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Autophagy Jan 2024Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt...
Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt to metabolic changes by degrading and recycling intracellular components. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained MTOR (mechanistic target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. MTOR inhibition by rapamycin treatment was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting MTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation. 5FU: fluoracil; AA: amino acids; AKT/PKB: thymoma viral proto-oncogene 1; ATF4: activating transcription factor 4; BafA: bafilomycin A; BM: bone marrow; EIF2: eukaryotic initiation factor 2; EIF4EBP1/4EBP1: eukaryotic translation initiation factor 4E binding protein 1; KIT/CD117/c-Kit: KIT proto-oncogene receptor tyrosine kinase; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; Kyn: kynurenine; LSK: lineage (Lin), LY6A/Sca-1, KIT/c-Kit/CD117; LY6A/Sca-1: lymphocyte antigen 6 family member A; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; OPP: O-propargyl-puromycin; PI3K: phosphoinositide 3-kinase; poly(I:C): polyinosinic:polycytidylic acid; RPS6/S6: ribosomal protein S6; tam: tamoxifen; TCA: tricarboxylic acid; TFEB: transcription factor EB; PTPRC/CD45: Protein Tyrosine Phosphatase Receptor Type C, CD45 antigen.
Topics: Mice; Animals; Mechanistic Target of Rapamycin Complex 1; Signal Transduction; Autophagy; Phosphatidylinositol 3-Kinases; Hematopoietic Stem Cells; Mechanistic Target of Rapamycin Complex 2; Sirolimus
PubMed: 37614038
DOI: 10.1080/15548627.2023.2247310 -
Poultry Science Oct 2023The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better...
The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.
Topics: Animals; Gene Knockout Techniques; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Chickens; Endonucleases
PubMed: 37562129
DOI: 10.1016/j.psj.2023.102970 -
Computational and Structural... 2023The central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase...
The central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase center (PTC). Experimental evidence shows that the catalytic activity is affected by the electrostatic environment around the peptidyl transferase center. Here, we set up a minimal geometrical model fitting the available x-ray solved structures of the ribonucleic cavity around the catalytic center of the large subunit of the ribosome. The purpose of this phenomenological model is to estimate quantitatively the electrostatic potential and electric field that are experienced during the peptidyl transfer reaction. At least two reasons motivate the need for developing this quantification. First, we inquire whether the electric field in this particular catalytic environment, made only of nucleic acids, is of the same order of magnitude as the one prevailing in catalytic centers of the proteic enzymes counterparts. Second, the protein synthesis rate is dependent on the nature of the amino acid sequentially incorporated in the nascent chain. The activation energy of the catalytic reaction and its detailed kinetics are shown to be dependent on the mechanical work exerted on the amino acids by the electric field, especially when one of the four charged amino acid residues (R, K, E, D) has previously been incorporated at the carboxy-terminal end of the peptidyl-tRNA. Physical values of the electric field provide quantitative knowledge of mechanical work, activation energy and rate of the peptide bond formation catalyzed by the ribosome. We show that our theoretical calculations are consistent with two independent sets of previously published experimental results. Experimental results for in the minimal case of the dipeptide bond formation when puromycin is used as the final amino acid acceptor strongly support our theoretically derived reaction time courses. Experimental Ribo-Seq results on and comparing the residence time distribution of ribosomes upon specific codons are also well accounted for by our theoretical calculations. The statistical queueing time theory was used to model the ribosome residence time per codon during nascent protein elongation and applied for the interpretation of the Ribo-Seq data. The hypo-exponential distribution fits the residence time observed distribution of the ribosome on a codon. An educated deconvolution of this distribution is used to estimate the rates of each elongation step in a codon specific manner. Our interpretation of all these results sheds light on the functional role of the electrostatic profile around the PTC and its impact on the ribosome elongation cycle.
PubMed: 37560126
DOI: 10.1016/j.csbj.2023.07.016 -
Nucleic Acids Research Sep 2023We describe a novel method for in vitro protein display-click display-that does not depend on maintaining RNA integrity during biopanning and yields covalently linked...
We describe a novel method for in vitro protein display-click display-that does not depend on maintaining RNA integrity during biopanning and yields covalently linked protein-cDNA complexes from double-stranded input DNA within 2 h. The display is achieved in a one-pot format encompassing transcription, translation and reverse transcription reactions in series. Stable linkage between proteins and the encoding cDNA is mediated by a modified DNA linker-ML-generated via a click chemistry reaction between a puromycin-containing oligo and a cDNA synthesis primer. Biopanning of a click-displayed mock library coupled with next-generation sequencing analysis revealed >600-fold enrichment of target binders within a single round of panning. A synthetic library of Designed Ankyrin Repeat Proteins (DARPins) with ∼1012 individual members was generated using click display in a 25-μl reaction and six rounds of library panning against a model protein yielded a panel of nanomolar binders. This study establishes click display as a powerful tool for protein binder discovery/engineering and provides a convenient platform for in vitro biopanning selection even in RNase-rich environments such as on whole cells.
Topics: DNA; DNA, Complementary; Peptide Library; Protein Engineering; Proteins; Directed Molecular Evolution
PubMed: 37548398
DOI: 10.1093/nar/gkad643 -
The Journal of Biological Chemistry Sep 2023Puromycin and its derivative O-propargyl puromycin (OPP) have recently found widespread use in detecting nascent proteins. Use of these metabolic labels in complex...
Puromycin and its derivative O-propargyl puromycin (OPP) have recently found widespread use in detecting nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here, we show how a widely used mammalian selection marker, puromycin N-acetyltransferase, can be repurposed for cell-specific metabolic labeling. This approach, which we named puromycin inactivation for cell-selective proteome labeling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing puromycin N-acetyltransferase and detection of translation in other cell types. Using cocultures of neurons and glial cells from the rat brain cortex, we show the application of PICSL for puromycin immunostaining, Western blot, and mass spectrometric identification of nascent proteins. By combining PICSL and OPP-mediated proteomics, cell type-enriched proteins can be identified based on reduced OPP labeling in the cell type of interest.
PubMed: 37543363
DOI: 10.1016/j.jbc.2023.105129 -
International Journal of Molecular... Jul 2023Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor () gene, leading to an...
Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused mainly by substitutions in the low-density lipoprotein receptor () gene, leading to an increased risk of premature cardiovascular diseases. Tremendous advances in sequencing techniques have resulted in the discovery of more than 3000 variants of the gene, but not all of them are clinically relevant. Therefore, functional studies of selected variants are needed for their proper classification. Here, a single-cell, kinetic, fluorescent LDL uptake assay was applied for the functional analysis of LDLR variants in a model of an LDLR-deficient human cell line. An LDLR-defective HEK293T cell line was established via a CRISPR/Cas9-mediated luciferase-puromycin knock-in. The expressing vector with the gene under the control of the regulated promoter and with a reporter gene has been designed to overproduce LDLR variants in the host cell. Moreover, an promoter-luciferase knock-in reporter system has been created in the human cell line to study transcriptional regulation of the gene, which can serve as a simple tool for screening and testing new HMG CoA reductase-inhibiting drugs for atherosclerosis therapy. The data presented here demonstrate that the obtained LDLR-deficient human cell line HEK293T-ldlrG1 and the dedicated pTetRedLDLRwt expression vector are valuable tools for studying LDL internalization and functional analysis of LDLR and its genetic variants. Using appropriate equipment, LDL uptake to a single cell can be measured in real time. Moreover, the luciferase gene knock-in downstream of the promotor allows the study of promoter regulation in response to diverse conditions or drugs. An analysis of four known LDLR variants previously classified as pathogenic and benign was performed to validate the LDLR-expressing system described herein with the dedicated LDLR-deficient human cell line, HEK293T-ldlrG1.
Topics: Humans; Atherosclerosis; HEK293 Cells; Hyperlipoproteinemia Type II; Lipoproteins, LDL; Receptors, LDL
PubMed: 37511194
DOI: 10.3390/ijms241411435 -
Virus Research Sep 2023The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of...
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the Δorf2-8, Δorf2.4, and Δorf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the Δorf2-8 and Δorf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the Δorf2.4-puro replicon by inserting a puromycin-resistant gene into the Δorf2.4 replicon. We observed the more sustained replication of the Δorf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.
Topics: Humans; Antiviral Agents; SARS-CoV-2; COVID-19; Drug Evaluation, Preclinical; Containment of Biohazards; Virus Replication; Replicon; Puromycin
PubMed: 37473963
DOI: 10.1016/j.virusres.2023.199176 -
BioRxiv : the Preprint Server For... Jul 2023parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug...
parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug targets are in critical need, along with novel assays to accelerate their identification. Given the essentiality of protein synthesis throughout the complex parasite lifecycle, translation inhibitors are a promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for prophylaxis. To identify compounds capable of inhibiting liver stage parasite translation, we developed an assay to visualize and quantify translation in the HepG2 infection model. After labeling infected monolayers with o-propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bioorthogonal addition of a fluorophore to each OPP-terminated nascent polypetide, we use automated confocal feedback microscopy followed by batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual liver stage parasites and host cells simultaneously. After validation, we demonstrate specific, concentration-dependent liver stage translation inhibition by both parasite-selective and pan-eukaryotic active compounds, and further show that acute pre-treatment and competition modes of the OPP assay can distinguish between direct and indirect translation inhibitors. We identify a Malaria Box compound, MMV019266, as a direct translation inhibitor in liver stages and confirm this potential mode of action in asexual blood stages.
PubMed: 37461595
DOI: 10.1101/2023.07.05.547872 -
Pharmacological Research Aug 2023Hypoxia-inducible factor-2α (HIF-2α) is a transcription factor responsible for regulating genes related to angiogenesis and metabolism. This study aims to explore the...
Hypoxia-inducible factor-2α (HIF-2α) is a transcription factor responsible for regulating genes related to angiogenesis and metabolism. This study aims to explore the effect of a previously unreported mutation c.C2473T (p.R825S) in the C-terminal transactivation domain (CTAD) of HIF-2α that we detected in tissue of patients with liver disease. We sequenced available liver and matched blood samples obtained during partial liver resection or liver transplantation performed for clinical indications including hepatocellular carcinoma and liver failure. In tandem, we constructed cell lines and a transgenic mouse model bearing the corresponding identified mutation in HIF-2α from which we extracted primary hepatocytes. Lipid accumulation was evaluated in these cells and liver tissue from the mouse model using Oil Red O staining and biochemical measurements. We identified a mutation in the CTAD of HIF-2α (c.C2473T; p.R825S) in 5 of 356 liver samples obtained from patients with hepatopathy and dyslipidemia. We found that introduction of this mutation into the mouse model led to an elevated triglyceride level, lipid droplet accumulation in liver of the mutant mice and in their extracted primary hepatocytes, and increased transcription of genes related to hepatic fatty acid transport and synthesis in the mutant compared to the control groups. In mutant mice and cells, the protein levels of nuclear HIF-2α and its target perilipin-2 (PLIN2), a lipid droplet-related gene, were also elevated. Decreased lipophagy was observed in mutant groups. Our study defines a subpopulation of dyslipidemia that is caused by this HIF-2α mutation. This may have implications for personalized treatment.
Topics: Animals; Humans; Mice; Basic Helix-Loop-Helix Transcription Factors; Dyslipidemias; Lipids; Liver Neoplasms; Mutation
PubMed: 37453673
DOI: 10.1016/j.phrs.2023.106851 -
PloS One 2023Puromycin-sensitive aminopeptidase (E.C. 3.4.11.14, UniProt P55786), a zinc metallopeptidase belonging to the M1 family, degrades a number of bioactive peptides as well...
Puromycin-sensitive aminopeptidase (E.C. 3.4.11.14, UniProt P55786), a zinc metallopeptidase belonging to the M1 family, degrades a number of bioactive peptides as well as peptides released from the proteasome, including polyglutamine. We report the crystal structure of PSA at 2.3 Ǻ. Overall, the enzyme adopts a V-shaped architecture with four domains characteristic of the M1 family aminopeptidases, but it is in a less compact conformation compared to most M1 enzymes of known structure. A microtubule binding sequence is present in a C-terminal HEAT repeat domain of the enzyme in a position where it might serve to mediate interaction with tubulin. In the catalytic metallopeptidase domain, an elongated active site groove lined with aromatic and hydrophobic residues and a large S1 subsite may play a role in broad substrate recognition. The structure with bound polyglutamine shows a possible interacting mode of this peptide, which is supported by mutation.
Topics: Aminopeptidases; Peptides; Metalloproteases; Binding Sites; Substrate Specificity
PubMed: 37440518
DOI: 10.1371/journal.pone.0287086