBα downregulation and inflammation. Collectively, these results indicated that the ß­TrCP/IκBα/inflammation pathway may act as a novel resistance factor of MLN4924, and targeting ß­TrCP may be beneficial for the treatment of liver cancer.

Acquired resistance to severe ethanol stress-induced inhibition of proteasomal proteolysis in Saccharomyces cerevisiae.

BACKGROUND: Although the budding yeast, Saccharomyces cerevisiae, produces ethanol via alcoholic fermentation, high-concentration ethanol is harmful to yeast cells. Severe ethanol stress (> 9% v/v) inhibits protein synthesis and increases the level of intracellular protein aggregates. However, its effect on proteolysis in yeast cells remains largely unknown. METHODS: We examined the effects of ethanol on proteasomal proteolysis in yeast cells through the cycloheximide-chase analysis of short-lived proteins. We also assayed protein degradation in the auxin-inducible degron system and the ubiquitin-independent degradation of Spe1 under ethanol stress conditions. RESULTS: We demonstrated that severe ethanol stress strongly inhibited the degradation of the short-lived proteins Rim101 and Gic2. Severe ethanol stress also inhibited protein degradation in the auxin-inducible degron system (Paf1-AID*-6FLAG) and the ubiquitin-independent degradation of Spe1. Proteasomal degradation of these proteins, which was inhibited by severe ethanol stress, resumed rapidly once the ethanol was removed. These results suggested that proteasomal proteolysis in yeast cells is reversibly inhibited by severe ethanol stress. Furthermore, yeast cells pretreated with mild ethanol stress (6% v/v) showed proteasomal proteolysis even with 10% (v/v) ethanol, indicating that yeast cells acquired resistance to proteasome inhibition caused by severe ethanol stress. However, yeast cells failed to acquire sufficient resistance to severe ethanol stress-induced proteasome inhibition when new protein synthesis was blocked with cycloheximide during pretreatment, or when Rpn4 was lost. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our results provide novel insights into the adverse effects of severe ethanol stress on proteasomal proteolysis and ethanol adaptability in yeast.

Epimedokoreanin B inhibits the growth of lung cancer cells through endoplasmic reticulum stress-mediated paraptosis accompanied by autophagosome accumulation.

Epimedokoreanin B (EKB) is a prenylated flavonoid isolated from Epimedium koreanum. In this article, we described the anti-cancerous effects of EKB and its underlying mechanism in human non-small cell lung cancer (NSCLC) A549 and NCI-H292 cells. EKB treatment inhibited cell proliferation and migration accompanied by cytoplasmic vacuolation in both cell lines. The cell death induced by EKB lacked the features of apoptosis like chromatin condensation, phosphatidyl serine exposure and caspase cleavage. The vacuoles stimulated by EKB predominantly derived from endoplasmic reticulum (ER) and mitochondria dilation, which are the characteristics of paraptosis. Down-regulation of Alix and up-regulation of ER stress-related proteins after EKB treatment further supported the occurrence of paraptosis. ER stress inhibitor 4-phenylbutyric acid (4-PBA) and protein synthesis inhibitor cycloheximide (CHX) treatment antagonized the vacuoles formation as well as cell death induced by EKB, indicating that ER stress was involved in EKB induced paraptosis. In addition, autophagosome accumulation accompanied with autophagy flux blocking was observed in EKB treated cells, this was consistent with the occurrence of ER stress. Collectively, EKB was demonstrated as a paraptosis-like cell death inducer in A549 and NCI-H292 cells. The inhibitory effect of EKB on lung cancer cell proliferation was further demonstrated in a zebrafish xenograft model. These findings raise the possibility that paraptosis inducers may be considered as alternative choices for lung cancer therapy.

[Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide].

The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a key transcriptional regulator of the proteasome in Saccharomycetes yeast and is also involved in the cellular response to various stresses. In addition to proteasomal genes, Rpn4 affects the expression of several hundred other genes, including genes involved in DNA repair and oxidative stress response. At the same time, the molecular mechanisms used by Rpn4 in controlling target genes and its functioning as a regulator of the cellular response to stress remain largely unclear. The aim of this work was to determine the Rpn4 domains required to ensure cell resistance to stress. It was shown that the N-terminal and central regions of the protein contain sites required for resistance to all types of stresses. The putative nuclear localization signal does not affect the functioning of Rpn4. Unexpectedly, a protein with the deletion of both zinc finger motifs that form the DNA-binding domain provides yeast resistance to oxidative stress and cycloheximide. Moreover, we showed that Rpn4 can be recruited to the promoter regions of the regulated genes even if they do not contain its binding sites. Based on these data, it can be assumed that Rpn4 is involved in gene regulation and the cellular response to stress due to protein-protein interactions.

The Multivalent Polyampholyte Domain of Nst1, a P-Body-Associated Saccharomyces cerevisiae Protein, Provides a Platform for Interacting with P-Body Components.

The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid-liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components.

Limonin stabilises sirtuin 6 (SIRT6) by activating ubiquitin specific peptidase 10 (USP10) in cardiac hypertrophy.

BACKGROUND AND PURPOSE: Limonin, a naturally occurring tetracyclic triterpenoid, has extensive pharmacological effects. Its role in cardiac hypertrophy remains to be elucidated. We investigated its effects on cardiac hypertrophy along with the potential mechanisms involved. EXPERIMENTAL APPROACH: The effects of limonin on cardiac hypertrophy in C57/BL6 mice caused by aortic banding, plus neonatal rat cardiac myocytes (NRCMs) stimulated with phenylephrine to induce cardiomyocyte hypertrophy in vitro were investigated. KEY RESULTS: Limonin markedly improved the cardiac function and heart weight in aortic banded mice. Limonin-treated mice and NRCMs also produced fewer cardiac hypertrophy markers than those treated with the vehicle in the hypertrophic groups. Sustained aortic banding- or phenylephrine-stimulation impaired cardiac sirtuin 6 (SIRT6) protein levels, which were partially reversed by limonin associated with enhanced activity of PPARα. Sirt6 siRNA inhibited the anti-hypertrophic effects of limonin in vitro. Interestingly, limonin did not influence Sirt6 mRNA levels, but regulated ubiquitin levels. Thus, the protein biosynthesis inhibitor, cycloheximide and proteasome inhibitor, MG-132, were used to determine SIRT6 protein expression levels. Under phenylephrine stimulation, limonin increased SIRT6 protein levels in the presence of cycloheximide, but it did not influence SIRT6 expression in the presence of MG-132, suggesting that limonin promotes SIRT6 levels by inhibiting its ubiquitination degradation. Furthermore, limonin inhibited the degradation of SIRT6 by activating ubiquitin-specific peptidase 10 (USP10), while Usp10 siRNA prevented the beneficial effects of limonin. CONCLUSION AND IMPLICATIONS: Limonin mediates the ubiquitination and degradation of SIRT6 by activating USP10, providing an attractive therapeutic target for cardiac hypertrophy.

Molecular basis of cycloheximide resistance in the Ophiostomatales revealed.

Resistance to the antibiotic Cycloheximide has been reported for a number of fungal taxa. In particular, some yeasts are known to be highly resistant to this antibiotic. Early research showed that this resulted from a transition mutation in one of the 60S ribosomal protein genes. In addition to the yeasts, most genera and species in the Ophiostomatales are highly resistant to this antibiotic, which is widely used to selectively isolate these fungi. Whole-genome sequences are now available for numerous members of the Ophiostomatales providing an opportunity to determine whether the mechanism of resistance in these fungi is the same as that reported for yeast genera such as Kluyveromyces. We examined all the available genomes for the Ophiostomatales and discovered that a transition mutation in the gene coding for ribosomal protein eL42, which results in the substitution of the amino acid Proline to Glutamine, likely confers resistance to this antibiotic. This change across all genera in the Ophiostomatales suggests that the mutation arose early in the evolution of these fungi.