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Although starch has been intensively studied as a raw material for 3D printing, the relationship between several important process parameters in the preparation of starch gels and the printing results is unclear. In this study, the relationship between different processing conditions and the gel printing performance of corn starch was evaluated by printing tests, rheological tests and low-field nuclear magnetic resonance (LF-NMR) tests, and a back-propagation artificial neural network (BP-ANN) model for predicting gel printing performance was developed. The results revealed that starch gels exhibited favorable printing performance when the gelatinization temperature ranged from 75 °C to 85 °C, and the starch content was maintained between 15 % and 20 %. The R2adj of the BP-ANN models were all reached 0.894, which indicated good predictive ability. The results of the study not only provide theoretical support for the application of corn starch gels in 3D food printing, but also present a novel approach for predicting the printing performance of related materials. This method contributes to the optimization of printing parameters, thereby enhancing printing efficiency and quality.
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Redes Neurales de la Computación , Impresión Tridimensional , Almidón , Zea mays , Almidón/química , Zea mays/química , Tinta , Reología , Geles/química , TemperaturaRESUMEN
TAX1BP1, a multifunctional autophagy adaptor, plays critical roles in different autophagy processes. As an autophagy receptor, TAX1BP1 can interact with RB1CC1, NAP1, and mammalian ATG8 family proteins to drive selective autophagy for relevant substrates. However, the mechanistic bases underpinning the specific interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins remain elusive. Here, we find that there are two distinct binding sites between TAX1BP1 and RB1CC1. In addition to the previously reported TAX1BP1 SKICH (skeletal muscle and kidney enriched inositol phosphatase (SKIP) carboxyl homology)/RB1CC1 coiled-coil interaction, the first coiled-coil domain of TAX1BP1 can directly bind to the extreme C-terminal coiled-coil and Claw region of RB1CC1. We determine the crystal structure of the TAX1BP1 SKICH/RB1CC1 coiled-coil complex and unravel the detailed binding mechanism of TAX1BP1 SKICH with RB1CC1. Moreover, we demonstrate that RB1CC1 and NAP1 are competitive in binding to the TAX1BP1 SKICH domain, but the presence of NAP1's FIP200-interacting region (FIR) motif can stabilize the ternary TAX1BP1/NAP1/RB1CC1 complex formation. Finally, we elucidate the molecular mechanism governing the selective interactions of TAX1BP1 with ATG8 family members by solving the structure of GABARAP in complex with the non-canonical LIR (LC3-interacting region) motif of TAX1BP1, which unveils a unique binding mode between LIR and ATG8 family protein. Collectively, our findings provide mechanistic insights into the interactions of TAX1BP1 with RB1CC1 and mammalian ATG8 family proteins and are valuable for further understanding the working mode and function of TAX1BP1 in autophagy.
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Autofagia , Proteínas de Ciclo Celular , Animales , Familia de las Proteínas 8 Relacionadas con la Autofagia , Sitios de Unión , Riñón , MamíferosRESUMEN
Objective: This study aimed to evaluate the impact of nonpharmaceutical interventions (NPIs) taken to combat COVID-19 on the prevalence of respiratory viruses (RVs) of acute respiratory infections (ARIs) in Shanghai. Methods: Samples from ARI patients were collected and screened for 17 respiratory viral pathogens using TagMan low density microfluidic chip technology in Shanghai from January 2019 to December 2020. Pathogen data were analyzed to assess changes in acute respiratory infections between 2019 and 2020. Results: A total of 2,744 patients were enrolled, including 1,710 and 1,034 in 2019 and 2020, respectively. The total detection rate of RVs decreased by 149.74% in 2020. However, detection rates for human respiratory syncytial virus B (RSVB), human coronavirus 229E (HCoV229E), human coronavirus NL63 (HCoVNL63), and human parainfluenza virus 3 (HPIV3) increased by 91.89, 58.33, 44.68 and 24.29%, in 2020. The increased positive rates of RSVB, HPIV3, resulted in more outpatients in 2020 than in 2019. IFV detection rates declined dramatically across gender, age groups, and seasons in 2020. Conclusion: NPIs taken to eliminate COVID-19 had an impact on the prevalence of respiratory viral pathogens, especially the IFVs in the early phases of the pandemic. Partial respiratory viruses resurged with the lifting of NPIs, leading to an increase in ARIs infection.
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COVID-19 , Infecciones del Sistema Respiratorio , Humanos , Pandemias , COVID-19/epidemiología , Prevalencia , China/epidemiología , Infecciones del Sistema Respiratorio/epidemiologíaRESUMEN
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a key regulator in inflammation and cell death and is involved in mediating a variety of inflammatory or degenerative diseases. A number of allosteric RIPK1 inhibitors (RIPK1i) have been developed, and some of them have already advanced into clinical evaluation. Recently, selective RIPK1i that interact with both the allosteric pocket and the ATP-binding site of RIPK1 have started to emerge. Here, we report the rational development of a new series of type-II RIPK1i based on the rediscovery of a reported but mechanistically atypical RIPK3i. We also describe the structure-guided lead optimization of a potent, selective, and orally bioavailable RIPK1i, 62, which exhibits extraordinary efficacies in mouse models of acute or chronic inflammatory diseases. Collectively, 62 provides a useful tool for evaluating RIPK1 in animal disease models and a promising lead for further drug development.
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BACKGROUND: Alveolar epithelial injury and dysfunction are the risk factors for radiation-induced pulmonary fibrosis (RIPF). However, it is not clear about the relationship between RIPF and the small extracellular vesicles (sEV) secreted by irradiated alveolar epithelial cells. Based on the activation of fibroblasts, this study explored the role of sEV derived from alveolar epithelial cells in RIPF and the potential mechanisms. METHODS: Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were used to characterize sEV. Western blotting was used to detect fibrosis-associated proteins. Cell counts and transwell assays were used to evaluate the proliferation and migration ability of fibroblasts. RT-PCR was used to observe the extracellular matrix (ECM) synthesized by fibroblasts, miRNA changes in the sEV were determined by second-generation sequencing. RESULTS: TEM, NTA, and western blotting showed the extracellular vesicles with a double-layer membrane structure of approximately 100 nm in diameter. The sEV derived from irradiated A549, HBEC3-KT, and MLE12 cells upregulated FN1 and alpha-SMA proteins expression in fibroblasts and drove the fibroblast to myofibroblast transition, and the sEV from irradiated mouse bronchoalveolar lavage fluid (BALF) affirmed the same results. In addition, the sEV derived from irradiated alveolar epithelial cells significantly increased the migration ability of fibroblasts and the expression of extracellular matrix proteins such as FN1. The results of miRNA sequencing of sEV in BALF of rats with RIPF showed that the metabolic pathway may be important for miRNA to regulate the activation of fibroblasts. CONCLUSION: The sEV derived from radiated pulmonary epithelial cells promote the activation, migration and extracellular matrix proteins expression of lung fibroblasts; miRNA in sEV may be an important molecular that affects the activation of lung fibroblasts.
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Vesículas Extracelulares , MicroARNs , Fibrosis Pulmonar , Ratas , Ratones , Animales , Fibrosis Pulmonar/etiología , Pulmón/metabolismo , Células Epiteliales/patología , MicroARNs/genética , MicroARNs/metabolismo , Fibroblastos/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas de la Matriz Extracelular/efectos adversos , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
Autophagy receptor NDP52 triggers bacterial autophagy against infection. However, the ability of NDP52 to protect against viral infection has not been established. We show that NDP52 binds to envelope proteins of hepatitis B virus (HBV) and triggers a degradation process that promotes HBV clearance. Inactivating NDP52 in hepatocytes results in decreased targeting of viral envelopes in the lysosome and increased levels of viral replication. NDP52 inhibits HBV at both viral entry and late replication stages. In contrast to NDP52-mediated bacterial autophagy, lysosomal degradation of HBV envelopes is independent of galectin 8 and ATG5. NDP52 forms complex with Rab9 and viral envelope proteins and links HBV to Rab9-dependent lysosomal degradation pathway. These findings reveal that NDP52 acts as a sensor for HBV infection, which mediates a unique antiviral response to eliminate the virus. This work also suggests direct roles for autophagy receptors in other lysosomal degradation pathways than canonical autophagy.
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Virus de la Hepatitis B , Hepatitis B , Humanos , Virus de la Hepatitis B/fisiología , Hepatocitos/metabolismo , Autofagia/fisiología , Lisosomas/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Antivirales/metabolismo , Replicación Viral/fisiologíaRESUMEN
HOIL-1L and SHARPIN are two essential regulatory subunits of the linear ubiquitin chain assembly complex (LUBAC), which is the only known E3 ligase complex generating linear ubiquitin chains. In addition to their LUBAC-dependent functions, HOIL-1L and SHARPIN alone play crucial roles in many LUBAC-independent cellular processes. Importantly, deficiency of HOIL-1L or SHARPIN leads to severe disorders in humans or mice. However, the mechanistic bases underlying the multi-functions of HOIL-1L and SHARPIN are still largely unknown. Here, we uncover that HOIL-1L and SHARPIN alone can form homo-dimers through their LTM motifs. We solve two crystal structures of the dimeric LTM motifs of HOIL-1L and SHARPIN, which not only elucidate the detailed molecular mechanism underpinning the dimer formations of HOIL-1L and SHARPIN, but also reveal a general mode shared by the LTM motifs of HOIL-1L and SHARPIN for forming homo-dimer or hetero-dimer. Furthermore, we elucidate that the polyglucosan body myopathy-associated HOIL-1L A18P mutation disturbs the structural folding of HOIL-1L LTM, and disrupts the dimer formation of HOIL-1L. In summary, our study provides mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN mediated by their LTM motifs, and expands our understandings of the multi-functions of HOIL-1L and SHARPIN as well as the etiology of relevant human disease caused by defective HOIL-1L.
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Ubiquitina-Proteína Ligasas , Ubiquitinas , Animales , Humanos , Ratones , Proteínas Portadoras/metabolismo , Dimerización , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
Heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) serves as a unique E3 ligase to catalyze the mono-ubiquitination of relevant protein or sugar substrates and plays vital roles in numerous cellular processes in mammals. However, the molecular mechanism underpinning the E3 activity of HOIL-1L and the related regulatory mechanism remain elusive. Here, we report the crystal structure of the catalytic core region of HOIL-1L and unveil the key catalytic triad residues of HOIL-1L. Moreover, we discover that HOIL-1L contains two distinct linear di-ubiquitin binding sites that can synergistically bind to linear tetra-ubiquitin, and the binding of HOIL-1L with linear tetra-ubiquitin can promote its E3 activity. The determined HOIL-1L/linear tetra-ubiquitin complex structure not only elucidates the detailed binding mechanism of HOIL-1L with linear tetra-ubiquitin but also uncovers a unique allosteric ubiquitin-binding site for the activation of HOIL-1L. In all, our findings provide mechanistic insights into the E3 activity of HOIL-1L and its regulation by the linear ubiquitin chain binding.
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Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Unión Proteica , Ubiquitina/metabolismo , Sitios de Unión , Mamíferos/metabolismoRESUMEN
Arl8b, a specific Arf-like family GTPase present on lysosome, and plays critical roles in many lysosome-related cellular processes such as autophagy. The active Arl8b can be specifically recognized by the RUN domains of two Arl8b-effectors PLEKHM1 and SKIP, thereby regulating the autophagosome/lysosome membrane fusion and the intracellular lysosome positioning, respectively. However, the mechanistic bases underlying the interactions of Arl8b with the RUN domains of PLEKHM1 and SKIP remain elusive. Here, we report the two high-resolution crystal structures of the active Arl8b in complex with the RUN domains of PLEKHM1 and SKIP. In addition to elucidating the detailed molecular mechanism governing the specific interactions of the active Arl8b with the RUN domains of PLEKHM1 and SKIP, the determined complex structures also reveal a general binding mode shared by the PLEKHM1 and SKIP RUN domains for interacting with the active Arl8b. Furthermore, we uncovered a competitive relationship between the RUN domains of PLEKHM1 and SKIP in binding to the active Arl8b as well as a unique small GTPase-binding mode adopted by the PLEKHM1 and SKIP RUN domains, thereby enriching the repertoire of the RUN domain/small GTPase interaction modes. In all, our findings provide new mechanistic insights into the interactions of the active Arl8b with PLEKHM1 and SKIP, and are valuable for further understanding the working modes of these proteins in relevant cellular processes.
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Factores de Ribosilacion-ADP , Proteínas Adaptadoras Transductoras de Señales , Proteínas Relacionadas con la Autofagia , Coactivadores de Receptor Nuclear , Dominios y Motivos de Interacción de Proteínas , Proteínas Adaptadoras Transductoras de Señales/química , Lisosomas/metabolismo , Fusión de Membrana , Factores de Ribosilacion-ADP/química , Proteínas Relacionadas con la Autofagia/química , Coactivadores de Receptor Nuclear/química , Cristalografía por Rayos X , HumanosRESUMEN
Diazo compounds are rare natural products possessing various biological activities. Kinamycin and lomaiviticin, two diazo natural products featured by the diazobenzofluorene core, exhibit exceptional potency as chemotherapeutic agents. Despite the extensive studies on their biosynthetic gene clusters and the assembly of their polyketide scaffolds, the formation of the characteristic diazo group remains elusive. L-Glutamylhydrazine was recently shown to be the hydrazine donor in kinamycin biosynthesis, however, the mechanism for the installation of the hydrazine group onto the kinamycin scaffold is still unclear. Here we describe an O-methyltransferase-like protein, AlpH, which is responsible for the hydrazine incorporation in kinamycin biosynthesis. AlpH catalyses a unique SAM-independent coupling of L-glutamylhydrazine and polyketide intermediate via a rare Mannich reaction in polyketide biosynthesis. Our discovery expands the catalytic diversity of O-methyltransferase-like enzymes and lays a strong foundation for the discovery and development of novel diazo natural products through genome mining and synthetic biology.
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Productos Biológicos , Policétidos , Metiltransferasas/genética , Metabolismo Secundario , CatálisisRESUMEN
Modification of organic molecules with fluorine functionalities offers a critical approach to develop new pharmaceuticals. Here, we report a multienzyme strategy for biocatalytic fluoroalkylation using S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) and fluorinated SAM cofactors prepared from ATP and fluorinated l-methionine analogues by an engineered human methionine adenosyltransferase hMAT2AI322A. This work introduces the first example of biocatalytic 3,3-difluoroallylation. Importantly, this strategy can be applied to late-stage site-selective fluoroalkylation of complex molecule vancomycin with conversions up to 99%.
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Metionina , S-Adenosilmetionina , Humanos , Metionina/metabolismo , S-Adenosilmetionina/metabolismo , Metiltransferasas/metabolismo , Racemetionina , BiocatálisisRESUMEN
The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.
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Necroptosis , Receptores Tipo I de Factores de Necrosis Tumoral , Humanos , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Hidroxilación , Hipoxia , Prolina/metabolismo , Inflamación , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
Adenosine monophosphate-activated protein kinase (AMPK) activity is stimulated to promote metabolic adaptation upon energy stress. However, sustained metabolic stress may cause cell death. The mechanisms by which AMPK dictates cell death are not fully understood. We report that metabolic stress promoted receptor-interacting protein kinase 1 (RIPK1) activation mediated by TRAIL receptors, whereas AMPK inhibited RIPK1 by phosphorylation at Ser415 to suppress energy stress-induced cell death. Inhibiting pS415-RIPK1 by Ampk deficiency or RIPK1 S415A mutation promoted RIPK1 activation. Furthermore, genetic inactivation of RIPK1 protected against ischemic injury in myeloid Ampkα1-deficient mice. Our studies reveal that AMPK phosphorylation of RIPK1 represents a crucial metabolic checkpoint, which dictates cell fate response to metabolic stress, and highlight a previously unappreciated role for the AMPK-RIPK1 axis in integrating metabolism, cell death, and inflammation.
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Proteínas Quinasas Activadas por AMP , Metabolismo Energético , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Estrés Fisiológico , Animales , Ratones , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Inflamación/metabolismo , Isquemia/metabolismoRESUMEN
Oxepinone rings represent one of structurally unusual motifs of natural products and the biosynthesis of oxepinones is not fully understood. 1,5-Seco-vibralactone (3) features an oxepinone motif and is a stable metabolite isolated from mycelial cultures of the mushroom Boreostereum vibrans. Cyclization of 3 forms vibralactone (1) whose ß-lactone-fused bicyclic core originates from 4-hydroxybenzoate, yet it remains elusive how 4-hydroxybenzoate is converted to 3 especially for the oxepinone ring construction in the biosynthesis of 1. In this work, using activity-guided fractionation together with proteomic analyses, we identify an NADPH/FAD-dependent monooxygenase VibO as the key enzyme performing a crucial ring-expansive oxygenation on the phenol ring to generate the oxepin-2-one structure of 3. The crystal structure of VibO reveals that it forms a dimeric phenol hydroxylase-like architecture featured with a unique substrate-binding pocket adjacent to the bound FAD. Computational modeling and solution studies provide insight into the likely VibO active site geometry, and suggest possible involvement of a flavin-C4a-OO(H) intermediate.
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Oxigenasas de Función Mixta , Proteómica , Lactonas/metabolismo , Flavinas , Flavina-Adenina DinucleótidoRESUMEN
Background: Urinary tract infections (UTIs) are some of the most common bacterial infections in the world. Nevertheless, as uncomplicated UTIs are treated empirically without culturing the urine, adequate knowledge of the resistance pattern of uropathogens is essential. Conventional urine culture and identification take at least 2 days. Here, we developed a platform based on LAMP and centrifugal disk system (LCD) to simultaneously detect the main pathogens and antibiotic resistant genes (ARGs) of urgent concern multidrug-resistant among UTIs. Methods: We designed specific primers to detect the target genes above and evaluated their sensitivity and specificity. We also assessed the result of our preload LCD platform on 645 urine specimens with a conventional culturing method and Sanger sequencing. Results: The results obtained with the 645 clinical samples indicated that the platform has high specificity (0.988-1) and sensitivity (0.904-1) for the studied pathogens and ARGs. Moreover, the kappa value of all pathogens was more than 0.75, revealing an excellent agreement between the LCD and culture method. Compared to phenotypic tests, the LCD platform is a practical and fast detection approach for methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter, carbapenem-resistant Pseudomonas aeruginosa (kappa value of all >0.75), and non-extended-spectrum ß-lactamase producers. Conclusion: We developed a detection platform that has high accuracy and that meets the need for rapid diagnosis, which can be completed within 1.5 h from specimen collection. It may be a powerful tool for evidence-based UTIs diagnosis, which has essential support for the rational use of antibiotics. More high-quality clinical studies are required to prove the effectiveness of our platform.
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The recruitment of ATG12-ATG5-ATG16L1 complex to phagophore mediated by the specific interaction between ATG16L1 and WIPI2, is pivotal to the formation of autophagosomes during macroautophagy. Recently, we reported that ATG16L1 contains two distinct WIPI2-binding sites, the previously reported WIPI2-binding site (WBS1), and the newly identified site (WBS2). By determining the crystal structures of WIPI2 with ATG16L1 WBS1 and WBS2 respectively, we uncovered that, unlike ATG16L1 WBS1, ATG16L1 WBS2 and its binding mechanism to WIPI2 are conserved from yeast to mammals. Using cell-based functional assays, we further demonstrated that the integrity of two WIPI2-binding sites of ATG16L1 is essential for normal autophagic flux. In summary, our study provided mechanistic insights into the interaction of two key autophagic proteins, ATG16L1 and WIPI2, and revealed a dual-binding-site mode adopted by ATG16L1 to associate with WIPI2.Abbreviations: ATG: autophagy-related protein; CCD: coiled-coil domain; ITC: isothermal titration calorimetry; PI3KC3-C1: class III phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; ULK: Unc-51-like kinase; WBS: WIPI2-binding site; WIPI: WD repeat domain phosphoinositide-interacting protein.
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Background: Osteoarthritis (OA) is one of the most prevalent chronic diseases, leading to degeneration of joints, chronic pain, and disability in the elderly. Little is known about the role of immune-related genes (IRGs) and immune cells in OA. Method: Hub IRGs of OA were identified by differential expression analysis and filtered by three machine learning strategies, including random forest (RF), least absolute shrinkage and selection operator (LASSO), and support vector machine (SVM). A diagnostic nomogram model was then constructed by using these hub IRGs, with receiver operating characteristic (ROC) curve, decision curve analysis (DCA), and clinical impact curve analysis (CICA) estimating its performance and clinical impact. Hierarchical clustering analysis was then conducted by setting the hub IRGs as input information. Differences in immune cell infiltration and activities of immune pathways were revealed between different immune subtypes. Result: Five hub IRGs of OA were identified, including TNFSF11, SCD1, PGF, EDNRB, and IL1R1. Of them, TNFSF11 and SCD1 contributed the most to the diagnostic nomogram model with area under the curve (AUC) values of 0.904 and 0.864, respectively. Two immune subtypes were characterized. The immune over-activated subtype showed excessively activated cellular immunity with a higher proportion of activated B cells and activated CD8 T cells. The two phenotypes were also seen in two validation cohorts. Conclusion: The present study comprehensively investigated the role of immune genes and immune cells in OA. Five hub IRGs and two immune subtypes were identified. These findings will provide novel insights into the diagnosis and treatment of OA.
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Dolor Crónico , Osteoartritis , Humanos , Osteoartritis/diagnóstico , Osteoartritis/genética , Área Bajo la Curva , Linfocitos B , Análisis por ConglomeradosRESUMEN
Macroautophagy plays crucial roles in the regulation of cellular physiology and requires de novo synthesis of double-membrane autophagosomes, which relies on a specific interaction between autophagy-related 16L1 (ATG16L1) and WD repeat domain phosphoinositide-interacting protein 2b (WIPI2b). However, the molecular mechanism governing the interaction of ATG16L1 with WIPI2b remains elusive. Here, we find that ATG16L1 has two distinct binding sites for interacting with WIPI2b, the previously reported WIPI2b-binding site (WBS1) and the previously unidentified site (WBS2). We determine the crystal structures of WIPI2b with ATG16L1 WBS1 and WBS2, respectively, and elucidate the molecular mechanism underpinning the recruitment of ATG16L1 by WIPI2b. Moreover, we uncover that ATG16L1 WBS2 and its binding mode with WIPI2b is well conserved from yeast to mammals, unlike ATG16L1 WBS1. Last, our cell-based functional assays demonstrate that both ATG16L1 WBS1 and WBS2 are required for the effective autophagic flux. In conclusion, our findings provide mechanistic insights into the key ATG16L1/WIPI2b interaction in autophagy.
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Autofagosomas , Autofagia , Animales , Sitios de Unión , Fosfatidilinositoles , Saccharomyces cerevisiae , MamíferosRESUMEN
Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.
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Melanoma , Microftalmía , Animales , Humanos , Factores de Transcripción/metabolismo , Microftalmía/genética , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Regulación de la Expresión Génica , Proteínas Oncogénicas/genética , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Cycloaddition reactions are among the most widely used reactions in chemical synthesis. Nature achieves these cyclization reactions with a variety of enzymes, including Diels-Alderases that catalyse concerted 4 + 2 cycloadditions, but biosynthetic enzymes with 2 + 2 cyclase activity have yet to be discovered. Here we report that PloI4, a ß-barrel-fold protein homologous to the exo-selective 4 + 2 cyclase that functions in the biosynthesis of pyrroindomycins, catalyses competitive 2 + 2 and 4 + 2 cycloaddition reactions. PloI4 is believed to catalyse an endo-4 + 2 cycloaddition in the biosynthesis of pyrrolosporin A; however, when the substrate precursor of pyrroindomycins was treated with PloI4, an exo-2 + 2 adduct was produced in addition to the exo- and endo-4 + 2 adducts. Biochemical characterizations, computational analyses, (co)crystal structures and mutagenesis outcomes have allowed the catalytic versatility of PloI4 to be rationalized. Mechanistic studies involved the directed engineering of PloI4 to variants that produced the exo-4 + 2, endo-4 + 2 or exo-2 + 2 product preferentially. This work illustrates an enzymatic thermal 2 + 2 cycloaddition and provides evidence of a process through which an enzyme evolves along with its substrate for specialization and activity improvement.