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Journal of Cellular Physiology Sep 2023Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The...
Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin-proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X-prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl-cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine-sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched-chain amino acid levels, and enhanced mTORC2-mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation.
Topics: Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Differentiation; Cell Line; Methionyl Aminopeptidases; Muscle Development; Muscle, Skeletal; Myoblasts; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Animals; Mice; Leucyl Aminopeptidase
PubMed: 37435895
DOI: 10.1002/jcp.31070 -
Journal of the American Chemical Society Nov 2022Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products...
Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products have emerged that interfere with the ribosomal function, such as puromycin, which mimics an aminoacyl tRNA and causes premature chain termination. Here, we introduce a photoswitchable version of puromycin that, in effect, puts translation under optical control. Our compound, termed , features a diazocine that allows for reversible and nearly quantitative isomerization and pharmacological modulation. Its synthesis involves a new photoswitchable amino acid building block. shows little activity in the dark and becomes substantially more active and cytotoxic, in a graded fashion, upon irradiation with various wavelengths of visible light. In vitro translation assays confirm that inhibits translation with a mechanism similar to that of puromycin itself. Once incorporated into nascent proteins, reacts with standard puromycin antibodies, which allows for tracking protein synthesis using western blots and immunohistochemistry. As a cell-permeable small molecule, can be used for nascent proteome profiling in a variety of cell types, including primary mouse neurons. We envision as a useful biochemical tool for the optical control of translation and for monitoring newly synthesized proteins in defined locations and at precise time points.
Topics: Animals; Mice; Puromycin; Light; Blotting, Western; RNA, Transfer, Amino Acyl; Amino Acids
PubMed: 36394560
DOI: 10.1021/jacs.2c07374 -
Journal of the American Society of... Feb 2021FSGS caused by mutations in is characterized by a podocytopathy with mistrafficked nephrin, an essential component of the slit diaphragm. Because INF2 is a formin-type...
BACKGROUND
FSGS caused by mutations in is characterized by a podocytopathy with mistrafficked nephrin, an essential component of the slit diaphragm. Because INF2 is a formin-type actin nucleator, research has focused on its actin-regulating function, providing an important but incomplete insight into how these mutations lead to podocytopathy. A yeast two-hybridization screen identified the interaction between INF2 and the dynein transport complex, suggesting a newly recognized role of INF2 in regulating dynein-mediated vesicular trafficking in podocytes.
METHODS
Live cell and quantitative imaging, fluorescent and surface biotinylation-based trafficking assays in cultured podocytes, and a new puromycin aminoglycoside nephropathy model of transgenic mice were used to demonstrate altered dynein-mediated trafficking of nephrin in INF2 associated podocytopathy.
RESULTS
Pathogenic mutations disrupt an interaction of INF2 with dynein light chain 1, a key dynein component. The best-studied mutation, R218Q, diverts dynein-mediated postendocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport can rescue this effect. Augmented dynein-mediated trafficking and degradation of nephrin underlies puromycin aminoglycoside-induced podocytopathy and FSGS .
CONCLUSIONS
mutations enhance dynein-mediated trafficking of nephrin to proteolytic pathways, diminishing its recycling required for maintaining slit diaphragm integrity. The recognition that dysregulated dynein-mediated transport of nephrin in R218Q knockin podocytes opens an avenue for developing targeted therapy for INF2-mediated FSGS.
Topics: Animals; Cell Culture Techniques; Cytoplasmic Dyneins; Formins; Glomerulosclerosis, Focal Segmental; Membrane Proteins; Mice; Mutation; Podocytes; Protein Transport
PubMed: 33443052
DOI: 10.1681/ASN.2020081109 -
Scientific Reports May 2023Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human...
Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.
Topics: Humans; Induced Pluripotent Stem Cells; Cells, Cultured; Cell Differentiation; Muscle, Skeletal; Muscular Diseases
PubMed: 37231024
DOI: 10.1038/s41598-023-34445-9 -
Journal of Genome Editing and Regulation 2022CRSPR/Cas9-mediated base editing introduces point mutations in cellular DNA by exploiting target-specific single guide RNA (sgRNA) along with a genetically modified...
CRSPR/Cas9-mediated base editing introduces point mutations in cellular DNA by exploiting target-specific single guide RNA (sgRNA) along with a genetically modified Cas9. Existing plasmid vectors for sgRNA expression in base editing contain either a fluorescent marker or an antibiotic resistance cassette but not both, preventing simultaneous monitoring and enrichment of transfected host cells. In this study, we introduced a fluorescent marker into pGL3-U6-sgRNA-PGK-puromycin, a popular sgRNA expression vector available at Addgene. Specifically, the cDNAs of mRFP and a T2A linker were inserted in between the hPGK promoter and the puromycin resistance gene (PuroR). After correct insertion was verified by DNA sequencing, this new plasmid, pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR, was utilized to generate a stop codon in the second exon of the Munc13-1 gene in RBL-2H3 cells. Both the mRFP fluorescent marker and the puromycin resistance marker functioned accordingly in the process. This new sgRNA vector therefore represents a useful addition to the CRISPR tool kit.
PubMed: 38076397
DOI: 10.32371/jger/246146 -
Gene Feb 2024Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will...
BACKGROUND
Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease.
METHODS
Initially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression.
RESULTS
We identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro.
CONCLUSIONS
Knock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.
Topics: Animals; Mice; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Gene Editing; Hemophilia A; RNA, Guide, CRISPR-Cas Systems; Factor VIII
PubMed: 38036077
DOI: 10.1016/j.gene.2023.148038 -
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 -
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 -
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 -
Frontiers in Microbiology 2021Recombinant DNA technology is a vital method in human hepatitis B virus (HBV), producing reporter viruses or vectors for gene transferring. Researchers have engineered...
Recombinant DNA technology is a vital method in human hepatitis B virus (HBV), producing reporter viruses or vectors for gene transferring. Researchers have engineered several genes into the HBV genome for different purposes; however, a systematic analysis of recombinant strategy is lacking. Here, using a 500-bp deletion strategy, we scanned the HBV genome and identified two regions, region I (from nt 2,118 to 2,814) and region II (from nt 99 to 1,198), suitable for engineering. Ten exogenous genes, including puromycin N-acetyl transferase gene (), blasticidin S deaminase gene (), Neomycin-resistance gene (), Gaussia luciferase (), NanoLuc (), , , , , and , were inserted into these two regions and fused into the open reading frames of hepatitis B core protein (HBC) and hepatitis B surface protein (HBS) via T2A peptide. Recombination of 9 of the 10 genes at region 99-1198 and 5 of the 10 genes at region 2118-2814 supported the formation of relaxed circular (RC) DNA. HBV DNA and HBV RNA assays implied that exogenous genes potentially abrogate RC DNA by inducing the formation of adverse secondary structures. This hypothesis was supported because sequence optimization of the UnaG gene based on HBC sequence rescued RC DNA formation. Findings from this study provide an informative basis and a valuable method for further constructing and optimizing recombinant HBV and imply that DNA sequence might be intrinsically a potential source of selective pressure in the evolution of HBV.
PubMed: 34858381
DOI: 10.3389/fmicb.2021.783040