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PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.
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Trastornos del Neurodesarrollo , Humanos , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Femenino , Masculino , Alelos , Autofagia/genética , Ubiquitina Tiolesterasa/genética , Recién Nacido , Complejo de la Endopetidasa Proteasomal/genética , Linaje , Homocigoto , Predisposición Genética a la Enfermedad , Mutación/genéticaRESUMEN
Dual left anterior descending artery (LAD) is a rare congenital coronary artery anomaly with a prevalence of approximately 1% in the general population. To date, 10 types of dual LAD artery anomalies have been reported. Among these, type 4 is one of the rarest. Knowledge and recognition of the dual LAD artery are important for correct diagnosis and planning of coronary bypass surgery and percutaneous coronary intervention. We report a case of a 59-year-old male with type 4 dual LAD artery who presented with dyspepsia and sweating for several months and had approximately 50%-70% stenosis in a major diagonal branch off the short LAD artery.
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Ubiquitin-specific protease 14 (USP14), one of the three major proteasome-associated deubiquitinating enzymes (DUBs), is known to be activated by the AKT-mediated phosphorylation at Ser432. Thereby, AKT can regulate global protein degradation by controlling the ubiquitin-proteasome system (UPS). However, the exact molecular mechanism of USP14 activation by AKT phosphorylation at the atomic level remains unknown. By performing the molecular dynamics (MD) simulation of the USP14 catalytic domain at three different states (inactive, active, and USP14-ubiquitin complex), we characterized the change in structural dynamics by phosphorylation. We observed that the Ser432 phosphorylation induced substantial conformational changes of USP14 in the blocking loop (BL) region to fold it from an open loop into a ß-sheet, which is critical for USP14 activation. Furthermore, phosphorylation also increased the frequency of critical hydrogen bonding and salt bridge interactions between USP14 and ubiquitin, which is essential for DUB activity. Structural dynamics insights from this study pinpoint the important local conformational landscape of USP14 by the phosphorylation event, which would be critical for understanding USP14-mediated proteasome regulation and designing future therapeutics.
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Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas c-akt , Ubiquitina Tiolesterasa , Fosforilación , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Humanos , Ubiquitina/metabolismo , Activación Enzimática , Dominio Catalítico , Unión Proteica , Conformación ProteicaRESUMEN
Aging is often accompanied by a decline in proteostasis, manifested as an increased propensity for misfolded protein aggregates, which are prevented by protein quality control systems, such as the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy. Although the role of the UPS and autophagy in slowing age-induced proteostasis decline has been elucidated, limited information is available on how these pathways can be activated in a collaborative manner to delay proteostasis-associated aging. Here, we show that activation of the UPS via the pharmacological inhibition of USP14 (ubiquitin specific peptidase 14) using IU1 improves proteostasis and autophagy decline caused by aging or proteostatic stress in Drosophila and human cells. Treatment with IU1 not only alleviated the aggregation of polyubiquitinated proteins in aging Drosophila flight muscles but also extended the fly lifespan with enhanced locomotive activity via simultaneous activation of the UPS and autophagy. Interestingly, the effect of this drug disappeared when proteasomal activity was inhibited, but was evident upon proteostasis disruption by foxo mutation. Overall, our findings shed light on potential strategies to efficiently ameliorate age-associated pathologies associated with perturbed proteostasis.Abbreviations: AAAs: amino acid analogs; foxo: forkhead box, sub-group O; IFMs: indirect flight muscles; UPS: ubiquitin-proteasome system; USP14: ubiquitin specific peptidase 14.
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Minimally invasive plate osteosynthesis is the most commonly used minimally invasive surgery technique for tibial fractures, possibly involving single or dual plate methods. Herein, we performed a finite element analysis to investigate plate strength according to the plate type, length, and presence of a fibula by constructing a three-dimensional tibia model. A thickness of 20 mm was cut 50 mm distal from the lateral plateau, and the ligaments were created. Plates were modeled with lengths of 150, 200, and 250 mm and mounted to the tibia. Screws were arranged to avoid overlapping in the dual plating. The von-Mises stress applied to the plates was measured by applying a load of 1 body weight. Dual plates showed the least stress with low displacement, followed by medial and lateral plates. As the plate length increased, the average stress gradually decreased, increasing plate safety. The difference in the influence of the fibula depending on the presence of proximal fibula osteotomy showed that the average stress increased by 35% following proximal fibula osteotomy in the D1(Plate type: Dual plate, Medial plate length: 150 mm, Lateral plate length: 200 mm, Non Proximal fibula osteotomy) and D1P(Plate type: Dual plate, Medial plate length: 150 mm, Lateral plate length: 200 mm, Proximal fibula osteotomy) models, confirming the necessity of the fibula model. There is no consensus guideline for treatment of this kind of fracture case. A single fracture plate can decrease the risk of skin damage, ligament damage, and wound infection, but because of its design, it cannot provide sufficient stability and satisfactory reduction of the condylar fragment, especially in cases of comminution or coronal fracture. So, these results will help clinicians make an informed choice on which plate to use in patients with tibial fractures.
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Placas Óseas , Peroné , Análisis de Elementos Finitos , Fijación Interna de Fracturas , Fracturas de la Tibia , Fracturas de la Tibia/cirugía , Humanos , Peroné/cirugía , Peroné/lesiones , Fijación Interna de Fracturas/métodos , Fijación Interna de Fracturas/instrumentación , Estrés Mecánico , Fenómenos Biomecánicos , Tibia/cirugía , Procedimientos Quirúrgicos Mínimamente Invasivos/métodosRESUMEN
Co-occurring mutations in KEAP1 in STK11/LKB1-mutant NSCLC activate NFE2L2/NRF2 to compensate for the loss of STK11-AMPK activity during metabolic adaptation. Characterizing the regulatory crosstalk between the STK11-AMPK and KEAP1-NFE2L2 pathways during metabolic stress is crucial for understanding the implications of co-occurring mutations. Here, we found that metabolic stress increased the expression and phosphorylation of SQSTM1/p62, which is essential for the activation of NFE2L2 and AMPK, synergizing antioxidant defense and tumor growth. The SQSTM1-driven dual activation of NFE2L2 and AMPK was achieved by inducing macroautophagic/autophagic degradation of KEAP1 and facilitating the AXIN-STK11-AMPK complex formation on the lysosomal membrane, respectively. In contrast, the STK11-AMPK activity was also required for metabolic stress-induced expression and phosphorylation of SQSTM1, suggesting a double-positive feedback loop between AMPK and SQSTM1. Mechanistically, SQSTM1 expression was increased by the PPP2/PP2A-dependent dephosphorylation of TFEB and TFE3, which was induced by the lysosomal deacidification caused by low glucose metabolism and AMPK-dependent proton reduction. Furthermore, SQSTM1 phosphorylation was increased by MAP3K7/TAK1, which was activated by ROS and pH-dependent secretion of lysosomal Ca2+. Importantly, phosphorylation of SQSTM1 at S24 and S226 was critical for the activation of AMPK and NFE2L2. Notably, the effects caused by metabolic stress were abrogated by the protons provided by lactic acid. Collectively, our data reveal a novel double-positive feedback loop between AMPK and SQSTM1 leading to the dual activation of AMPK and NFE2L2, potentially explaining why co-occurring mutations in STK11 and KEAP1 happen and providing promising therapeutic strategies for lung cancer.Abbreviations: AMPK: AMP-activated protein kinase; BAF1: bafilomycin A1; ConA: concanamycin A; DOX: doxycycline; IP: immunoprecipitation; KEAP1: kelch like ECH associated protein 1; LN: low nutrient; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NSCLC: non-small cell lung cancer; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PPP2/PP2A: protein phosphatase 2; ROS: reactive oxygen species; PPP3/calcineurin: protein phosphatase 3; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TCL: total cell lysate; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; V-ATPase: vacuolar-type H+-translocating ATPase.
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Monolayered transition-metal dichalcogenides (TMDs) are easily exposed to air, and their crystal quality can often be degraded via oxidation, leading to poor electronic and optical device performance. The degradation becomes more severe in the presence of defects, grain boundaries, and residues. Here, we report crack propagation in pristine TMD monolayers grown by chemical vapor deposition under ambient conditions and light illumination. Under a high relative humidity (RH) of â¼60% and white light illumination, the cracks appear randomly. Photo-oxidative cracks gradually propagated along the grain boundaries of the TMD monolayers. In contrast, under low RH conditions of â¼2%, cracks were scarcely observed. Crack propagation is predominantly attributed to the accumulation of water underneath the TMD monolayers, which is preferentially absorbed by hygroscopic alkali metal-based precursor residues. Crack propagation is further accelerated by the cyclic process of photo-oxidation in a basic medium, leading to localized tensile strain. We also found that such crack propagation is prevented after the removal of alkali metals via the transfer of the sample to other substrates.
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Nonsteroidal anti-inflammatory drug (NSAID) use is associated with adverse consequences, including hepatic injury. The detrimental hepatotoxicity of diclofenac, a widely used NSAID, is primarily connected to oxidative damage in mitochondria, which are the primary source of reactive oxygen species (ROS). The primary ROS responsible for inducing diclofenac-related hepatocellular toxicity and the principal antioxidant that mitigates these ROS remain unknown. Peroxiredoxin III (PrxIII) is the most abundant and potent H2O2-eliminating enzyme in the mitochondria of mammalian cells. Here, we investigated the role of mitochondrial H2O2 and the protective function of PrxIII in diclofenac-induced mitochondrial dysfunction and apoptosis in hepatocytes. Mitochondrial H2O2 levels were differentiated from other types of ROS using a fluorescent H2O2 indicator. Upon diclofenac treatment, PrxIII-knockdown HepG2 human hepatoma cells showed higher levels of mitochondrial H2O2 than PrxIII-expressing controls. PrxIII-depleted cells exhibited higher mitochondrial dysfunction as measured by a lower oxygen consumption rate, loss of mitochondrial membrane potential, cardiolipin oxidation, and caspase activation, and were more sensitive to apoptosis. Ectopic expression of mitochondrially targeted catalase in PrxIII-knockdown HepG2 cells or in primary hepatocytes derived from PrxIII-knockout mice suppressed the diclofenac-induced accumulation of mitochondrial H2O2 and decreased apoptosis. Thus, we demonstrated that mitochondrial H2O2 is a key mediator of diclofenac-induced hepatocellular damage driven by mitochondrial dysfunction and apoptosis. We showed that PrxIII loss results in the critical accumulation of mitochondrial H2O2 and increases the harmful effects of diclofenac. PrxIII or other antioxidants targeting mitochondrial H2O2 could be explored as potential therapeutic agents to protect against the hepatotoxicity associated with NSAID use.