-
Biological Research May 2024Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to...
BACKGROUND
Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to the key enzyme gulonolactone oxidase (Gulo) unlike humans. In the current investigation, we used Gulo mice, which cannot synthesize their own ascorbate to determine the impact of this vitamin on both the transcriptomics and proteomics profiles in the whole liver. The study included Gulo mouse groups treated with either sub-optimal or optimal ascorbate concentrations in drinking water. Liver tissues of females and males were collected at the age of four months and divided for transcriptomics and proteomics analysis. Immunoblotting, quantitative RT-PCR, and polysome profiling experiments were also conducted to complement our combined omics studies.
RESULTS
Principal component analyses revealed distinctive differences in the mRNA and protein profiles as a function of sex between all the mouse cohorts. Despite such sexual dimorphism, Spearman analyses of transcriptomics data from females and males revealed correlations of hepatic ascorbate levels with transcripts encoding a wide array of biological processes involved in glucose and lipid metabolisms as well as in the acute-phase immune response. Moreover, integration of the proteomics data showed that ascorbate modulates the abundance of various enzymes involved in lipid, xenobiotic, organic acid, acetyl-CoA, and steroid metabolism mainly at the transcriptional level, especially in females. However, several proteins of the mitochondrial complex III significantly correlated with ascorbate concentrations in both males and females unlike their corresponding transcripts. Finally, poly(ribo)some profiling did not reveal significant enrichment difference for these mitochondrial complex III mRNAs between Gulo mice treated with sub-optimal and optimal ascorbate levels.
CONCLUSIONS
Thus, the abundance of several subunits of the mitochondrial complex III are regulated by ascorbate at the post-transcriptional levels. Our extensive omics analyses provide a novel resource of altered gene expression patterns at the transcriptional and post-transcriptional levels under ascorbate deficiency.
Topics: Animals; Ascorbic Acid; Liver; Female; Male; Proteomics; Mice; L-Gulonolactone Oxidase; Gene Expression Profiling; Transcriptome; Principal Component Analysis; Antioxidants
PubMed: 38735981
DOI: 10.1186/s40659-024-00509-x -
Communications Biology May 2024The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are...
The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.
Topics: Cryoelectron Microscopy; Capsid; Cell Extracts; Saccharomyces cerevisiae; RNA, Viral; RNA, Messenger
PubMed: 38730276
DOI: 10.1038/s42003-024-06204-7 -
Communications Biology Apr 2024Protein diffusion is a critical factor governing the functioning and organization of a cell's cytoplasm. In this study, we investigate the influence of (poly)ribosome...
Protein diffusion is a critical factor governing the functioning and organization of a cell's cytoplasm. In this study, we investigate the influence of (poly)ribosome distribution, cell aging, protein aggregation, and biomolecular condensate formation on protein mobility within the E. coli cytoplasm. We employ nanoscale single-molecule displacement mapping (SMdM) to determine the spatial distribution of the proteins and to meticulously track their diffusion. We show that the distribution of polysomes does not impact the lateral diffusion coefficients of proteins. However, the degradation of mRNA induced by rifampicin treatment leads to an increase in protein mobility within the cytoplasm. Additionally, we establish a significant correlation between cell aging, the asymmetric localization of protein aggregates and reduced diffusion coefficients at the cell poles. Notably, we observe variations in the hindrance of diffusion at the poles and the central nucleoid region for small and large proteins, and we reveal differences between the old and new pole of the cell. Collectively, our research highlights cellular processes and mechanisms responsible for spatially organizing the bacterial cytoplasm into domains with different structural features and apparent viscosity.
Topics: Escherichia coli; Cytoplasm; Escherichia coli Proteins; Diffusion
PubMed: 38678067
DOI: 10.1038/s42003-024-06216-3 -
Journal of Experimental & Clinical... Apr 2024Radiation therapy stands to be one of the primary approaches in the clinical treatment of malignant tumors. Nasopharyngeal Carcinoma, a malignancy predominantly treated...
BACKGROUND
Radiation therapy stands to be one of the primary approaches in the clinical treatment of malignant tumors. Nasopharyngeal Carcinoma, a malignancy predominantly treated with radiation therapy, provides an invaluable model for investigating the mechanisms underlying radiation therapy resistance in cancer. While some reports have suggested the involvement of circRNAs in modulating resistance to radiation therapy, the underpinning mechanisms remain unclear.
METHODS
RT-qPCR and in situ hybridization were used to detect the expression level of circCDYL2 in nasopharyngeal carcinoma tissue samples. The effect of circCDYL2 on radiotherapy resistance in nasopharyngeal carcinoma was demonstrated by in vitro and in vivo functional experiments. The HR-GFP reporter assay determined that circCDYL2 affected homologous recombination repair. RNA pull down, RIP, western blotting, IF, and polysome profiling assays were used to verify that circCDYL2 promoted the translation of RAD51 by binding to EIF3D protein.
RESULTS
We have identified circCDYL2 as highly expressed in nasopharyngeal carcinoma tissues, and it was closely associated with poor prognosis. In vitro and in vivo experiments demonstrate that circCDYL2 plays a pivotal role in promoting radiotherapy resistance in nasopharyngeal carcinoma. Our investigation unveils a specific mechanism by which circCDYL2, acting as a scaffold molecule, recruits eukaryotic translation initiation factor 3 subunit D protein (EIF3D) to the 5'-UTR of RAD51 mRNA, a crucial component of the DNA damage repair pathway to facilitate the initiation of RAD51 translation and enhance homologous recombination repair capability, and ultimately leads to radiotherapy resistance in nasopharyngeal carcinoma.
CONCLUSIONS
These findings establish a novel role of the circCDYL2/EIF3D/RAD51 axis in nasopharyngeal carcinoma radiotherapy resistance. Our work not only sheds light on the underlying molecular mechanism but also highlights the potential of circCDYL2 as a therapeutic sensitization target and a promising prognostic molecular marker for nasopharyngeal carcinoma.
Topics: Humans; Nasopharyngeal Carcinoma; Rad51 Recombinase; Mice; Animals; Radiation Tolerance; Recombinational DNA Repair; RNA, Circular; Nasopharyngeal Neoplasms; Cell Line, Tumor; Female; Male; Prognosis; Mice, Nude
PubMed: 38654320
DOI: 10.1186/s13046-024-03049-0 -
Scientific Reports Apr 2024Protein synthesis is a highly energy-consuming process that is downregulated in response to many environmental stresses or adverse conditions. Studies in the yeast...
Protein synthesis is a highly energy-consuming process that is downregulated in response to many environmental stresses or adverse conditions. Studies in the yeast Saccharomyces cerevisiae have shown that bulk translation is inhibited during adaptation to iron deficiency, which is consistent with its requirement for ribosome biogenesis and recycling. Although iron deficiency anemia is the most common human nutritional disorder, how iron modulates translation in mammals is poorly understood. Studies during erythropoiesis have shown that iron bioavailability is coordinated with globin synthesis via bulk translation regulation. However, little is known about the control of translation during iron limitation in other tissues. Here, we investigated how iron depletion affects protein synthesis in human osteosarcoma U-2 OS cells. By adding an extracellular iron chelator, we observed that iron deficiency limits cell proliferation, induces autophagy, and decreases the global rate of protein synthesis. Analysis of specific molecular markers indicates that the inhibition of bulk translation upon iron limitation occurs through the eukaryotic initiation factor eIF2α and mechanistic target of rapamycin (mTOR) pathways. In contrast to other environmental and nutritional stresses, iron depletion does not trigger the assembly of messenger ribonucleoprotein stress granules, which typically form upon polysome disassembly.
Topics: Animals; Humans; Iron; Phosphorylation; Protein Biosynthesis; Iron Deficiencies; Saccharomyces cerevisiae; Eukaryotic Initiation Factor-2; Mammals
PubMed: 38605136
DOI: 10.1038/s41598-024-59003-9 -
G3 (Bethesda, Md.) Jun 2024Sse1 is a cytosolic Hsp110 molecular chaperone of yeast, Saccharomyces cerevisiae. Its multifaceted roles in cellular protein homeostasis as a nucleotide exchange factor...
Sse1 is a cytosolic Hsp110 molecular chaperone of yeast, Saccharomyces cerevisiae. Its multifaceted roles in cellular protein homeostasis as a nucleotide exchange factor (NEF), as a protein-disaggregase and as a chaperone linked to protein synthesis (CLIPS) are well documented. In the current study, we show that SSE1 genetically interacts with IRE1 and HAC1, the endoplasmic reticulum-unfolded protein response (ER-UPR) sensors implicating its role in ER protein homeostasis. Interestingly, the absence of this chaperone imparts unusual resistance to tunicamycin-induced ER stress which depends on the intact Ire1-Hac1 mediated ER-UPR signaling. Furthermore, cells lacking SSE1 show inefficient ER-stress-responsive reorganization of translating ribosomes from polysomes to monosomes that drive uninterrupted protein translation during tunicamycin stress. In consequence, the sse1Δ strain shows prominently faster reversal from ER-UPR activated state indicating quicker restoration of homeostasis, in comparison to the wild-type (WT) cells. Importantly, Sse1 plays a critical role in controlling the ER-stress-mediated cell division arrest, which is escaped in sse1Δ strain during chronic tunicamycin stress. Accordingly, sse1Δ strain shows significantly higher cell viability in comparison to WT yeast imparting the stark fitness following short-term as well as long-term tunicamycin stress. These data, all together, suggest that cytosolic chaperone Sse1 is an important modulator of ER stress response in yeast and it controls stress-induced cell division arrest and cell death during overwhelming ER stress induced by tunicamycin.
Topics: Endoplasmic Reticulum Stress; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Tunicamycin; Unfolded Protein Response; HSP110 Heat-Shock Proteins; Signal Transduction; Basic-Leucine Zipper Transcription Factors; Protein Biosynthesis; Molecular Chaperones; Endoplasmic Reticulum; Protein Serine-Threonine Kinases; Membrane Glycoproteins; Repressor Proteins; HSP70 Heat-Shock Proteins
PubMed: 38577891
DOI: 10.1093/g3journal/jkae075 -
Neurobiology of Disease Jun 2024Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein...
Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington's disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease.
Topics: Animals; Huntington Disease; Disease Models, Animal; Mice; Polyribosomes; Ribosomes; Corpus Striatum; Mice, Transgenic; Disease Progression; Huntingtin Protein; Peptide Initiation Factors
PubMed: 38565397
DOI: 10.1016/j.nbd.2024.106488 -
International Journal of Molecular... Mar 2024Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years,...
Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.
Topics: Humans; Ribosomal Proteins; Myocytes, Cardiac; Ribosomes; Polyribosomes; Cytosol; RNA, Messenger; Internal Ribosome Entry Sites; Protein Biosynthesis
PubMed: 38542224
DOI: 10.3390/ijms25063250 -
BioRxiv : the Preprint Server For... Mar 2024Stress granules form via co-condensation of RNA binding proteins with prion-like low complexity domains (PLCDs) and RNA molecules released by stress-induced polysomal...
Stress granules form via co-condensation of RNA binding proteins with prion-like low complexity domains (PLCDs) and RNA molecules released by stress-induced polysomal runoff. Homotypic interactions among PLCDs can drive amyloid fibril formation and this is enhanced by ALS-associated mutations. We find that homotypic interactions that drive condensation versus fibril formation are separable for A1-LCD, the PLCD of hnRNPA1. These separable interactions lead to condensates that are metastable versus fibrils that are globally stable. Metastable condensates suppress fibril formation, and ALS-associated mutations enhance fibril formation by weakening condensate metastability. Mutations designed to enhance A1-LCD condensate metastability restore wild-type behaviors of stress granules in cells even when ALS-associated mutations are present. This suggests that fibril formation can be suppressed by enhancing condensate metastability through condensate-driving interactions.
PubMed: 38464104
DOI: 10.1101/2024.02.28.582569 -
Nature Communications Feb 2024Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial...
Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.
Topics: Escherichia coli; Cryoelectron Microscopy; Ribosomes; Protein Biosynthesis; Polyribosomes
PubMed: 38409277
DOI: 10.1038/s41467-024-46092-3