Role of ERG11 and MDR1 genes in cycloheximide and multidrug resistance in Candida species.

Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs.

Inhibition of Protein Synthesis Attenuates Formation of Traumatic Memory and Normalizes Fear-Induced c-Fos Expression in a Mouse Model of Posttraumatic Stress Disorder.

Posttraumatic stress disorder (PTSD) is a debilitating psychosomatic condition characterized by impairment of brain fear circuits and persistence of exceptionally strong associative memories resistant to extinction. In this study, we investigated the neural and behavioral consequences of inhibiting protein synthesis, a process known to suppress the formation of conventional aversive memories, in an established PTSD animal model based on contextual fear conditioning in mice. Control animals were subjected to the conventional fear conditioning task. Utilizing c-Fos neural activity mapping, we found that the retrieval of PTSD and normal aversive memories produced activation of an overlapping set of brain structures. However, several specific areas, such as the infralimbic cortex and the paraventricular thalamic nucleus, showed an increase in the PTSD group compared to the normal aversive memory group. Administration of protein synthesis inhibitor before PTSD induction disrupted the formation of traumatic memories, resulting in behavior that matched the behavior of mice with usual aversive memory. Concomitant with this behavioral shift was a normalization of brain c-Fos activation pattern matching the one observed in usual fear memory. Our findings demonstrate that inhibiting protein synthesis during traumatic experiences significantly impairs the development of PTSD in a mouse model. These data provide insights into the neural underpinnings of protein synthesis-dependent traumatic memory formation and open prospects for the development of new therapeutic strategies for PTSD prevention.

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.

Real-time measurements of ATP dynamics via ATeams in Plasmodium falciparum reveal drug-class-specific response patterns.

Malaria tropica, caused by the parasite Plasmodium falciparum (P. falciparum), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.

Hydroxymethylnitrofurazone lymphatic uptake with nanostructured lipid carrier after oral administration in rats.

Background: Leishmaniasis, caused by the protozoan Leishmania sp., infects phagocyte cells present in lymphatic organs. This study demonstrates the influence of nanostructured lipid carrier-loaded hydroxymethylnitrofurazone (NLC-NFOH) on lymphatic uptake using a chylomicron-blocking flow model in rats. Method: Lymphatic uptake of NFOH was assessed 1 h after oral administration of dimethyl sulfoxide with NFOH or NLC-NFOH with and without cycloheximide pretreatment. Result: Dimethyl sulfoxide with NFOH and NLC-NFOH showed NFOH serum concentrations of 0.0316 and 0.0291 µg/ml, respectively. After chylomicron blocking, NFOH was not detected. Conclusion: Despite log P below 5, NFOH was successfully taken up by the lymphatic system. Long-chain fatty acids and particle size might be main factors in these findings. NLC-NFOH is a promising and convenient platform for treating leishmaniasis via oral administration.

Puerarin Attenuates Cycloheximide-Induced Oxidative Damage and Memory-Consolidation Impairment in Rats.

BACKGROUND: Cycloheximide (CXM), an antifungal antibiotic, causes impaired memory consolidation as a side effect partially by disturbing the activities of the central catecholaminergic and cholinergic system. Some reports indicated that puerarin prevented memory impairment in various models in rodents. However, the protective effects of puerarin on the side effects of cycloheximide for memory consolidation impairment have not yet been investigated. METHODS: The protective effects of puerarin on CXM-induced memory-consolidation impairment, and memory impairment produced by central administration of AF64A neurotoxin, were investigated using a passive avoidance task in rats. A combination of transmitter receptor agonists and antagonists was used to explore the effects of puerarin on nervous system function. The activity of antioxidant defense systems and neurotransmitter systems in the prefrontal cortex and hippocampus were assayed. RESULTS: Systemic (25 and 50 mg/kg, i.p.) or central (5 and 10 µg/brain, i.c.v.) administration of puerarin attenuated CXM-induced memory-consolidation impairment produced by 1.5 mg/kg CXM (s.c.) in rats. The improvements produced by 50 mg/kg puerarin were blocked by cholinergic antagonists, a 5-HT2 receptor agonist, and an adrenergic receptor antagonist. Puerarin (only at 50 mg/kg, i.p.) reversed the CXM-induced alterations of the levels of norepinephrine in the prefrontal cortex and the levels of monoamines in the hippocampus. Puerarin also increased antioxidant-defense-system activities in the prefrontal cortex and hippocampus, which had been decreased by CXM. CONCLUSIONS: We suggested that the attenuating effects of puerarin on CXM-induced memory-consolidation impairment may be due to decrease oxidative damage and the normalition of the neurotransmitter function in the prefrontal cortex and hippocampus.

P21 resists ferroptosis in osteoarthritic chondrocytes by regulating GPX4 protein stability.

Ferroptosis is involved in the pathogenesis of osteoarthritis (OA) while suppression of chondrocyte ferroptosis has a beneficial effect on OA. However, the molecular mechanism of ferroptosis in OA remains to be elucidated. P21, an indicator of aging, has been reported to inhibit ferroptosis, but the relationship between P21 and ferroptosis in OA remains unclear. Here, we aimed to investigate the expression and function of P21 in OA chondrocytes, and the involvement of P21 in the regulation of ferroptosis in chondrocytes. First, we demonstrated that high P21 expression was observed in the cartilage from OA patients and destabilized medial meniscus (DMM) mice, and in osteoarthritic chondrocytes induced by IL-1ß, FAC and erastin. P21 knockdown exacerbated the reduction of Col2a1 and promoted the upregulation of MMP13 in osteoarthritic chondrocytes. Meanwhile, P21 knockdown exacerbated cartilage degradation in DMM-induced OA mouse models and decreased GPX4 expression in vivo. Furthermore, P21 knockdown sensitized chondrocytes to ferroptosis induced by erastin, which was closely associated with the accumulation of lipid peroxides. In mechanism, we demonstrated that P21 regulated the stability of GPX4 protein, and the regulation was independent of NRF2. Meanwhile, we found that P21 significantly affected the recruitment of GPX4 to linear ubiquitin chain assembly complex (LUBAC) and regulated the level of M1-linked ubiquitination of GPX4. Overall, our results suggest that P21 plays an essential anti-ferroptosis role in OA by regulating the stability of GPX4.

Insights into Some Onygenalean Fungi from Freshwater Sediments in Spain and Description of Novel Taxa.

During the course of a project investigating culturable Ascomycota diversity from freshwater sediments in Spain, we isolated 63 strains of cycloheximide-resistant fungi belonging to the order Onygenales. These well-known ascomycetes, able to infect both humans and animals, are commonly found in terrestrial habitats, colonizing keratin-rich soils or dung. Little is known about their diversity in aquatic environments. Combining morphological features and sequence analyses of the ITS and LSU regions of the nrDNA, we identified 14 species distributed in the genera Aphanoascus, Arachniotus, Arthroderma, Arthropsis, Emmonsiellopsis, Gymnoascoideus, Leucothecium, Malbranchea, and Myriodontium. Furthermore, three novel species for the genus Malbranchea are proposed as M. echinulata sp. nov., M. irregularis sp. nov., and M. sinuata sp. nov. The new genera Albidomyces and Neoarthropsis are introduced based on Arachniotus albicans and Arthropsis hispanica, respectively. Neoarthropsis sexualis sp. nov. is characterized and differentiated morphologically from its counterpart by the production of a sexual morph. The novel family Neoarthropsidaceae is proposed for the genera Albidomyes, Apinisia, Arachnotheca, Myriodontium, and Neoarthropsis, based on their phylogenetic relationships and phenotypic and ecological traits. Pseudoamaurascopsis gen. nov. is introduced to accommodate P. spiralis sp. nov., a fungus with unclear taxonomy related to Amaurascopsis and Polytolypa. We traced the ecology and global distribution of the novel fungi through ITS environmental sequences deposited in the GlobalFungi database. Studying the fungal diversity from freshwater sediments not only contributes to filling gaps in the relationships and taxonomy of the Ascomycota but also gives us insights into the fungal community that might represent a putative risk to the health of animals and humans inhabiting or transient in aquatic environments.

Reversible translocation of acyl-CoA:cholesterol acyltransferase (ACAT) between the endoplasmic reticulum and vesicular structures.

The enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is normally localized in the endoplasmic reticulum (ER) where it can esterify cholesterol for storage in lipid droplets and/or the formation of lipoproteins. Here, we report that ACAT can translocate from the ER into vesicular structures in response to different ACAT inhibitors. The translocation was fast (within minutes), reversible and occurred in different cell types. Interestingly, oleic acid was able to fasten the re-translocation from vesicles back into the reticular ER network. The process of ACAT translocation could also be induced by cyclodextrins, cholesterol, lanosterol (but not 4-cholestene-3 one), 25-hydroxycholesterol, and by certain stress stimuli such as hyperosmolarity (sucrose treatment), temperature change, or high-density cultivation. In vitro esterification showed that ACAT remains fully active after it has been translocated to vesicles in response to hyperosmotic sucrose treatment of the cells. The translocation process was not accompanied by changes in the electrophoretic mobility of ACAT, even after chemical crosslinking. Interestingly, the protein synthesis inhibitor cycloheximide showed a stimulating effect on ACAT activity and prevented the translocation of ACAT from the ER into vesicles.

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.

Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA.

Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.

Tetrandrine synergizes with MAPK inhibitors in treating KRAS-mutant pancreatic ductal adenocarcinoma via collaboratively modulating the TRAIL-death receptor axis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.

Rifampicin-induced ER stress and excessive cytoplasmic vacuolization instigate hepatotoxicity via alternate programmed cell death paraptosis in vitro and in vivo.

AIMS: Rifampicin-induced hepatotoxicity is a primary cause of drug-induced liver injury (DILI), posing a significant challenge to its continued clinical application. Moreover, the mechanism underlying rifampicin-induced hepatotoxicity remains unclear. MAIN METHODS: Human hepatocyte line-17 (HHL-17) cells were treated with an increasing dose of rifampicin for 24 h, and male Wistar rats were given rifampicin [150 mg/kg body weight (bw)] orally for 28 days. Viability assay, protein expression, and cell death assays were analyzed in vitro. Moreover, liver serum markers, body/organ weight, H&E staining, protein expression, etc., were assayed in vivo. KEY FINDINGS: Rifampicin induced a dose-dependent hepatotoxicity in HHL-17 cells (IC50; 600 µM), and increased the serum levels of liver injury markers, e.g., alanine transaminase (ALT) and aspartate transaminase (AST) in rats. Rifampicin-induced cell death was non-apoptotic and non-necroptotic both in vitro and in vivo. Further, excessive cellular vacuolization and reduced expression of Alix protein confirmed the induction of paraptosis both in vitro and in vivo. In addition, a significant increase in the endoplasmic reticulum (ER) stress markers (e.g., BiP, CHOP, and total polyubiquitinated proteins) was detected, demonstrating the induction of ER stress and altered protein homeostasis. Interestingly, rifampicin-induced hepatotoxicity was associated with the inhibition of autophagy and enhanced reactive oxygen species (ROS) generation in HHL-17 cells. Furthermore, inhibition of protein synthesis by cycloheximide (CHX) suppressed paraptosis by alleviating rifampicin-induced ER stress and ROS generation. SIGNIFICANCE: Rifampicin-induced hepatotoxicity involves ER stress-driven paraptosis as a novel mechanism of its toxicity that may be targeted to protect liver cells from rifampicin toxicity.

Transcriptomic Analysis Reveals Novel Regulators of the Scots Pine Stilbene Pathway.

Stilbenes accumulate in Scots pine heartwood where they have important roles in protecting wood from decaying fungi. They are also part of active defense responses, and their production is induced by different (a)biotic stressors. The specific transcriptional regulators as well as the enzyme responsible for activating the stilbene precursor cinnamate in the pathway are still unknown. UV-C radiation was the first discovered artificial stress activator of the pathway. Here, we describe a large-scale transcriptomic analysis of pine needles in response to UV-C and treatment with translational inhibitors, both activating the transcription of stilbene pathway genes. We used the data to identify putative candidates for the missing CoA ligase and for pathway regulators. We further showed that the pathway is transcriptionally activated by phosphatase inhibitor, ethylene and jasmonate treatments, as in grapevine, and that the stilbene synthase promoter retains its inducibility in some of the tested conditions in Arabidopsis, a species that normally does not synthesize stilbenes. Shared features between gymnosperm and angiosperm regulation and partially retained inducibility in Arabidopsis suggest that pathway regulation occurs not only via ancient stress-response pathway(s) but also via species-specific regulators. Understanding which genes control the biosynthesis of stilbenes in Scots pine aids breeding of more resistant trees.

In vitro Analysis of Stalled Ribosomes Using Puromycin Incorporation.

Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via a ribosome-binding protein (e.g., FMRP). Stalled ribosomes can act as physical roadblocks for trailing ribosomes and ultimately can cause ribosome collisions that stimulate no-go mRNA decay. Detecting stalled or slowed ribosomes in cells by ribosome footprint profiling or classic polysome profiling is laborious, technically challenging, and low throughput. Here, we present a protocol to assay for stalled ribosomes on in vitro-transcribed reporter mRNAs using a robust, commercially available mammalian in vitro translation lysate and an optimized low-speed sucrose cushion. In short, we take advantage of the ability of puromycin to incorporate into the nascent polypeptide and cause the ribosome to dissociate from the mRNA during active elongation, as well as the ability to selectively pellet ribosomes through a low-speed sucrose cushion due to their large molecular weight. Stalled ribosomes are not actively elongating and do not incorporate puromycin, allowing the ribosome-bound mRNA to pellet in the low-speed sucrose cushion. RT-qPCR is used to quantify the amount of ribosome-bound reporter mRNA in the pellet. This workflow allows for direct assessment of stalled ribosomes and is fully amendable to insertion of putative stalling motifs in the target mRNA, as well as supplementation with recombinant proteins or small molecule inhibitors that target translation elongation. Key features This protocol is optimized for cap-dependent in vitro translation in the dynamic linear range. Details for generating capped reporter mRNA in one day are provided. Requires as little as one day to complete if starting with in vitro-transcribed mRNA. This protocol requires access to an ultracentrifuge and a real-time PCR system.

Cycloheximide (CHX) Chase Assay to Examine Protein Half-life.

Cycloheximide (CHX) is a small molecule derived from Streptomyces griseus that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay. Graphical overview.

A lipid scramblase TMEM41B is involved in the processing and transport of GPI-anchored proteins.

Protein modification by glycosylphosphatidylinositol (GPI) takes place in the endoplasmic reticulum (ER). GPI-anchored proteins (GPI-APs) formed in the ER are transported to the cell surface through the Golgi apparatus. During transport, the GPI-anchor structure is processed. In most cells, an acyl chain modified to the inositol of GPI is removed by a GPI-inositol deacylase, PGAP1, in the ER. Inositol-deacylated GPI-APs become sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). We previously reported that GPI-APs are partially resistant to PI-PLC when PGAP1 activity is weakened by the deletion of selenoprotein T (SELT) or cleft lip and palate transmembrane protein 1 (CLPTM1). In this study, we found that the loss of TMEM41B, an ER-localized lipid scramblase, restored PI-PLC sensitivity of GPI-APs in SELT-knockout (KO) and CLPTM1-KO cells. In TMEM41B-KO cells, the transport of GPI-APs as well as transmembrane proteins from the ER to the Golgi was delayed. Furthermore, the turnover of PGAP1, which is mediated by ER-associated degradation, was slowed in TMEM41B-KO cells. Taken together, these findings indicate that inhibition of TMEM41B-dependent lipid scrambling promotes GPI-AP processing in the ER through PGAP1 stabilization and slowed protein trafficking.

Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation.

A character of active protein translation is formation of multiple ribosomes, or polysomes, on translating mRNAs. Polysome intensity reflects global cellular translation activity and can be assessed after biochemical fractionations of polysomes. Polysome fractionation begins with immobilizing ribosomes on mRNAs using inhibitors of translation elongation, for example, cycloheximide. Nuclei-free cell lysates are then isolated and layered on the top of a sucrose gradient for ultracentrifugation to separate ribosomal subunits, monosome, and multiple fractions of polysomes by their different sedimentation rates along the sucrose gradient. A density gradient fractionation system including a spectrophotometer reads the RNA absorbance of the flowed gradient and generates the fractions. These fractions can be subjected to further RNA and protein analyses, for example, polysome profiling and mass spectrometry. Here, we present a detailed protocol of polysome fractionation for mammalian cells.

Corrigendum: A novel assisted oocyte activation method improves fertilization in patients with recurrent fertilization failure.

[This corrects the article DOI: 10.3389/fcell.2021.672081.].

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.