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BioRxiv : the Preprint Server For... Apr 2024E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external...
E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external cues to regulate protein homeostasis. However, the regulation of membrane protein ubiquitination by E3s within distinct cell membrane compartments or organelles is not well understood. We show that FBXO10, the interchangeable component of the SKP1/CUL1/F-box ubiquitin ligase complex (SCF-E3), undergoes lipid-modification with geranylgeranyl isoprenoid at Cysteine953 (C953), facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide does not contain a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and the interaction with two cytosolic factors, PDE6δ (a prenyl group-binding protein), and HSP90 (a mitochondrial chaperone) orchestrate specific OMM targeting of prenyl-FBXO10 across diverse membrane compartments. The geranylgeranylation-deficient FBXO10(C953S) mutant redistributes away from the OMM, leading to impaired mitochondrial ATP production, decreased mitochondrial membrane potential, and increased mitochondrial fragmentation. Phosphoglycerate mutase 5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative mass spectrometry analyses of enriched mitochondria (LFQ-MS/MS), leveraging the redistribution of FBXO10(C953S). FBXO10, but not FBXO10(C953S), promoted polyubiquitylation and degradation of PGAM5. Examination of the role of this pathway in a physiological context revealed that the loss of FBXO10 or expression of prenylation-deficient-FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human iPSCs and murine myoblasts. Our studies identify a mechanism for selective E3-ligase mediated regulation of mitochondrial membrane proteostasis and metabolic health, potentially amenable to therapeutic intervention.
PubMed: 38659932
DOI: 10.1101/2024.04.16.589745 -
Stem Cell Research & Therapy Apr 2024The detection rate of superficial non-ampullary duodenal epithelial tumors (SNADETs) has recently been increasing. Large tumors may contain malignant lesions and early...
BACKGROUND
The detection rate of superficial non-ampullary duodenal epithelial tumors (SNADETs) has recently been increasing. Large tumors may contain malignant lesions and early therapeutic intervention is recommended. Endoscopic mucosal dissection (ESD) is considered a feasible treatment modality, however, the anatomical and physiological characteristics of the duodenum create a risk of postoperative perforation after ESD.
METHODS
To explore whether myoblast sheet transplantation could prevent delayed perforation after ESD, a first-in-human (FIH) clinical trial of laparoscopic autologous myoblast sheet transplantation after duodenal ESD was launched. Autologous myoblast sheets fabricated from muscle tissue obtained seven weeks before ESD were transplanted laparoscopically onto the serous side of the ESD. The primary endpoints were the onset of peritonitis due to delayed perforation within three days after surgery and all adverse events during the follow-up period.
RESULTS
Three patients with SNADETs ≥ 20 mm in size underwent transplantation of a myoblast sheet onto the serous side of the duodenum after ESD. In case 1, The patient's postoperative course was uneventful. Endoscopy and abdominal computed tomography revealed no signs of delayed perforation. Despite incomplete mucosal closure in case 2, and multiple micro perforations during ESD in case 3, cell sheet transplantation could prevent the postoperative massive perforation after ESD, and endoscopy on day 49 after transplantation revealed no stenosis.
CONCLUSIONS
This clinical trial showed the safety, efficacy, and procedural operability of this novel regenerative medicine approach involving transplanting an autologous myoblast sheet laparoscopically onto the serosa after ESD in cases with a high risk of delayed perforation. This result indicates the potential application of cell sheet medicine in treating various abdominal organs and conditions with minimal invasiveness in the future.
TRIAL REGISTRATION
jRCT, jRCT2073210094. Registered November 8 2021, https://jrct.niph.go.jp/latest-detail/jRCT2073210094 .
Topics: Humans; Laparoscopy; Male; Female; Myoblasts; Transplantation, Autologous; Middle Aged; Duodenum; Aged; Intestinal Mucosa; Endoscopic Mucosal Resection; Duodenal Neoplasms; Intestinal Perforation
PubMed: 38654373
DOI: 10.1186/s13287-024-03730-3 -
BioRxiv : the Preprint Server For... Apr 2024Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates;...
Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; and encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, Pkm1 and Pkm2, function in glycolysis, but Pkm2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of Pkm1 and Pkm2 during myoblast differentiation. RNA-seq analysis revealed that Pkm2 promotes the expression of and . Dpf2 and Baf250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for activation of myogenic gene expression during differentiation. Pkm2 also mediated the incorporation of Dpf2 and Baf250a into the regulatory sequences controlling myogenic gene expression. Pkm1 did not affect expression but was required for nuclear localization of Dpf2. Additionally, Pkm2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters, but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for Pkm2 and a novel function for Pkm1 in gene expression and chromatin regulation during myoblast differentiation.
PubMed: 38645038
DOI: 10.1101/2024.04.10.588959 -
Nanoscale Advances Apr 2024Due to their biocompatibility and biodegradability and their unique structural and physicochemical properties, laser-synthesized silicon nanoparticles (Si-NPs) are one...
Due to their biocompatibility and biodegradability and their unique structural and physicochemical properties, laser-synthesized silicon nanoparticles (Si-NPs) are one of the nanomaterials which have been most studied as potential theragnostic tools for non-invasive therapeutic modalities. However, their ability to modulate cell behavior and to promote proliferation and differentiation is still very little investigated or unknown. In this work, ultrapure ligand free Si-NPs of 50 ± 11.5 nm were prepared by femtosecond (fs) laser ablation in liquid. After showing the ability of Si-NPs to be internalized by murine C2C12 myoblasts, the cytotoxicity of the Si-NPs on these cells was evaluated at concentrations ranging from 14 to 224 μg mL. Based on these findings, three concentrations of 14, 28 and 56 μg mL were thus considered to study the effect on myoblast differentiation, proliferation and motility at the molecular and phenotypical levels. It was demonstrated that up to 28 μg mL, the Si-NPs are able to promote the proliferation of myoblasts and their subsequent differentiation. Scratch tests were also performed revealing the positive Si-NP effect on cellular motility at 14 and 28 μg mL. Finally, gene expression analysis confirmed the ability of Si-NPs to promote proliferation, differentiation and motility of myoblasts even at very low concentration. This work opens up novel exciting prospects for Si-NPs made by the laser process as innovative tools for skeletal muscle tissue engineering in view of developing novel therapeutic protocols for regenerative medicine.
PubMed: 38633050
DOI: 10.1039/d3na01020a -
Scientific Reports Apr 2024HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop...
HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop have not been clearly elucidated. Here, we sought to elucidate these mechanisms using the murine myoblast cell line C2C12 and disease-specific human induced pluripotent stem cells (hiPSCs). Myotubes differentiated from HOIL-1L-KO C2C12 cells exhibited deteriorated differentiation and mitotic cell accumulation. CMs differentiated from patient-derived hiPSCs had an abnormal morphology with a larger size and were excessively multinucleated compared with CMs differentiated from control hiPSCs. Further analysis of hiPSC-derived CMs showed that HOIL-1L deficiency caused cell cycle alteration and mitotic cell accumulation. These results demonstrate that abnormal cell maturation possibly contribute to the development of myopathy and DCM. In conclusion, HOIL-1L is an important intrinsic regulator of cell cycle-related myotube and CM maturation and cell proliferation.
Topics: Animals; Humans; Mice; Cell Differentiation; Cell Line; Induced Pluripotent Stem Cells; Muscle, Skeletal; Muscular Diseases; Myocytes, Cardiac; Ubiquitin-Protein Ligases; Transcription Factors; Cell Cycle
PubMed: 38632277
DOI: 10.1038/s41598-024-57504-1 -
Nature Communications Apr 2024Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation...
Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.
Topics: Humans; Myotonic Dystrophy; Heterochromatin; Cell Differentiation; DNA Methylation; Epigenesis, Genetic
PubMed: 38627364
DOI: 10.1038/s41467-024-47217-4 -
Ecotoxicology and Environmental Safety May 2024According to the International Agency for Research on Cancer (IARC), aflatoxin B1 (AFB1) has been recognized as a major contaminant in food and animal feed and which is...
According to the International Agency for Research on Cancer (IARC), aflatoxin B1 (AFB1) has been recognized as a major contaminant in food and animal feed and which is a common mycotoxin with high toxicity. Previous research has found that AFB1 inhibited zebrafish muscle development. However, the potential mechanism of AFB1 on fish muscle development is unknown, so it is necessary to conduct further investigation. In the present research, the primary myoblast of grass carp was used as a model, we treated myoblasts with AFB1 for 24 h. Our results found that 5 μM AFB1 significantly inhibited cell proliferation and migration (P < 0.05), and 10 μM AFB1 promoted lactate dehydrogenase (LDH) release (P < 0.05). Reactive oxygen species (ROS), protein carbonyl (PC) and malondialdehyde (MDA) levels were increased in 15, 5 and 10 μM AFB1 (P < 0.05), respectively. Catalase (CAT), glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activities were decreased in 10, 10 and 15 μM AFB1 (P < 0.05), respectively. Furthermore, 15 μM AFB1 induced oxidative damage by Nrf2 pathway, also induced apoptosis in primary myoblast of grass carp. Meanwhile, 15 μM AFB1 decreased MyoD gene and protein expression (P < 0.05). Importantly, 15 μM AFB1 decreased the protein expression of collagen Ⅰ and fibronectin (P < 0.05), and increased the protein levels of urokinase plasminogen activator (uPA), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and p38 mitogen-activated protein kinase (p38MAPK) (P < 0.05). As a result, our findings suggested that AFB1 damaged the cell morphology, induced oxidative damage and apoptosis, degraded ECM components, in turn inhibiting myoblast development by activating the p38MAPK/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase (MMPs)/extracellular matrix (ECM) signaling pathway.
Topics: Animals; Carps; Aflatoxin B1; Myoblasts; Extracellular Matrix; Cell Proliferation; Reactive Oxygen Species; Oxidative Stress; Cell Movement
PubMed: 38626608
DOI: 10.1016/j.ecoenv.2024.116332 -
Proceedings of the National Academy of... Apr 2024The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody...
The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.
Topics: APOBEC Deaminases; APOBEC-1 Deaminase; Cell Differentiation; Chromatin; Cytidine Deaminase; DNA; Muscle Fibers, Skeletal; Muscle Proteins; Myoblasts; RNA, Messenger; Animals; Mice
PubMed: 38625936
DOI: 10.1073/pnas.2312330121 -
International Journal of Molecular... Apr 2024A novel organic-inorganic gliclazide-loaded composite bead was developed by an ionic gelation process using acidified CaCl, chitosan and tetraethylorthosilicate (TEOS)...
A novel organic-inorganic gliclazide-loaded composite bead was developed by an ionic gelation process using acidified CaCl, chitosan and tetraethylorthosilicate (TEOS) as a crosslinker. The beads were manufactured by crosslinking an inorganic silicone elastomer (-OH terminated polydimethylsiloxane, PDMS) with TEOS at different ratios before grafting onto an organic backbone (Na-alginate) using a 3 factorial experimental design. Gliclazide's encapsulation efficiency (EE%) and drug release over 8 h (% DR 8 h) were set as dependent responses for the optimisation of a pharmaceutical formula (herein referred to as 'G op') by response surface methodology. EE % and %DR 8 h of G op were 93.48% ± 0.19 and 70.29% ± 0.18, respectively. G op exhibited a controlled release of gliclazide that follows the Korsmeyer-Peppas kinetic model (R = 0.95) with super case II transport and pH-dependent swelling behaviour. In vitro testing of G op showed 92.17% ± 1.18 cell viability upon testing on C2C12 myoblasts, indicating the compatibility of this novel biomaterial platform with skeletal muscle drug delivery.
Topics: Gliclazide; Dimethylpolysiloxanes; Alginates; Biocompatible Materials
PubMed: 38612802
DOI: 10.3390/ijms25073991 -
International Journal of Molecular... Mar 2024In this study, gilthead sea bream () fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to...
Exploring the Integrated Role of miRNAs and lncRNAs in Regulating the Transcriptional Response to Amino Acids and Insulin-like Growth Factor 1 in Gilthead Sea Bream () Myoblasts.
In this study, gilthead sea bream () fast muscle myoblasts were stimulated with two pro-growth treatments, amino acids (AA) and insulin-like growth factor 1 (Igf-1), to analyze the transcriptional response of mRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and to explore their possible regulatory network using bioinformatic approaches. AA had a higher impact on transcription (1795 mRNAs changed) compared to Igf-1 (385 mRNAs changed). Both treatments stimulated the transcription of mRNAs related to muscle differentiation (GO:0042692) and sarcomere (GO:0030017), while AA strongly stimulated DNA replication and cell division (GO:0007049). Both pro-growth treatments altered the transcription of over 100 miRNAs, including muscle-specific miRNAs (myomiRs), such as , , , , and . Among 111 detected lncRNAs (>1 FPKM), only 30 were significantly changed by AA and 11 by Igf-1. Eight lncRNAs exhibited strong negative correlations with several mRNAs, suggesting a possible regulation, while 30 lncRNAs showed strong correlations and interactions with several miRNAs, suggesting a role as sponges. This work is the first step in the identification of the ncRNAs network controlling muscle development and growth in gilthead sea bream, pointing out potential regulatory mechanisms in response to pro-growth signals.
Topics: Animals; Amino Acids; Sea Bream; RNA, Long Noncoding; Insulin-Like Peptides; Insulin-Like Growth Factor I; MicroRNAs; Myoblasts; Antifibrinolytic Agents; RNA, Messenger; Sarcomeres
PubMed: 38612703
DOI: 10.3390/ijms25073894