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Journal of Biological Rhythms Feb 2024Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are...
Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration.
Topics: Circadian Clocks; Circadian Rhythm; Protein Synthesis Inhibitors; Nuclear Receptor Subfamily 1, Group D, Member 1; Heart
PubMed: 37872767
DOI: 10.1177/07487304231202561 -
Toxicology and Applied Pharmacology Nov 2023Chronic arsenic exposure through drinking water is a global health issue, affecting >200 million people. Arsenic is a group I human carcinogen and causes chromosomal...
Clonal variability in chromosomal instability as a potential driver in the acquisition of tumorigenic phenotype in chronic arsenic-exposed and hsa-miR-186 overexpressing human keratinocytes.
Chronic arsenic exposure through drinking water is a global health issue, affecting >200 million people. Arsenic is a group I human carcinogen and causes chromosomal instability (CIN). Arsenic exposure is the second most common cause of skin cancer after UV radiation. hsa-miR-186 is overexpressed in arsenic-induced squamous cell carcinoma relative to premalignant hyperkeratosis. Among predicted targets of hsa-miR-186 are cell cycle regulators including regulators of mitotic progression. Disruption of mitotic progression can contribute to CIN. Thus, we hypothesized that hsa-miR-186 overexpression contributes to malignant transformation of arsenic exposed HaCaT cells by induction of CIN. Stable clones of HaCaT cells transfected with pEP-hsa-miR-186 expression vector or empty vector were maintained under puromycin selection and exposed to 0 or 100 nM NaAsO and cultured for 29 weeks. HaCaT clones overexpressing hsa-miR-186 and exposed to NaAsO showed increased CIN and anchorage independent growth at 29 weeks in a stochastic manner, in contrast to unexposed empty vector transfected clones. These results suggest that clonal variability mediates arsenic-induced carcinogenesis in hsa-miR-186 overexpressing human keratinocytes.
Topics: Humans; Arsenic; Cell Line; MicroRNAs; Carcinogenesis; Keratinocytes; Clone Cells; Phenotype; Chromosomal Instability
PubMed: 37866707
DOI: 10.1016/j.taap.2023.116730 -
STAR Protocols Dec 2023Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using...
Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging. We also provide details of quantification analysis. Significantly, OPP addition to methionine-containing media enables polypeptide labeling in living cells. Here, we study wing imaginal discs, an excellent model system for investigating growth, proliferation, pattern formation, differentiation, and cell death. For complete details on the use and execution of this protocol, please refer to Lee et al. (2018), Ji et al. (2019), and Kiparaki et al. (2022)..
Topics: Animals; Drosophila; Imaginal Discs; Larva; Puromycin
PubMed: 37862174
DOI: 10.1016/j.xpro.2023.102653 -
American Journal of Physiology. Renal... Jan 2024As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have...
As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes (p16INK4a/p14ARF), (p19INK4d), and (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of , , and In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated β-galactosidase (SA-β-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span. Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.
Topics: Middle Aged; Humans; Mice; Animals; Aged; Podocytes; Glomerulosclerosis, Focal Segmental; Kidney Glomerulus; Kidney Diseases; Aging; Doxorubicin
PubMed: 37855038
DOI: 10.1152/ajprenal.00261.2023 -
Genes & Genetic Systems Oct 2023Polyglutamine (polyQ) diseases are rare autosomal-dominant neurodegenerative diseases associated with the expansion of glutamine-encoding triplet repeats in certain...
Polyglutamine (polyQ) diseases are rare autosomal-dominant neurodegenerative diseases associated with the expansion of glutamine-encoding triplet repeats in certain genes. To investigate the functional influence of repeat expansion on disease mechanisms, we applied a biallelic genome-engineering platform that we recently established, called Universal Knock-in System or UKiS, to develop a human cell trio, a set of three isogenic cell lines that are homozygous for two different numbers of repeats (first and second lines) or heterozygous for the two repeat numbers (third line). As an example of a polyQ disease, we chose spinocerebellar ataxia type 2 (SCA2). In a pseudodiploid human cell line, both alleles of the glutamine-encoding triplet repeat in the SCA2-causing gene, ataxin 2 or ATXN2, were first knocked in with a donor sequence encoding both thymidine kinase and either puromycin or blasticidin resistance proteins under dual drug selection. The knocked-in donor alleles were then substituted with a payload having either 22 or 76 triplet repeats in ATXN2 by ganciclovir negative selection. The two-step substitution and subsequent SNP typing and genomic sequencing confirmed that the SCA2-modeling isogenic cell trio was obtained: three clones of 22-repeat homozygotes, two clones of 22/76-repeat heterozygotes and two clones of 76-repeat homozygotes. Finally, RT-PCR and immunoblotting using the obtained clones showed that, consistent with previous observations, glutamine tract expansion reduced transcriptional and translational expression of ATXN2. The cell clones with homozygous long-repeat alleles, which are rarely obtained from patients with SCA2, showed more drastic reduction of ATXN2 expression than the heterozygous clones. This study thus demonstrates the potential of UKiS, which is a beneficial platform for the efficient development of cell models not only for polyQ diseases but also for any other genetic diseases, which may accelerate our deeper understanding of disease mechanisms and cell-based screening for therapeutic drugs.
Topics: Humans; Glutamine; Peptides; Spinocerebellar Ataxias; Proteins
PubMed: 37821389
DOI: 10.1266/ggs.22-00030 -
Proceedings of the National Academy of... Oct 2023Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many...
Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac Na1.5 () and hERG1 (), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of , , , and transcripts collectively copurifying with nascent hERG1, Na1.5, Ca1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for and , thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when I was reduced by the selective blocker dofetilide at IC concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.
Topics: Humans; KCNQ1 Potassium Channel; ERG1 Potassium Channel; In Situ Hybridization, Fluorescence; Arrhythmias, Cardiac; RNA, Messenger; RNA, Small Interfering; NAV1.5 Voltage-Gated Sodium Channel
PubMed: 37816059
DOI: 10.1073/pnas.2305295120 -
Proceedings of the National Academy of... Oct 2023The two main steps of translation, peptidyl transfer, and translocation are accompanied by counterclockwise and clockwise rotations of the large and small ribosomal...
The two main steps of translation, peptidyl transfer, and translocation are accompanied by counterclockwise and clockwise rotations of the large and small ribosomal subunits with respect to each other. Upon peptidyl transfer, the small ribosomal subunit rotates counterclockwise relative to the large subunit, placing the ribosome into the rotated conformation. Simultaneously, tRNAs move into the hybrid conformation, and the L1 stalk moves inward toward the P-site tRNA. The conformational dynamics of pretranslocation ribosomes were extensively studied by ensemble and single-molecule methods. Different experimental modalities tracking ribosomal subunits, tRNAs, and the L1 stalk showed that pretranslocation ribosomes undergo spontaneous conformational transitions. Thus, peptidyl transfer unlocks the ribosome and decreases an energy barrier for the reverse ribosome rotation during translocation. However, the tracking of translation with ribosomes labeled at rRNA helices h44 and H101 showed a lack of spontaneous rotations in pretranslocation complexes. Therefore, reverse intersubunit rotations occur during EF-G catalyzed translocation. To reconcile these views, we used high-speed single-molecule microscopy to follow translation in real time. We showed spontaneous rotations in puromycin-released h44-H101 dye-labeled ribosomes. During elongation, the h44-H101 ribosomes undergo partial spontaneous rotations. Spontaneous rotations in h44-H101-labeled ribosomes are restricted prior to aminoacyl-tRNA binding. The pretranslocation h44-H101 ribosomes spontaneously exchanged between three different rotational states. This demonstrates that peptidyl transfer unlocks spontaneous rotations and pretranslocation ribosomes can adopt several thermally accessible conformations, thus supporting the Brownian model of translocation.
Topics: Fluorescence Resonance Energy Transfer; Ribosomes; RNA, Transfer; Nucleic Acid Conformation; Peptide Elongation Factor G; Protein Biosynthesis
PubMed: 37801472
DOI: 10.1073/pnas.2114979120 -
Bio-protocol Sep 2023The study of translation is important to the understanding of gene expression. While genome-wide measurements of translation efficiency (TE) rely upon ribosome...
The study of translation is important to the understanding of gene expression. While genome-wide measurements of translation efficiency (TE) rely upon ribosome profiling, classical approaches to address translation of individual genes of interest rely on biochemical methods, such as polysome fractionation and immunoprecipitation (IP) of ribosomal components, or on reporter constructs, such as luciferase reporters. Methods to investigate translation have been developed that, however, require considerable research effort, including addition of numerous features to mRNA regions, genomic integration of reporters, and complex data analysis. Here, we describe a simple biochemical reporter assay to study TE of mRNAs expressed from a transiently transfected plasmid, which we term Nascent Chain Immunoprecipitation (NC IP). The assay is based on a plasmid expressing an N-terminally Flag-tagged protein and relies on the IP of Flag-tagged nascent chains from elongating ribosomes, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) quantification of eluted mRNA. We report that elution of mRNA following IP can be achieved by treatment with puromycin, which releases ribosome-mRNA complexes, or with purified Flag peptide, which instead releases nascent chain-ribosome-mRNA complexes. In the example described in this protocol, untranslated regions (UTRs) of a gene of interest were used to flank a FlagVenus coding sequence, with the method allowing to infer UTR-dependent regulation of TE. Importantly, our method enables discrimination of translating from non-translating mRNAs. Additionally, it requires simple procedures and standard laboratory equipment. Our method can be used to test the effect of regulators, such as microRNAs or therapeutic drugs or of various genetic backgrounds, on translation of any user-selected mRNA. Key features • The novel NC IP protocol builds upon a previously published method for detection of mRNA-binding proteins (Williams et al., 2022). • The NC IP protocol is adapted for detecting mRNA actively undergoing translation. • The method uses mammalian cell culture but could be adapted to multiple organisms, including budding yeast ().
PubMed: 37753466
DOI: 10.21769/BioProtoc.4821 -
STAR Protocols Dec 2023Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present...
Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present an optimized protocol for assessing the translation rate in mouse adult microglia and cultured bone-marrow-derived macrophages. We describe steps for isolating cells, treating them with a puromycin-analog probe, and fluorescently labeling the puromycylated-polypeptide chains. We then detail their quantification by flow cytometry or with a fluorescent plate reader. For complete details on the use and execution of this protocol, please refer to Keane et al. (2021)..
Topics: Animals; Mice; Microglia; Bone Marrow; Macrophages; Coloring Agents; Protein Biosynthesis
PubMed: 37713309
DOI: 10.1016/j.xpro.2023.102559 -
Cancer Biology & Therapy Dec 2023Lung adenocarcinoma is one of the leading causes of cancer-related mortality globally. Various treatment approaches and drugs had little influence on overall survival;... (Meta-Analysis)
Meta-Analysis
Lung adenocarcinoma is one of the leading causes of cancer-related mortality globally. Various treatment approaches and drugs had little influence on overall survival; thus, new drugs and treatment strategies are needed. Drug repositioning (repurposing) seems a favorable approach considering that developing new drugs needs much more time and costs. We performed a meta-analysis on 6 microarray datasets to obtain the main genes with significantly altered expression in lung adenocarcinoma. Following that, we found major gene clusters and hub genes. We assessed their enrichment in biological pathways to get insight into the underlying biological process involved in lung adenocarcinoma pathogenesis. The L1000 database was explored for drug perturbations that might reverse the expression of differentially expressed genes in lung adenocarcinoma. We evaluated the potential drug combinations that interact the most with hub genes and hence have the most potential to reverse the disease process. A total of 2148 differentially expressed genes were identified. Six main gene clusters and 27 significant hub genes mainly involved in cell cycle regulation have been identified. By assessing the interaction between 3 drugs and hub genes and information gained from previous clinical investigations, we suggested the three possible repurposed drug combinations, Vorinostat - Dorsomorphin, PP-110 - Dorsomorphin, and Puromycin - Vorinostat with a high chance of success in clinical trials.
Topics: Humans; Drug Repositioning; Vorinostat; Adenocarcinoma of Lung; Drug Combinations; Lung Neoplasms
PubMed: 37710391
DOI: 10.1080/15384047.2023.2253586