ABSTRACT
Coronaviruses (CoV) are highly pathogenic single-strand RNA viruses. CoV infections cause fatal respiratory symptoms and lung injuries in humans and significant economic losses in livestock. Since the SARS-2 outbreak in 2019, the highly conserved main protease (Mpro), also termed 3-chymotrypsin-like protease (3CLpro), has been considered an attractive drug target for treating CoV infections. Mpro mediates the proteolytic cleavage of eleven sites in viral polypeptides necessary for virus replication. Here, we report that disulfiram, an FDA-approved drug for alcoholic treatment, exhibits a broad-spectrum inhibitory effect on CoV Mpros. Analytical ultracentrifugation and circular dichroism analyses indicated that disulfiram treatment blocks the dimeric formation of SARS and PEDV Mpros and decreases the thermostability of SARS, SARS-2, and PEDV Mpros, whereas it facilitates the dimerization and stability of MERS Mpro. Furthermore, mass spectrometry and structural alignment revealed that disulfiram targets the Cys44 residue of Mpros, which is located at the substrate entrance and close to the catalytic His41. In addition, molecular docking analysis suggests that disulfiram conjugation interferes with substrate entry to the catalytic center. In agreement, mutation of Cys44 modulates the disulfiram sensitivity of CoV Mpros. Our study suggests a broad-spectrum inhibitory function of disulfiram against CoV Mpros.
ABSTRACT
TFIID, one of the general transcription factor (GTF), regulates transcriptional initiation of protein-coding genes through direct binding to promoter elements and subsequent recruitment of other GTFs and RNA polymerase II. Although generally required for most protein-coding genes, accumulated studies have also demonstrated promoter-specific functions for several TFIID subunits in gene activation. Here, we report that TBP-associated factor 2 (TAF2) specifically regulates TFIID binding to a small subset of protein-coding genes and is essential for cell growth of multiple cancer lines. Co-immunoprecipitation assays revealed that TAF2 may be sub-stoichiometrically associated with the TFIID complex, thus indicating a minor fraction of TAF2-containing TFIID in cells. Consistently, integrated genome-wide profiles show that TAF2 binds to and regulates only a small subset of protein-coding genes. Furthermore, through the use of an inducible TAF2 degradation system, our results reveal a reduction of TBP/TFIID binding to several ribosomal genes upon selective ablation of TAF2. In addition, depletion of TAF2, as well as the TAF2-regulated ribosomal protein genes RPL30 and RPL39, decreases ribosome assembly and global protein translation. Collectively, this study suggests that TAF2 within the TFIID complex is of functional importance for TBP/TFIID binding to and expression of a small subset of protein-coding genes, thus establishing a previously unappreciated promoter-selective function for TAF2.
ABSTRACT
In recent years, mounting evidence has highlighted a global decline in male semen quality, paralleling an increase in male infertility problems. Such developments in the male reproductive system are likely due to a range of environmental factors, which could negatively affect the outcomes of pregnancy, reproductive health, and the well-being of fetuses. Different environmental contaminants ultimately accumulate in riverbed sediments due to gravity, so these sediments are frequently considered hotspots for pollutants. Therefore, understanding the detrimental effects of river sediment pollution on human reproductive health is crucial. This study indicates male germ cells' high vulnerability to environmental contaminants. There is a strong positive correlation between the concentration of complex accumulated pollutants from human activities and the reproductive toxicity observed in human testicular embryonic cell lines NCCIT and NTERA-2. This toxicity is characterized by increased levels of reactive oxygen species, disruption of critical cellular functions, genotoxic impacts, and the induction of cell apoptosis. This research marks a significant step in providing in vitro evidence of the damaging effects of environmental pollutants on the human male germline.
Subject(s)
Geologic Sediments , Male , Humans , Geologic Sediments/chemistry , Water Pollutants, Chemical/toxicity , Spermatozoa/drug effects , DNA Damage , Cell Line , Reactive Oxygen Species/metabolism , Testis/drug effectsABSTRACT
Alpha-pinene is one of the main defensive components in conifers. Monochamus alternatus (Coleoptera: Cerambycidae), a wood borer feeding on Pinaceae plants, relies on its detoxifying enzymes to resist the defensive terpenoids. Here, we assayed the peroxide level and GST activity of M. alternatus larvae treated with different concentrations of α-pinene. Meanwhile, a gst gene (MaGSTe3) was isolated and analyzed. We determined its expression level and verified its function. The results showed that α-pinene treatment led to membrane lipid peroxidation and thus increased the GST activity. Expression of MaGSTe3 was significantly upregulated in guts following exposure to α-pinene, which has a similar pattern with the malonaldehyde level. In vitro expression and disk diffusion assay showed that the MaGSTe3 protein had high antioxidant capacity. However, RNAi treatment of MaGSTe3 did not reduce the hydrogen peroxide and malonaldehyde levels, while GST activity was significantly reduced. These results suggested MaGSTe3 takes part in α-pinene adaptation, but it does not play a great role in the resistance of M. alternatus larvae to α-pinene.
Subject(s)
Coleoptera , Glutathione Transferase , Animals , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Coleoptera/genetics , Coleoptera/metabolism , Bicyclic Monoterpenes , Larva/genetics , Larva/metabolism , MalondialdehydeABSTRACT
Ataxia telangiectasia and Rad3-related protein (ATR) is a critical component of the DNA damage response and a potential target in the treatment of cancers. An ATR inhibitor, BAY 1895344, was evaluated for its use in differentiated thyroid cancer (DTC) therapy. BAY 1895344 inhibited cell viability in four DTC cell lines (TPC1, K1, FTC-133, and FTC-238) in a dose-dependent manner. BAY 1895344 treatment arrested DTC cells in the G2/M phase, increased caspase-3 activity, and caused apoptosis. BAY 1895344 in combination with either sorafenib or lenvatinib showed mainly synergistic effects in four DTC cell lines. The combination of BAY 1895344 with dabrafenib plus trametinib revealed synergistic effects in K1 cells that harbor BRAFV600E. BAY 1895344 monotherapy retarded the growth of K1 and FTC-133 tumors in xenograft models. The combinations of BAY 1895344 plus lenvatinib and BAY 1895344 with dabrafenib plus trametinib were more effective than any single therapy in a K1 xenograft model. No appreciable toxicity appeared in animals treated with either a single therapy or a combination treatment. Our findings provide the rationale for the development of clinical trials of BAY 1895344 in the treatment of DTC.
Subject(s)
Adenocarcinoma , Thyroid Neoplasms , Animals , Humans , Thyroid Neoplasms/pathology , Phenylurea Compounds/pharmacology , Phenylurea Compounds/therapeutic use , Sorafenib/pharmacology , Adenocarcinoma/drug therapy , Ataxia Telangiectasia Mutated ProteinsABSTRACT
The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-ß (Kap-ß) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-ß that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBPC). Remarkably, Kap114p wraps around the yTBPC N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network.
Subject(s)
Saccharomyces cerevisiae , Transcription Factors , Active Transport, Cell Nucleus , TATA-Box Binding Protein/genetics , Saccharomyces cerevisiae/genetics , Cryoelectron Microscopy , Transcription Factors/genetics , Receptors, Cytoplasmic and Nuclear/genetics , beta Karyopherins/geneticsABSTRACT
The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the ability to reprogram brain astrocytes into neurons in vivo. Here, we demonstrate that in adult mice, NeuroD1 can reprogram Müller cells, the principal glial cell type in the retina, to become retinal neurons. Most strikingly, ectopic expression of NeuroD1 using two different viral vectors converted Müller cells into different cell types. Specifically, AAV7m8 GFAP681::GFP-ND1 converted Müller cells into inner retinal neurons, including amacrine cells and ganglion cells. In contrast, AAV9 GFAP104::ND1-GFP converted Müller cells into outer retinal neurons such as photoreceptors and horizontal cells, with higher conversion efficiency. Furthermore, we demonstrate that Müller cell conversion induced by AAV9 GFAP104::ND1-GFP displayed clear dose- and time-dependence. These results indicate that Müller cells in adult mice are highly plastic and can be reprogrammed into various subtypes of retinal neurons.
ABSTRACT
Background: Post-stroke hemiparesis strongly affects stroke patients' activities of daily living and health-related quality of life. Scalp acupuncture (SA) is reportedly beneficial for post-stroke hemiparesis. However, there is still no standard of SA for the treatment of post-stroke hemiparesis. Apriori algorithm-based association rule analysis is a kind of "if-then" rule-based machine learning method suitable for investigating the underlying rules of acupuncture point/location selections. This study aimed to investigate the core SA combinations for the treatment of post-stroke hemiparesis by using a systematic review and Apriori algorithm-based association rule analysis. Methods: We conducted a systematic review to include relevant randomized controlled trial (RCT) studies investigating the effects of SA treatment in treating patients with post-stroke hemiparesis, assessed by the Fugl-Meyer Assessment (FMA) score. We excluded studies using herbal medicine or manual acupuncture. Results: We extracted 33 SA locations from the 35 included RCT studies. The following SA styles were noted: International Standard Scalp Acupuncture (ISSA), WHO Standard Acupuncture Point Locations (SAPL), Zhu's style SA, Jiao's style SA, and Lin's style SA. Sixty-one association rules were investigated based on the integrated SA location data. Conclusions: SAPL_GV20 (Baihui), SAPL_GV24 (Shenting), ISSA_MS6_i (ISSA Anterior Oblique Line of Vertex-Temporal, lesion-ipsilateral), ISSA_MS7_i (ISSA Posterior Oblique Line of Vertex-Temporal, lesion-ipsilateral), ISSA_PR (ISSA Parietal region, comprised of ISSA_MS5, ISSA_MS6, ISSA_MS7, ISSA_MS8, and ISSA_MS9), and SAPL_Ex.HN3 (Yintang) can be considered the core SA location combination for the treatment of post-stroke hemiparesis. We recommend a core SA combination for further animal studies, clinical trials, and treatment strategies.
ABSTRACT
Purpose: To develop a zebrafish cataract model for screening potential anti-cataract compounds. Methods: Living zebrafish were anesthetized and exposed to ultraviolet-C (UV-C) irradiation at a dosage of 3250 mJ/cm2/d until they developed severe cataracts. These cataracts were graded based on photographs analyzed with ImageQuant TL version 7.0. Fish with severe cataracts were used to evaluate a range of compounds for cataract treatment, including the previously demonstrated hit compound lanosterol. For the initial evaluation, fish were divided into four groups: no treatment, balanced salt solution, ß-cyclodextrin (ß-CD), and lanosterol dissolved in ß-CD. The treatments were performed for 10 days, and the clarity of lenses was evaluated. To assess the persistence of treatment, fish were treated with ß-CD and lanosterol dissolved in ß-CD for seven consecutive days followed by monitoring for three days without treatment. Results: The average time for zebrafish to develop severe cataracts using the present UV-C irradiation protocol was 7.8 days (range 4-15 days). Both study designs required only another 10 days to determine the effect of hit compounds. The total experimental period could be completed within one month, and the entire experiment was economical. Conclusions: We could assay a large number of hit compounds at a reasonable cost and within a short time using this newly developed zebrafish cataract model. These assays may allow development of an efficient platform for screening potential anti-cataract compounds. Translational Relevance: The results may facilitate the development of ani-cataract medication for humans after further experiments and investigations.
Subject(s)
Cataract , Lens, Crystalline , Animals , Cataract/drug therapy , Disease Models, Animal , Humans , Lanosterol/pharmacology , Lanosterol/therapeutic use , Lens, Crystalline/radiation effects , ZebrafishABSTRACT
BACKGROUND: CYP11A1 is a protein located in the inner membrane of mitochondria catalyzing the first step of steroid synthesis. As a marker gene for steroid-producing cells, the abundance of CYP11A1 characterizes the extent of steroidogenic cell differentiation. Besides, the mitochondria of fully differentiated steroidogenic cells are specialized with tubulovesicular cristae. The participation of CYP11A1 in the change of mitochondrial structure and the differentiation of steroid-producing cells, however, has not been investigated. METHODS: We engineered nonsteroidogenic monkey kidney COS1 cells to express CYP11A1 upon doxycycline induction and examined the mitochondrial structure of these cells. We also mapped the CYP11A1 domains that confer structural changes of mitochondria. We searched for CYP11A1-interacting proteins and investigated the role of this interacting protein in shaping mitochondrial structure. Finally, we examined the effect of CYP11A1 overexpression on the amount of mitochondrial contact site and cristae organizing system. RESULTS: We found that CYP11A1 overexpression led to the formation of tubulovesicular cristae in mitochondria. We also identified the A'-helix located at amino acid #57-68 to be sufficient for membrane insertion and crista remodeling. We identified heat shock protein 60 (Hsp60) as the CYP11A1-interacting protein and showed that Hsp60 is required for CYP11A1 accumulation and crista remodeling. Finally, we found that the small MIC10 subcomplex of the mitochondrial contact site and cristae organizing system was reduced when CYP11A1 was overexpressed. CONCLUSIONS: CYP11A1 participates in the formation of tubulovesicular cristae in the mitochondria of steroidogenic cells. Its A'-helix is sufficient for the formation of tubulovesicular cristae and for protein integration into the membrane. CYP11A1 interacts with Hsp60, which is required for CYP11A1 accumulation. The accumulation of CYP11A1 leads to the reduction of MIC10 complex and changes mitochondrial structure.
Subject(s)
Cholesterol Side-Chain Cleavage Enzyme , Mitochondrial Membranes , Cholesterol Side-Chain Cleavage Enzyme/analysis , Cholesterol Side-Chain Cleavage Enzyme/genetics , Cholesterol Side-Chain Cleavage Enzyme/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/genetics , Steroids/analysis , Steroids/metabolismABSTRACT
This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.
Subject(s)
Immunologic Deficiency Syndromes , R-Loop Structures , Animals , Centromere/genetics , Centromere/metabolism , DNA/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Damage/genetics , DNA Methylation , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/metabolism , Mutation , DNA Methyltransferase 3BABSTRACT
Bioallethrin belongs to the family of pyrethroid insecticides. Previous studies have shown that bioallethrin affected the function of muscarinic receptor and subsequently induced neurotoxicity in different brain models. Reactive oxygen species (ROS) are generated in the metabolic course of the human body, which can cause human damage when overactivated. However, whether bioallethrin evokes cytotoxicity through ROS signaling and whether the antioxidant Vitamin E (VE) protects these cytotoxic responses in human glial cell model are still elusive. This study investigated the effect of bioallethrin on cytotoxicity through ROS signaling and evaluated the protective effect of the antioxidant VE in DBTRG-05MG human glioblastoma cells. The cell counting kit-8 (CCK-8) was used to measure cell viability. Intracellular ROS and glutathione (GSH) levels were measured by a cellular assay kit. The levels of apoptosis- and antioxidant-related protein were analyzed by Western blotting. In DBTRG-05MG cells, bioallethrin (25-75 µM) concentration-dependently induced cytotoxicity by increasing ROS productions, decreasing GSH contents, and regulating protein expressions related to apoptosis or antioxidation. Furthermore, these cytotoxic effects were partially reversed by VE (20 µM) pretreatment. Together, VE partially lessened bioallethrin-induced apoptosis through oxidative stress in DBTRG-05MG cells. The data assist us in identifying the toxicological mechanism of bioallethrin and offer future development of the antioxidant VE to reduce brain damage caused by bioallethrin.
Subject(s)
Antineoplastic Agents , Glioblastoma , Pyrethrins , Allethrins , Antineoplastic Agents/pharmacology , Antioxidants/metabolism , Apoptosis , Cell Survival , Glioblastoma/drug therapy , Glioblastoma/metabolism , Humans , Oxidative Stress , Pyrethrins/therapeutic use , Pyrethrins/toxicity , Reactive Oxygen Species/metabolism , Vitamin E/pharmacologyABSTRACT
Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2-PRC2) and noncanonical (EZH2-TAD-cMyc-coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.
Subject(s)
Enhancer of Zeste Homolog 2 Protein , Neoplasms , Carcinogenesis/genetics , Cytoskeletal Proteins/metabolism , E1A-Associated p300 Protein , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Humans , Proteolysis , Transcriptional ActivationABSTRACT
Acute myeloid leukemias (AMLs) with the NUP98-NSD1 or mixed lineage leukemia (MLL) rearrangement (MLL-r) share transcriptomic profiles associated with stemness-related gene signatures and display poor prognosis. The molecular underpinnings of AML aggressiveness and stemness remain far from clear. Studies with EZH2 enzymatic inhibitors show that polycomb repressive complex 2 (PRC2) is crucial for tumorigenicity in NUP98-NSD1+ AML, whereas transcriptomic analysis reveal that Kdm5b, a lysine demethylase gene carrying "bivalent" chromatin domains, is directly repressed by PRC2. While ectopic expression of Kdm5b suppressed AML growth, its depletion not only promoted tumorigenicity but also attenuated anti-AML effects of PRC2 inhibitors, demonstrating a PRC2-|Kdm5b axis for AML oncogenesis. Integrated RNA sequencing (RNA-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and Cleavage Under Targets & Release Using Nuclease (CUT&RUN) profiling also showed that Kdm5b directly binds and represses AML stemness genes. The anti-AML effect of Kdm5b relies on its chromatin association and/or scaffold functions rather than its demethylase activity. Collectively, this study describes a molecular axis that involves histone modifiers (PRC2-|Kdm5b) for sustaining AML oncogenesis.
Subject(s)
Jumonji Domain-Containing Histone Demethylases/metabolism , Leukemia, Myeloid, Acute/pathology , Nuclear Proteins/metabolism , Polycomb Repressive Complex 2/metabolism , Repressor Proteins/metabolism , Animals , Carcinogenesis , Gene Expression Profiling , Histone Demethylases/metabolism , Humans , Leukemia, Myeloid, Acute/metabolism , Mice , Oncogene Proteins/metabolism , Polycomb Repressive Complex 2/antagonists & inhibitors , Protein Binding , Sequence Analysis, RNA/methodsABSTRACT
INTRODUCTION: Learning through international experience has been well documented in USA literature, yet not well explored in Australia. The aim of this study is to understand Australian dental students' experiences and perceptions of international dental experience by: (i) capturing past participation; (ii) identifying potential interests for participants who have not been on a trip and (iii) ascertaining perceived benefits of the trip/s. MATERIALS AND METHODS: A 19 question paper survey was distributed to 310 eligible undergraduate dental students of The University of Queensland (UQ). An online counterpart survey was created via Google Forms and promoted on relevant Facebook groups targeted at current dental students of UQ. RESULTS: A total of 203 of the 310 eligible UQ undergraduate dental students responded to the distributed paper and electronic surveys making the response rate 65.5%. A total of 13 undergraduate respondents participated in an international experience trip. The majority of respondents who had not participated in an international dental experience trip were interested in participating in one. Education (84.6%) and improved dental skills (84.6%) were the most reported perceived benefits by the students who had participated in an international dental experience. CONCLUSION: A majority of respondents were interested in participating in international dental experiences. Despite this, only 1 in 15 respondents had participated in an international dental experience. Further exploration of this concept is needed; however, dental schools within Australia could potentially explore integration of international short-term dental experiences into formal curricula in the future.
Subject(s)
Education, Dental , Students, Dental , Australia , Humans , Surveys and Questionnaires , UniversitiesABSTRACT
Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.
Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Porcine epidemic diarrhea virus/chemistry , Coronavirus/chemistry , Coronavirus/classification , Coronavirus/enzymology , Coronavirus Papain-Like Proteases/genetics , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Mutation , Porcine epidemic diarrhea virus/enzymology , Porcine epidemic diarrhea virus/genetics , Protein Binding , Protein Domains , Structure-Activity Relationship , Substrate Specificity , Ubiquitin/chemistry , Ubiquitin/metabolismABSTRACT
BACKGROUND: Frailty may increase the risk of complications and mortality in patients undergoing cardiac surgery. Few studies on frailty and its associated factors have been conducted in these patients. OBJECTIVE: The aim of this study was to explore frailty and related factors in patients undergoing cardiac surgery. METHODS: A total of 154 patients undergoing cardiac surgery in northern Taiwan were recruited using a longitudinal study design and interviewed using structured questionnaires assessing physical activity, anxiety and depression, and social support before surgery and at 1 month and 3 months after surgery. RESULTS: The prevalence of frailty in patients undergoing cardiac surgery was 16.2%, 20.5%, and 16.6% before surgery and at 1 month and 3 months after surgery, respectively. Frail and prefrail patients undergoing cardiac surgery were more likely to be unemployed, have gout, have a higher New York Heart Association class, have preoperative dysrhythmia, undergo cardiopulmonary bypass, have a lower functional ability, have a higher European System for Cardiac Operative Risk Evaluation score, have a longer anesthesia time, have longer endotracheal tube and extracorporeal circulation times, have longer intensive care unit and hospital stays, have lower hemoglobin and albumin levels, have higher anxiety and depression levels, and have lower Mini-Mental State Examination scores. The significant predictors of prefrailty and frailty included unemployment, the presence of gout, higher New York Heart Association classes, less independence in activities of daily living, lower hemoglobin levels, and higher levels of depression. CONCLUSIONS: Frailty was associated with patients' functional status, perioperative conditions and psychosocial factors. Preoperative assessments of frailty and appropriate interventions are needed to improve frailty in patients undergoing cardiac surgery.
Subject(s)
Cardiac Surgical Procedures , Frailty , Gout , Activities of Daily Living , Aged , Cardiac Surgical Procedures/adverse effects , Frail Elderly , Frailty/complications , Frailty/epidemiology , Geriatric Assessment , Gout/complications , Hemoglobins , Humans , Longitudinal Studies , Postoperative Complications/epidemiology , Postoperative Complications/etiology , Risk FactorsABSTRACT
USP7, one of the most abundant ubiquitin-specific proteases (USP), plays multifaceted roles in many cellular events, including oncogenic pathways. Accumulated studies have suggested that USP7, through modulating the MDM2/MDMX-p53 pathway, is a promising target for cancer treatment; however, little is known about the function of USP7 in p53-deficient tumors. Here we report that USP7 regulates the autoregulation of SMAD3, a key regulator of transforming growth factor ß (TGFß) signaling, that represses the cell progression of p53-deficient lung cancer. CRISPR/Cas9-mediated inactivation of USP7 in p53-deficient lung cancer H1299 line resulted in advanced cell proliferation in vitro and in xenograft tumor in vivo. Genome-wide analyses (ChIP-seq and RNA-seq) of USP7 KO H1299 cells reveal a dramatic reduction of SMAD3 autoregulation, including decreased gene expression and blunted function of associated super-enhancer (SE). Furthermore, biochemical assays show that SMAD3 is conjugated by mono-ubiquitin, which negatively regulates the DNA-binding function of SMAD3, in USP7 KO cells. In addition, cell-free and cell-based analyses further demonstrate that the deubiquitinase activity of USP7 mediates the removal of mono-ubiquitin from SMAD3 and facilitates the DNA-binding of SMAD3-SMAD4 dimer at SMAD3 locus, and thus enhance the autoregulation of SMAD3. Collectively, our study identified a novel mechanism by which USP7, through catalyzing the SMAD3 de-monoubiquitination, facilitates the positive autoregulation of SMAD3, and represses the cancer progression of p53-deficient lung cancer.