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Advances in next-generation sequencing technologies have led to elucidation of sensorineural hearing loss genetics and associated clinical impacts. However, studies on the functional pathogenicity of variants of uncertain significance (VUS), despite their close association with clinical phenotypes, are lacking. Here we identified compound heterozygous variants in ESRRB transcription factor gene linked to DFNB35, specifically a novel splicing variant (NM_004452.4(ESRRB): c.397 + 2T>G) in trans with a missense variant (NM_004452.4(ESRRB): c.1144C>T p.(Arg382Cys)) whose pathogenicity remains unclear. The splicing variant (c.397 + 2T>G) caused exon 4 skipping, leading to premature stop codon formation and nonsense-mediated decay. The p.(Arg382Cys) variant was classified as a VUS due to its particularly higher allele frequency among East Asian population despite disease-causing in-silico predictions. However, functional assays showed that p.(Arg382Cys) variant disrupted key intramolecular interactions, leading to protein instability. This variant also reduced transcriptional activity and altered expression of downstream target genes essential for inner ear function, suggesting genetic contribution to disease phenotype. This study expanded the phenotypic and genotypic spectrum of ESRRB in DFNB35 and revealed molecular mechanisms underlying ESRRB-associated DFNB35. These findings suggest that variants with high allele frequencies can also possess functional pathogenicity, providing a breakthrough for cases where VUS, previously unexplored, could be reinterpreted by elucidating their functional roles and disease-causing characteristics.
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Perda Auditiva Neurossensorial , Receptores de Estrogênio , Feminino , Humanos , Masculino , Códon sem Sentido/genética , Frequência do Gene , Predisposição Genética para Doença , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Mutação de Sentido Incorreto , Linhagem , Splicing de RNA/genética , Receptores de Estrogênio/genéticaRESUMO
Mutations in nuclear genes regulating mitochondrial DNA (mtDNA) replication are associated with mtDNA depletion syndromes. Using whole-genome sequencing, we identified a heterozygous mutation (c.272G>A:p.Arg91Gln) in single-stranded DNA-binding protein 1 (SSBP1), a crucial protein involved in mtDNA replisome. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy. This mutation impeded multimer formation and DNA-binding affinity, leading to reduced efficiency of mtDNA replication, altered mitochondria dynamics, and compromised mitochondrial function. To correct this mutation, we tested two adenine base editor (ABE) variants on patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), showed higher editing efficacy (≤30%) and enhanced mitochondrial replication and function, despite off-target editing frequencies; however, risks from bystander editing were limited due to silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (≤10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. Taken together, base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.
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Branchio-oto-renal (BOR) and branchio-otic (BO) syndromes are characterized by anomalies affecting the ears, often accompanied by hearing loss, as well as abnormalities in the branchial arches and renal system. These syndromes exhibit a broad spectrum of phenotypes and a complex genomic landscape, with significant contributions from the EYA1 gene and the SIX gene family, including SIX1 and SIX5. Due to their diverse phenotypic presentations, which can overlap with other genetic syndromes, molecular genetic confirmation is essential. As sequencing technologies advance, whole-genome sequencing (WGS) is increasingly used in rare disease diagnostics. We explored the genomic landscape of 23 unrelated Korean families with typical or atypical BOR/BO syndrome using a stepwise approach: targeted panel sequencing and exome sequencing (Step 1), multiplex ligation-dependent probe amplification (MLPA) with copy number variation screening (Step 2), and WGS (Step 3). Integrating WGS into our diagnostic pipeline detected structure variations, including cryptic inversion and complex genomic rearrangement, eventually enhancing the diagnostic yield to 91%. Our findings expand the genomic architecture of BOR/BO syndrome and highlight the need for WGS to address the genetic diagnosis of clinically heterogeneous rare diseases.
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Síndrome Brânquio-Otorrenal , Variações do Número de Cópias de DNA , Sequenciamento Completo do Genoma , Humanos , Síndrome Brânquio-Otorrenal/genética , República da Coreia , Sequenciamento Completo do Genoma/métodos , Feminino , Masculino , Variações do Número de Cópias de DNA/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doenças Raras/genética , Proteínas Nucleares/genética , Proteínas de Homeodomínio/genética , Criança , Proteínas Tirosina Fosfatases/genética , Pré-Escolar , Adulto , Genômica/métodos , Fenótipo , Linhagem , Adolescente , LactenteRESUMO
Pathogenic structure variations (SVs) are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements, focusing on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto-renal/branchio-oto syndrome. To address this, two CRISPR-based approaches were tested. First, we used Cas9 nuclease and paired gRNAs tailored to the patient's genome. The dual CRISPR-Cas9 system induced efficient correction of paracentric inversion in patient-derived fibroblast, and effectively restored the expression of EYA1 mRNA and protein, along with its transcriptional activity required to regulate the target gene expression. Additionally, we used CRISPR activation (CRISPRa), which leads to the upregulation of EYA1 mRNA expression in patient-derived fibroblasts. Moreover, CRISPRa significantly improved EYA1 protein expression and transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies could offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. Our findings demonstrate the potential of CRISPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.
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The 26S proteasome comprises 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in cells exist as free 20S complexes; however, our mechanistic understanding of what determines the ratio of 26S to 20S species remains incomplete. Here, we show that glucose starvation uncouples 26S holoenzymes into 20S and 19S subcomplexes. Subcomplex affinity purification and quantitative mass spectrometry reveal that Ecm29 proteasome adaptor and scaffold (ECPAS) mediates this structural remodeling. The loss of ECPAS abrogates 26S dissociation, reducing degradation of 20S proteasome substrates, including puromycylated polypeptides. In silico modeling suggests that ECPAS conformational changes commence the disassembly process. ECPAS is also essential for endoplasmic reticulum stress response and cell survival during glucose starvation. In vivo xenograft model analysis reveals elevated 20S proteasome levels in glucose-deprived tumors. Our results demonstrate that the 20S-19S disassembly is a mechanism adapting global proteolysis to physiological needs and countering proteotoxic stress.
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Complexo de Endopeptidases do Proteassoma , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Citoplasma/metabolismo , Proteólise , Espectrometria de MassasRESUMO
The spatiotemporal sequestration of misfolded proteins is a mechanism by which cells counterbalance proteome homeostasis upon exposure to various stress stimuli. Chronic inhibition of proteasomes results in a large, juxtanuclear, membrane-less inclusion, known as the aggresome. Although the molecular mechanisms driving its formation, clearance, and pathophysiological implications are continuously being uncovered, the biophysical aspects of aggresomes remain largely uncharacterized. Using fluorescence recovery after photobleaching and liquid droplet disruption assays, we found that the aggresomes are a homogeneously blended condensates with liquid-like properties similar to droplets formed via liquid-liquid phase separation. However, unlike fluidic liquid droplets, aggresomes have more viscosity and hydrogel-like characteristics. We also observed that the inhibition of aggresome formation using microtubule-disrupting agents resulted in less soluble and smaller cytoplasmic speckles, which was associated with marked cytotoxicity. Therefore, the aggresome appears to be cytoprotective and serves as a temporal reservoir for dysfunctional proteasomes and substrates that need to be degraded. Our results suggest that the aggresome assembles through distinct and potentially sequential processes of energy-dependent retrograde transportation and spontaneous condensation into a hydrogel.
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Hidrogéis , Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Hidrogéis/metabolismo , Proteínas/metabolismo , Corpos de Inclusão/metabolismo , Microtúbulos/metabolismoRESUMO
The 26S proteasome irreversibly hydrolyzes polyubiquitylated substrates to maintain protein homeostasis; it also regulates immune responses by generating antigenic peptides. An alternative form of the 26S proteasome is the immunoproteasome, which contains substituted catalytic subunits (ß1i/PSMB9, ß2i/PSMB10, and ß5i/PSMB8) instead of constitutively expressed counterparts (ß1/PSMB6, ß2/PSMB7, and ß5/PSMB5). The immunoproteasome expands the peptide repertoire presented on MHC class I molecules. However, how its activity changes in this context is largely elusive, possibly due to the lack of a standardized methodology to evaluate its specific activity. Here, we describe an assay protocol that measures the immunoproteasome activity of whole-cell lysates using commercially available fluorogenic peptide substrates. Our results showed that the most accurate assessment of immunoproteasome activity could be achieved by combining ß5i-targeting substrate Ac-ANW-AMC and immunoproteasome inhibitor ONX-0914. This simple and reliable protocol may contribute to future studies of immunoproteasomes and their pathophysiological roles during viral infection, inflammation, and tumorigenesis.
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The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenance of cellular homeostasis and cell growth. mTOR also functions as an endogenous inhibitor of autophagy. Under nutrient-rich conditions, mTOR complex 1 (mTORC1) phosphorylates the ULK1 complex, preventing its activation and subsequent autophagosome formation, while inhibition of mTORC1 using either rapamycin or nutrient deprivation induces autophagy. Autophagy and proteasomal proteolysis provide amino acids necessary for protein translation. Although the connection between mTORC1 and autophagy is well characterized, the association of mTORC1 inhibition with proteasome biogenesis and activity has not been fully elucidated yet. Proteasomes are long-lived cellular organelles. Their spatiotemporal rather than homeostatic regulation could be another adaptive cellular mechanism to respond to starvation. Here, we reviewed several published reports and the latest research from our group to examine the connection between mTORC1 and proteasome. We have also investigated and described the effect of mTORC1 inhibition on proteasome activity using purified proteasomes. Since mTORC1 inhibitors are currently evaluated as treatments for several human diseases, a better understanding of the link between mTORC1 activity and proteasome function is of utmost importance. [BMB Reports 2022; 55(4): 161-165].
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Complexo de Endopeptidases do Proteassoma , Serina-Treonina Quinases TOR , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
This study aimed to evaluate the quality of low-dose-rate (LDR) prostate brachytherapy (BT) based on treatment-related dosimetric outcomes. Data of 100 patients treated using LDR BT with stranded seeds from November 2012 to November 2017 were collected. The prescription dose for the prostate was 145 Gy. The dose constraints for the preoperative plan were: V100% ≥ 95%, V150% ≤ 60%, V200% ≤ 20% for the prostate; V100% for rectum, ≤ 1 cc; and V200 Gy for urethra, 0.0 cc. Intraoperative real-time dose calculation and postoperative dose distribution analysis on days 0 and 30 were performed. Median dosimetric outcomes on days 0 and 30 respective were: V100% 92.28% and 92.23%, V200% 18.63% and 25.02%, and D90% 150.88 Gy and 151.46 Gy for the prostate; V100% for the rectum, 0.11 cc and 0.22 cc; and V200 Gy for the urethra, 0.00 cc and 0.00 cc, respectively. Twenty patients underwent additional seed implantation to compensate for insufficient dose coverage of the prostate. No loss or substantial migration of seeds or severe toxicity was reported. With stranded seed implantation and intraoperative optimization, appropriate dose delivery to the prostate without excessive dose to the organs at risk could be achieved.
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Braquiterapia , Neoplasias da Próstata , Braquiterapia/efeitos adversos , Humanos , Radioisótopos do Iodo/uso terapêutico , Masculino , Próstata/cirurgia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/cirurgia , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador , RetoRESUMO
Proteostasis is primarily a function of protein synthesis and degradation. Although the components and processes involved in intracellular proteostasis have been studied extensively, it is apparent that extracellular proteostasis is equitably crucial for the viability of organisms. The 26S proteasome, a unique ATP-dependent proteolytic complex in eukaryotic cells, contributes to the majority of intracellular proteolysis. Accumulating evidence suggests the presence of intact 20S proteasomes in the circulatory system (c-proteasomes), and similar to other plasma proteins, the levels of these c-proteasomes may vary, potentially reflecting specific pathophysiological conditions. Under normal conditions, the concentration of c-proteasomes has been reported to be in the range of ~0.2-2 µg/mL, which is ~2-4-fold lower than that of functional plasma proteins but markedly higher than that of signaling proteins. The characterization of c-proteasomes, such as their origin, structure, role, and clearance, has been delayed mainly due to technical limitations. In this review, we summarize the current perspectives pertaining to c-proteasomes, focusing on the methodology, including our experimental understanding. We believe that once the pathological relevance of c-proteasomes is revealed, these unique components may be utilized in the diagnosis and prognosis of diverse human diseases.
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Complexo de Endopeptidases do Proteassoma , Proteínas , Células Eucarióticas , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , ProteóliseRESUMO
The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3ß and CHIP, respectively. The resulting phospho-ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one-three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho-ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho-ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a "multiple-hit model," where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.
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BACKGROUND: Although the existence of proteasomes in human blood, termed circulating proteasomes (c-proteasomes), has been reported previously, their origin and pathophysiological functions remain largely unknown. OBJECTIVE: Given that c-proteasome activity was significantly reduced in Alzheimer's disease model mice and relatively high frequency of mild cognitive impairment (MCI) is accompanied by chronic tinnitus in aged patients, we examined whether c-proteasome activity in human plasma was associated with cognitive function in patients with chronic tinnitus. METHODS: c-Proteasome activity in the plasma of tinnitus patients (Nâ=â55) was measured with fluorogenic reporter substrate, suc-LLVY-AMC. To assess MCI, the Montreal Cognitive Assessment was conducted with a cut-off score of 22/23. All patients underwent audiological and psychoacoustic analyses. Levels of c-proteasomes, Aß42, and Aß40 were measured using ELISA, and their association with c-proteasome activity was evaluated. RESULTS: The activity of circulating proteasomes was significantly lower in patients with chronic tinnitus and MCI (pâ=â0.042), whereas activities of other plasma enzymes showed little correlation. In addition, c-proteasome activity was negatively associated with the level of plasma Aß and was directly dependent on its own concentration in the plasma of patients with chronic tinnitus. CONCLUSION: Our current work provides a new perspective for understanding the potential relationship between circulating proteasomes in the plasma and cognitive dysfunction, suggesting a novel, non-invasive biomarker in the context of MCI diagnosis.
Assuntos
Disfunção Cognitiva/sangue , Complexo de Endopeptidases do Proteassoma/sangue , Zumbido/sangue , Idoso , Doença de Alzheimer/sangue , Animais , Biomarcadores/sangue , Feminino , Humanos , Masculino , Testes de Estado Mental e Demência , Camundongos , Pessoa de Meia-Idade , República da CoreiaRESUMO
The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.
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Macroautofagia , Organelas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Humanos , Organelas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
BACKGROUND: We evaluated the optimal radiotherapy (RT) plan for synchronous bilateral breast cancer (SBBC), especially treatment plans including the regional lymph node (LN) area. METHODS: This study was conducted using 15 patients with SBBC (5 with small breasts, 5 with large breasts, and 5 who underwent a left total mastectomy). The clinical target volume (CTV) was defined as the volume enveloping the bilateral whole breasts/chest wall and left regional LN area. We established the following plans: 1) volumetric-modulated arc therapy (VMAT)-the only plan using two pairs of partial arcs for the whole target volume, 2) VMAT using one partial arc for the left CTV followed by a 3D tangential technique for the right breast (primary hybrid plan), and 3) VMAT for the left CTV followed by a tangential technique using an automatically calculated prescription dose for the right breast, considering the background dose from the left breast VMAT plan (modified hybrid plan). The Tukey test and one-way analysis of variance were used to compare the target coverage and doses to organs at risk (OARs) of the three techniques. RESULTS: For target coverage, the VMAT-only and modified hybrid plans showed comparable target coverage in terms of Dmean (50.33 Gy vs. 50.53 Gy, p = 0.106). The primary hybrid plan showed the largest distribution of the high-dose volume, with V105% of 25.69% and V110% of 6.37% for the planning target volume (PTV) (p < 0.001). For OARs including the lungs, heart, and left anterior descending artery, the percentages of volume at various doses (V5Gy, V10Gy, V20Gy, V30Gy) and Dmean were significantly lower in both the primary and modified hybrid plans compared to those of the VMAT-only plan. These results were consistent in subgroup analyses of breast size and morphological variation. CONCLUSIONS: The modified hybrid plan, using an automatically calculated prescription dose for the right breast and taking into consideration the background dose from the left breast VMAT plan, showed comparable target coverage to that of the VMAT-only plan, and was superior for saving OARs. However, considering that VMAT can be adjusted according to the physician's intention, further evaluation is needed for developing a better plan.
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Neoplasias da Mama/radioterapia , Linfonodos/efeitos da radiação , Órgãos em Risco/efeitos da radiação , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia de Intensidade Modulada/métodos , Feminino , Humanos , Dosagem RadioterapêuticaRESUMO
BACKGROUND: The current standard treatment for resectable pancreatic cancer is surgical resection followed by adjuvant chemotherapy. Local recurrence rates are high even after curative resection; thus, the long-term outcome of locally advanced pancreatic cancer remains poor. Intraoperative radiotherapy (IORT) uses a low-energy x-ray source to deliver a single fraction of high-dose radiation to the tumor bed during a surgical procedure, while effectively sparing the surrounding normal tissues. IORT has the potential to improve the efficacy of radiation therapy for pancreatic cancer. METHODS/DESIGN: This prospective, one-armed, phase II study will investigate the role of IORT in improving local control in patients with resectable pancreatic adenocarcinoma. The patients will receive surgery and IORT of 10 Gy prescribed at a 5-mm depth of the tumor bed, followed by adjuvant gemcitabine chemotherapy according to the current standard of care. The aim is to enroll 42 patients. DISCUSSION: The primary endpoint of this trial is to evaluate the feasibility of IORT and the local recurrence rate after one year. The secondary endpoints include the acute and late toxicities, and disease-free survival and overall survival rates. TRIAL REGISTRATION: The trial was prospectively registered at Clinicaltrials.gov NCT03273374 on September 6, 2017.
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Adenocarcinoma/radioterapia , Neoplasias Pancreáticas/radioterapia , Adenocarcinoma/cirurgia , Terapia Combinada , Humanos , Cuidados Intraoperatórios , Neoplasias Pancreáticas/cirurgia , Estudos Prospectivos , Radioterapia AdjuvanteRESUMO
Five new tripeptides, acidiphilamides A-E (1-5), were discovered along with two previously reported compounds, l-isoleucinamide (6) and l-valinamide (7), from Streptacidiphilus rugosus AM-16, an acidophilic actinobacterial strain isolated from acidic forest soil. The structures of 1-5 were elucidated as modified tripeptides bearing phenylalaninol or methioninol fragments with C3-C5 acyl chains based mainly on NMR and mass spectroscopic data. The absolute configurations of the amine units were established by advanced Marfey's method and GITC (2,3,4,6-tetra- O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization followed by LC/MS analysis. Acidiphilamides A and B (1 and 2), the first secondary metabolites isolated from the rare actinobacterial genus Streptacidiphilus, significantly inhibited autophagic flux but not proteasome activity in HeLa cells. These compounds appeared to block mainly the autophagosome-lysosome fusion step in the late stage of cellular autophagy.
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Actinobacteria/metabolismo , Autofagia/efeitos dos fármacos , Oligopeptídeos/isolamento & purificação , Células HeLa , Humanos , Oligopeptídeos/química , Oligopeptídeos/farmacologiaRESUMO
The ubiquitin-proteasome system and the autophagy-lysosome system are two major intracellular proteolytic pathways in eukaryotes. Although several biochemical mechanisms underlying the crosstalk between them have been suggested, little is known about the effect of enhanced proteasome activity on autophagic flux. Here, we found that upregulation of proteasome activity, which was achieved through the inhibition of USP14, significantly impaired cellular autophagic flux, especially at the autophagosome-lysosome fusion step. UVRAG appeared to function as a crucial checkpoint for the proper progression of autophagic flux. Although proteasome activation through USP14 inhibition facilitated the clearance of microtubule-associated protein tau (MAPT) and reduced the amount of its oligomeric forms, the same conditions increased the formation of inclusion bodies from nonproteasomal substrates such as huntingtin with long polyglutamine repeats. Our results collectively indicate that USP14 may function as a common denominator in the compensatory negative feedback between the two major proteolytic processes in the cell.
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Autofagia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Retroalimentação Fisiológica , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Fusão de Membrana , Camundongos , Modelos Biológicos , Proteólise , Especificidade por Substrato , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidores , Proteínas tau/metabolismoRESUMO
BACKGROUND/AIMS: Radiation-induced skin fibrosis is a common side effect of clinical radiotherapy. Our previous next-generation sequencing (NGS) study demonstrated the reduced expression of the regulatory α subunit of phosphatidylinositol 3-kinase (PIK3r1) in irradiated murine skin. Metformin has been reported to target the PIK3-FOXO3 pathway. In this study, we investigated the effects of metformin on radiation-induced skin fibrosis. METHODS: Metformin was orally administered to irradiated mice. Skin fibrosis was analyzed by staining with H&E and Masson's trichrome stain. The levels of cytokines and chemokines associated with fibrosis were analyzed by immunohistochemistry and quantitative RT-PCR. The roles of PIK3rl and FOXO3 in radiation-induced skin fibrosis were studied by overexpressing PIK3rl and transfecting FOXO3 siRNA in NIH3T3 cells and mouse-derived dermal fibroblasts (MDF). RESULTS: The oral administration of metformin significantly reduced radiation-induced skin thickening and collagen accumulation and significantly reduced the radiation-induced expression of FOXO3 in murine skin. Additionally, the overexpression of PIK3r1 reduced the radiation-induced expression of FOXO3, while FOXO3 silencing decreased the radiation-induced expression of TGFß in vitro. CONCLUSIONS: The results indicated that metformin suppresses radiation-induced skin injuries by modulating the expression of FOXO3 through PIK3r1. Collectively, the data obtained in this study suggested that metformin could be a potent therapeutic agent for alleviating radiation-induced skin fibrosis.
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Regulação para Baixo/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Metformina/farmacologia , Animais , Células Cultivadas , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Regulação para Baixo/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Proteína Forkhead Box O3/antagonistas & inibidores , Proteína Forkhead Box O3/genética , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Estresse Oxidativo/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/prevenção & controle , Radiação Ionizante , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Pele/efeitos dos fármacos , Pele/patologia , Pele/efeitos da radiação , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND/AIMS: The 26S proteasome is the key proteolytic complex for recognition and degradation of polyubiquitinated target substrates in eukaryotes. Among numerous proteasome-associated proteins, a deubiquitinating enzyme (DUB) USP14 has been identified as an endogenous inhibitor of the proteasome. Here, we explored the complex regulatory functions of USP14 that involve ubiquitin (Ub) homeostasis and substrate degradation in flies and mammals. METHODS: USP14-null primary and immortalized mouse embryonic fibroblasts (MEFs) and USP14 knocked-down Drosophila were analyzed in this study. We measured proteasome and DUB activities using fluorogenic reporter substrates and adduct-forming probes. To examine the levels of ubiquitin, we performed immunoblotting and immunohistochemistry. Mass spectrometry (MS) was used to examine polyUb chain linkages and USP14-interacing proteins. Cell cycle was analyzed by flow cytometry, BrdU labeling, and phospho-histone H3 staining. RESULTS: The homeostasis of Ub in USP14-/-MEFs was markedly perturbed because of facilitated clearance of Ub. This phenomenon was recapitulated in muscles of USP14-deficient Drosophila with old ages. Absolute quantitation using MS also revealed that USP14-/- MEFs contained significantly increased amounts of Ub, compared with wild-type. The key phenotype of USP14-/- MEFs was their delayed proliferation originated from prolonged interphase possibly through aberrant degradation of cyclins A and B1. We found that knocking down USP14 in Drosophila resulted in delayed eye development associated with reduced mitotic activity. CONCLUSION: Our study identifies novel cellular functions of USP14 not only in cellular Ub hometostasis but also in cell cycle progression. USP14 was also essential for proper Drosophila eye development. These results strongly suggest that the USP14-mediated proteasome activity regulation may be directly related to various human diseases including cancer.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Fibroblastos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Animais , Ciclo Celular , Linhagem Celular , Células Cultivadas , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Fibroblastos/citologia , Técnicas de Silenciamento de Genes , Homeostase , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina Tiolesterase/genética , UbiquitinaçãoRESUMO
An intronic hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 gene is the most common cause of familial ALS and frontotemporal dementia (FTD) and is found in â¼7% of individuals with apparently sporadic disease. Several different diamino acid peptides can be generated from the HRE by noncanonical translation (repeat-associated non-ATG translation, or RAN translation), and some of these peptides can be toxic. Here, we studied the effects of two arginine containing RAN translation products [proline/arginine repeated 20 times (PR20) and glycine/arginine repeated 20 times (GR20)] in primary rat spinal cord neuron cultures grown on an astrocyte feeder layer. We find that PR20 kills motor neurons with an LD50 of 2 µM, but in contrast to the effects of other ALS-causing mutant proteins (i.e., SOD or TDP43), PR20 does not evoke the biochemical signature of mitochondrial dysfunction, ER stress, or mTORC down-regulation. PR20 does result in a time-dependent build-up of ubiquitylated substrates, and this is associated with a reduction of flux through both autophagic and proteasomal degradation pathways. GR20, however, does not have these effects. The effects of PR20 on the proteasome are likely to be direct because (1) PR20 physically associates with proteasomes in biochemical assays, and (2) PR20 inhibits the degradation of a ubiquitylated test substrate when presented to purified proteasomes. Application of a proteasomal activator (IU1) blocks the toxic effects of PR20 on motor neuron survival. This work suggests that proteasomal activators have therapeutic potential in individuals with C9ORF72 HRE.