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Brain Research Bulletin Apr 2021Memory formation is a fundamental function of the nervous system that enables the experience-based adaptation of behaviour. The formation, recall and updating of... (Review)
Review
Memory formation is a fundamental function of the nervous system that enables the experience-based adaptation of behaviour. The formation, recall and updating of long-term memory (LTM) requires new protein synthesis through its direct involvement in neuronal processes, such as long-term potentiation (LTP), long-term depression (LTD) and synaptic scaling. We discuss the advantages and limitations of several emerging techniques which enable the tagging of newly synthesised proteins, including stable isotope labelling with amino acids in cell culture (SILAC), puromycin labelling, and non-canonical amino acid (NCAA) labelling. We further present how these methods allow for the identification and visualisation of proteins which are newly synthesised during different stages of memory formation. These emerging techniques will continue to expand our understanding of how memories are formed, consolidated and retrieved.
Topics: Animals; Brain; Hippocampus; Memory, Long-Term; Neuronal Plasticity; Proteomics; Synapses
PubMed: 33465403
DOI: 10.1016/j.brainresbull.2020.12.015 -
Animal Cells and Systems 2023Puromycin treatment can cause glomerular injury to the kidney, leading to proteinuria. However, the pathogenesis of acute kidney injury and subsequent regeneration after...
Puromycin treatment can cause glomerular injury to the kidney, leading to proteinuria. However, the pathogenesis of acute kidney injury and subsequent regeneration after puromycin administration in animal models remain unclear. In this work, we examined the characteristics of kidney injury and subsequent regeneration following puromycin treatment in adult zebrafish. We intraperitoneally injected 100 μg of puromycin into zebrafish; sacrificed them at 1, 3, 5, 7, or 14 days post-injection (dpi); and examined the morphological, functional, and molecular changes in the kidney. Puromycin-treated zebrafish presented more rapid clearance of rhodamine dextran than control animals. Morphological changes were observed immediately after the puromycin injection (1-7 dpi) and had recovered by 14 dpi. The mRNA production of , a renal progenitor marker, increased during recovery from kidney injury. Levels of NFκB, TNFα, Nampt, and p-ERK increased significantly during nephron injury and regeneration, and Sirt1, FOXO1, pax2, and wt1b showed an increasing tendency. However, TGF-β1 and smad5 production did not show any changes after puromycin treatment. This study provides evidence that puromycin-induced injury in adult zebrafish kidneys is a potential tool for evaluating the mechanism of nephron injury and subsequent regeneration.
PubMed: 37089626
DOI: 10.1080/19768354.2023.2203211 -
Current Opinion in Chemical Biology Feb 2022Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry-based... (Review)
Review
Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry-based proteomics being an excellent method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging-based, and stable isotope labeling by amino acids in cell culture-based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.
Topics: Amino Acids; Isotope Labeling; Mass Spectrometry; Proteome; Proteomics
PubMed: 34364788
DOI: 10.1016/j.cbpa.2021.07.001 -
Nephrology, Dialysis, Transplantation :... Jun 2024Glucocorticoids are the treatment of choice for proteinuric patients with minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS)....
BACKGROUND
Glucocorticoids are the treatment of choice for proteinuric patients with minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes.
METHODS
We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone.
RESULTS
Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria.
CONCLUSIONS
Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids.
Topics: Animals; Receptors, Glucocorticoid; Mice; Proteinuria; Humans; Rats; Podocytes; Zebrafish; Male; Mifepristone; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Female; Kidney Diseases; Puromycin Aminonucleoside; Hormone Antagonists; Nephrosis, Lipoid; Mice, Inbred C57BL; Mice, Transgenic
PubMed: 38037533
DOI: 10.1093/ndt/gfad254 -
Small Methods Jul 2023Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the...
Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Cell Line; Mammals
PubMed: 37156748
DOI: 10.1002/smtd.202300069 -
Biomarker Research Sep 2022Neuroblastoma (NBL) is the most common extra-cranial solid tumour in childhood, with prognosis ranging from spontaneous remission to high risk for rapid and fatal...
BACKGROUND
Neuroblastoma (NBL) is the most common extra-cranial solid tumour in childhood, with prognosis ranging from spontaneous remission to high risk for rapid and fatal progression. Despite existing therapy approaches, the 5-year event-free survival (EFS) for patients with advanced NBL remains below 30%, emphasizing urgent necessary for novel therapeutic strategies. Studies have shown that epigenetic disorders play an essential role in the pathogenesis of NBL. However, the function and mechanism of N7-methylguanosine (mG) methyltransferase in NBL remains unknown.
METHODS
The expression levels of mG tRNA methyltransferase Methyltransferase-like 1 (METTL1) were analyzed by querying the Gene Expression Omnibus (GEO) database and further confirmed by immunohistochemistry (IHC) assay. Kaplan-Meier, univariate and multivariate cox hazard analysis were performed to reveal the prognostic role of METTL1. Cell function assays were performed to evaluate how METTL1 works in proliferation, apoptosis and migration in cell lines and xenograft mouse models. The role of METTL1 on mRNA translation activity of NBL cells was measured using puromycin intake assay and polysome profiling assay. The mG modified tRNAs were identified by tRNA reduction and cleavage sequencing (TRAC-seq). Ribosome nascent-chain complex-bound mRNA sequencing (RNC-seq) was utilized to identify the variation of gene translation efficiency (TE). Analyzed the codon frequency decoded by mG tRNA to clarify the translation regulation and mechanism of mG modification in NBL.
RESULTS
This study found that METTL1 were significantly up-regulated in advanced NBL, which acted as an independent risk factor and predicted poor prognosis. Further in NBL cell lines and BALB/c-nu female mice, we found METTL1 played a crucial role in promoting NBL progression. Furthermore, mG profiling and translation analysis revealed downregulation of METTL1 would inhibit puromycin intake efficiency of NBL cells, indicating that METTL1 did count crucially in regulation of NBL cell translation. With all tRNAs with mG modification identified in NBL cells, knockdown of METTL1 would significantly reduce the levels of both mG modification and mG tRNAs expressions. Result of RNC-seq shew there were 339 overlapped genes with impaired translation in NBL cells upon METTL1 knockdown. Further analysis revealed these genes contained higher frequency of codons decoded by mG-modified tRNAs and were enriched in oncogenic pathways.
CONCLUSION
This study revealed the critical role and mechanism of METTL1-mediated tRNA mG modification in regulating NBL progression, providing new insights for developing therapeutic approaches for NBL patients.
PubMed: 36071474
DOI: 10.1186/s40364-022-00414-z -
IScience Jul 2022Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the...
Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.
PubMed: 35754732
DOI: 10.1016/j.isci.2022.104516 -
FEBS Open Bio Jun 2021In this issue of FEBS Open Bio, Shen Li et al., in the laboratory of Hector L. Franco (University of North Carolina), provide a proof-of-principle solution for...
In this issue of FEBS Open Bio, Shen Li et al., in the laboratory of Hector L. Franco (University of North Carolina), provide a proof-of-principle solution for correcting all copies of a gene in the widely used MCF7 breast cancer cell line. The gene for the FOXA1 pioneer transcription factor is localised on chromosome 14, which is present at least 4-5 times in MCF7 cells. To achieve their goal, the authors used a 'classical' version of the CRISPR/Cas9 system. Both sgRNA and Cas9 components were expressed from a single vector, which also has a puromycin resistance cassette; this is an essential module for the chosen strategy, because it ensures expression of both sgRNA and Cas9 in selected cells. A targeting template in the form of nonlinearised plasmid was shown to have the best efficiency and was used to introduce a substitution at position 295 in the gene encoding FOXA1 to change a codon encoding lysine into a codon encoding glutamine (K295Q). The strategy suggested by Li and co-authors is an important development towards genome editing of multiple copy genes in a polyploid environment like cancer cells. One important application of the technique could be in creating models to study the role of single nucleotide polymorphisms in cancer progression and metastasis. Isogenic cancer lines carrying polymorphic variants of key drug targets could be used to optimise anticancer treatment protocols, laying a foundation for personalised therapy.
Topics: CRISPR-Cas Systems; Gene Editing; Plasmids
PubMed: 34060719
DOI: 10.1002/2211-5463.13195 -
Biological Research May 2023Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation....
BACKGROUND
Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation. This event causes muscle mass loss and decreased strength and muscle function, a syndrome known as sarcopenia. Recently, our laboratory described secondary sarcopenia in a chronic cholestatic liver disease (CCLD) mouse model. Interestingly, the administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is an effective therapy for cholestatic hepatic alterations. However, the effect of UDCA on skeletal muscle mass and functionality has never been evaluated, nor the possible involved mechanisms.
METHODS
We assessed the ability of UDCA to generate sarcopenia in C57BL6 mice and develop a sarcopenic-like phenotype in CC myotubes and isolated muscle fibers. In mice, we measured muscle strength by a grip strength test, muscle mass by bioimpedance and mass for specific muscles, and physical function by a treadmill test. We also detected the fiber's diameter and content of sarcomeric proteins. In CC myotubes and/or isolated muscle fibers, we determined the diameter and troponin I level to validate the cellular effect. Moreover, to evaluate possible mechanisms, we detected puromycin incorporation, p70S6K, and 4EBP1 to evaluate protein synthesis and ULK1, LC3 I, and II protein levels to determine autophagic flux. The mitophagosome-like structures were detected by transmission electron microscopy.
RESULTS
UDCA induced sarcopenia in healthy mice, evidenced by decreased strength, muscle mass, and physical function, with a decline in the fiber's diameter and the troponin I protein levels. In the CC myotubes, we observed that UDCA caused a reduction in the diameter and content of MHC, troponin I, puromycin incorporation, and phosphorylated forms of p70S6K and 4EBP1. Further, we detected increased levels of phosphorylated ULK1, the LC3II/LC3I ratio, and the number of mitophagosome-like structures. These data suggest that UDCA induces a sarcopenic-like phenotype with decreased protein synthesis and autophagic flux.
CONCLUSIONS
Our results indicate that UDCA induces sarcopenia in mice and sarcopenic-like features in CC myotubes and/or isolated muscle fibers concomitantly with decreased protein synthesis and alterations in autophagic flux.
Topics: Mice; Animals; Sarcopenia; Ursodeoxycholic Acid; Ribosomal Protein S6 Kinases, 70-kDa; Troponin I; Mice, Inbred C57BL; Muscle, Skeletal
PubMed: 37237400
DOI: 10.1186/s40659-023-00431-8 -
Chemical Society Reviews Aug 2021mRNA display is a powerful biological display platform for the directed evolution of proteins and peptides. mRNA display libraries covalently link the displayed peptide... (Review)
Review
mRNA display is a powerful biological display platform for the directed evolution of proteins and peptides. mRNA display libraries covalently link the displayed peptide or protein (phenotype) with the encoding genetic information (genotype) through the biochemical activity of the small molecule puromycin. Selection for peptide/protein function is followed by amplification of the linked genetic material and generation of a library enriched in functional sequences. Iterative selection cycles are then performed until the desired level of function is achieved, at which time the identity of candidate peptides can be obtained by sequencing the genetic material. The purpose of this review is to discuss the development of mRNA display technology since its inception in 1997 and to comprehensively review its use in the selection of novel peptides and proteins. We begin with an overview of the biochemical mechanism of mRNA display and its variants with a particular focus on its advantages and disadvantages relative to other biological display technologies. We then discuss the importance of scaffold choice in mRNA display selections and review the results of selection experiments with biological (e.g., fibronectin) and linear peptide library architectures. We then explore recent progress in the development of "drug-like" peptides by mRNA display through the post-translational covalent macrocyclization and incorporation of non-proteogenic functionalities. We conclude with an examination of enabling technologies that increase the speed of selection experiments, enhance the information obtained in post-selection sequence analysis, and facilitate high-throughput characterization of lead compounds. We hope to provide the reader with a comprehensive view of current state and future trajectory of mRNA display and its broad utility as a peptide and protein design tool.
Topics: Animals; Directed Molecular Evolution; Humans; Ligands; Peptide Library; Peptides; Proteins; RNA, Messenger
PubMed: 34165126
DOI: 10.1039/d1cs00160d