RESUMEN
The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. After binding to genomic lesions, these enzymes use NAD+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues5-10. Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.
Asunto(s)
ADP-Ribosilación , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Daño del ADN , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/química , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regulación Alostérica , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Biocatálisis , Proteínas Portadoras/genética , Dominio Catalítico , Células HEK293 , Humanos , Modelos Moleculares , Mutación , NAD/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Anémonas de MarRESUMEN
AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.
Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Dominios Proteicos , ADP-Ribosilación , ARN/metabolismoRESUMEN
Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.
RESUMEN
Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1's function remained obscure; inherent dynamics of SSBs and PARP-1's multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1's signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodification in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.
Asunto(s)
Roturas del ADN de Cadena Simple , ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , ADN/química , Reparación del ADN , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Poli(ADP-Ribosa) Polimerasa-1 , Pliegue de Proteína , Dedos de ZincRESUMEN
PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.
Asunto(s)
Poli(ADP-Ribosa) Polimerasa-1/química , Regulación Alostérica , Amidas/química , Dominio Catalítico , Cristalografía por Rayos X , Daño del ADN , Activación Enzimática , Modelos Moleculares , Mutación , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Dominios ProteicosRESUMEN
Ratiometric fluorescence detection attracts much attention because of its decreased environmental influence and easy-to-differentiate color and intensity change. Herein, a guest-encapsulation metal-organic framework (MOF), Ru@MIL-NH2, is prepared with 2-aminoterephthalic acid, AlCl3, and Ru(bpy)32+ by a simple one-pot method for ratiometric fluorescence sensing of water in organic solvents. The rational selection of the excitation wavelength provides dual emission at 465 and 615 nm from Ru@MIL-NH2 under a single excitation of 300 nm. High sensitivity, low detection limit (0.02% v/v), wide response range (0-100%), and fast response (less than 1 min) are obtained for ratiometric fluorescence sensing of water under single excitation with Ru@MIL-NH2 as the probe. Moreover, the result of water content is independent of the concentration of Ru@MIL-NH2 as the merit of ratiometric fluorescence detection. The response mechanism reveals that the protonation of the nitrogen atom of the MIL-NH2, the π-conjugation system, and the stable fluorescence of Ru(bpy)32+ achieve the ratiometric fluorescence. The analysis of real spirit samples confirms the proposed method. A test strip is prepared with Ru@MIL-NH2 for convenient use. We believe that such turn-on ratiometric host-guest MOFs and the rational selection of excitation wavelength will offer guidance for ratiometric fluorescence detection with wide applications.
RESUMEN
For predicting the temperature distribution within skin tissue in 980-nm laser-evoked potentials (LEPs) experiments, a five-layer finite element model (FEM-5) was constructed based on Pennes bio-heat conduction equation and the Lambert-Beer law. The prediction results of the FEM-5 model were verified by ex vivo pig skin and in vivo rat experiments. Thirty ex vivo pig skin samples were used to verify the temperature distribution predicted by the model. The output energy of the laser was 1.8, 3, and 4.4 J. The laser spot radius was 1 mm. The experiment time was 30 s. The laser stimulated the surface of the ex vivo pig skin beginning at 10 s and lasted for 40 ms. A thermocouple thermometer was used to measure the temperature of the surface and internal layers of the ex vivo pig skin, and the sampling frequency was set to 60 Hz. For the in vivo experiments, nine adult male Wistar rats weighing 180 ± 10 g were used to verify the prediction results of the model by tail-flick latency. The output energy of the laser was 1.4 and 2.08 J. The pulsed width was 40 ms. The laser spot radius was 1 mm. The Pearson product-moment correlation and Kruskal-Wallis test were used to analyze the correlation and the difference of data. The results of all experiments showed that the measured and predicted data had no significant difference (P > 0.05) and good correlation (r > 0.9). The safe laser output energy range (1.8-3 J) was also predicted. Using the FEM-5 model prediction, the effective pain depth could be accurately controlled, and the nociceptors could be selectively activated. The FEM-5 model can be extended to guide experimental research and clinical applications for humans.
Asunto(s)
Análisis de Elementos Finitos , Rayos Láser , Dolor/patología , Temperatura Cutánea/efectos de la radiación , Temperatura , Adulto , Animales , Humanos , Masculino , Ratas Wistar , Reproducibilidad de los Resultados , Piel/efectos de la radiación , Sus scrofa , Cola (estructura animal)RESUMEN
Plutella xylostella (P. xylostella) is a highly migratory, cosmopolitan species and one of the most important pest of cruciferous crops worldwide. Pyridalyl as a novel class of insecticides has good efficacy against P. xylostella. On the basis of the commercial insecticide pyridalyl, a series of new aryloxy dihalopropene derivatives were designed and synthesized by using Intermediate Derivatization Methods. Their chemical structures were confirmed by (1)H NMR, high-resolution mass spectrum (HRMS), and single-crystal X-ray diffraction analysis. The insecticidal activities of the new compounds against P. xylostella were evaluated. The results of bioassays indicated that most of the compounds showed moderate to high activities at the tested concentration, especially compounds 10e and 10g displayed more than 75% insecticidal activity against P. xylostella at 6.25mg/L, while pyridalyl showed 50% insecticidal activity at the same concentration. The field trials result of the insecticidal activities showed that compound 10e as a 10% emulsifiable concentrate (EC) was effective in the control of P. xylostella at 75-150g a.i./ha, and the mortality of P. xylostella for treatment with compound 10e at 75g a.i./ha was equivalent to pyridalyl at 105g a.i./ha.
Asunto(s)
Compuestos Alílicos/farmacología , Diseño de Fármacos , Insecticidas/síntesis química , Insecticidas/farmacología , Mariposas Nocturnas/efectos de los fármacos , Piridinas/farmacología , Compuestos Alílicos/síntesis química , Compuestos Alílicos/química , Animales , Relación Dosis-Respuesta a Droga , Insecticidas/química , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
In the past decades, diabetes, in particular type 2 diabetes (T2D)mainly characterized by global insulin resistance and pancreatic beta cell failure, had become epidemic and a severe public health threat worldwide with the development of economy and change of lifestyle.The interactions between environment factors and genetic background play vital roles in the development and progression of T2D.More recently, it had been revealed that non-coding RNA including microRNA (miRNA)and long noncoding RNA (LncRNA)are widely involved inthe regulation of glucose and lipid metabolism. So far, it had been established that deregulated miRNA and LncRNA profile in main metabolic tissues is tightly associated with T2D,and intensive studies on non-coding RNAs had shed light on understanding the pathogen-esis of T2D.The current review aimed to briefly summarize and discuss the latest findings regarding the role and mechanism of miRNAs and LncRNAs in the regulation hepatic glucose and lipid metabolism.
Asunto(s)
Metabolismo de los Lípidos , Diabetes Mellitus Tipo 2 , Glucosa , Humanos , Resistencia a la Insulina , Células Secretoras de Insulina , Hígado , ARN no TraducidoRESUMEN
Establishing the binding topology of structural zinc ions in proteins is an essential part of their structure determination by NMR spectroscopy. Using (113)Cdâ NMR experiments with (113)Cd-substituted samples is a useful approach but has previously been limited mainly to very small protein domains. Here we used (113)Cdâ NMR spectroscopy during structure determination of Bud31p, a 157-residue yeast protein containing an unusual Zn3Cys9 cluster, demonstrating that recent hardware developments make this approach feasible for significantly larger systems.
Asunto(s)
Espectroscopía de Resonancia Magnética , Metales/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Cadmio/química , Isótopos , Metales/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Soluciones/química , Electricidad Estática , Zinc/química , Zinc/metabolismoRESUMEN
Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed ß-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.
Asunto(s)
Factor 3 de Iniciación Eucariótica/química , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Saccharomyces cerevisiae/química , Codón Iniciador , Cristalografía por Rayos X , Factor 1 Eucariótico de Iniciación/genética , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Dosificación de Gen , Modelos Moleculares , Mutación , Fenotipo , Estructura Terciaria de Proteína , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Levaduras/genética , Levaduras/crecimiento & desarrolloRESUMEN
BACKGROUND: Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet ß cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed. METHODS: db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays. RESULTS: In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers. CONCLUSIONS: PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A's suppressive effects on hepatic gluconeogenesis and lipogenesis.
Asunto(s)
Adenosina Trifosfato , Conexinas , Gluconeogénesis , Lipogénesis , Hígado , Proteínas del Tejido Nervioso , Animales , Conexinas/metabolismo , Ratones , Gluconeogénesis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Adenosina Trifosfato/metabolismo , Lipogénesis/fisiología , Hígado/metabolismo , Ratones Noqueados , Masculino , Humanos , Dieta Alta en Grasa/efectos adversos , CitocinasRESUMEN
TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.
Asunto(s)
Lisina , Ubiquitina-Proteína Ligasas , Animales , Lisina/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Mamíferos/metabolismoRESUMEN
Rds3p, a component of the U2 snRNP subcomplex SF3b, is essential for pre-mRNA splicing and is extremely well conserved in all eukaryotic species. We report here the solution structure of Rds3p, which reveals an unusual knotted fold unrelated to previously known knotted proteins. Rds3p has a triangular shape with a GATA-like zinc finger at each vertex. Pairs of cysteines contributing to each finger are arranged nonsequentially in a permuted arrangement reminiscent of domain-swapping but which here involves segments of subdomains within a single chain. We suggest that the structure arose through a process of segment swapping after gene duplication. The fingers are connected through beta-strands and loops, forming an overall topology strongly resembling a "triquetra knot." The conservation and surface properties of Rds3p suggest that it functions as a platform for protein assembly within the multiprotein SF3b complex of U2 snRNP. The recombinant protein used for structure determination is biologically active, as it restores splicing activity in a yeast splicing extract depleted of native Rds3p.
Asunto(s)
Proteínas Portadoras/química , Ribonucleoproteína Nuclear Pequeña U2/química , Proteínas de Saccharomyces cerevisiae/química , Dedos de Zinc , Cisteína/química , Conformación Proteica , SolucionesRESUMEN
Prostaglandin(PG) E2 is an important metabolic product of arachidonic acid. PGE2 plays important roles in regulation of fever, inflammatory responses and blood pressure via four functionally antagonistic E-prostanoid (EP) receptors, which are designated as EP1, EP2, EP3 and EP4, respectively. Recently, there is increasing evidence that PGE2 also regulates the maturation of immune cells and immune response. This review aims to briefly summarize and discuss the recent findings regarding the role of PGE2 in regulation of immunity.
Asunto(s)
Dinoprostona/fisiología , Inmunidad/fisiología , Inflamación/fisiopatología , Animales , Humanos , Receptores de Prostaglandina E/metabolismoRESUMEN
VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.
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Evolución Molecular , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Zinc/metabolismo , Microscopía por Crioelectrón , Cisteína/química , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación Proteica , Proteínas de Transporte Vesicular/genética , Dedos de ZincRESUMEN
OBJECTIVE: To study the role of the carbohydrate response element binding protein (ChREBP) in excessive lipid deposition in the liver of db/db mouse. METHODS: The deposition of neutral lipids in the liver was evaluated by Oil Red O staining. Immunohistochemical assay was utilized to determine the localization of ChREBP protein expression in mouse liver. The expressions of ChREBP and its target genes including acetyl-coenzyme A carboxylase 1 (Acc-1), fatty acid synthase (Fas), glycerol-3-phosphate acyltransferase (Gpat) were analyzed by Real-time PCR and Western blot. RESULTS: Significant lipid droplet deposition was detected in the livers of db/db mice. ChREBP was diffusely expressed in heptocytes with relative higher expression levels around portal and central veins. ChREBP was predominantly located in the cytosol in non-diabetic db/m mice, but was translocated to the nucleus in db/db mice. Nuclear ChREBP protein levels were 8.2-fold higher in db/db mice than in db/m mice(P<0.01). In contrast, another lipogenic transcription factor, sterol regulatory element binding protein-1(SREBP-1), remained unchanged. Consistent with increased nuclear ChREBP levels, expressions of ChREBP target genes involved in lipogenesis including Acc-1, Fas and Gpat were upregulated by 2-fold(P<0.05),1.7-fold (P<0.05) and 4.2-fold(P<0.05), respectively, in db/db mice. CONCLUSION: The db/db mouse exhibits significantly higher liver ChREBP activity, which may be associated with the development of hepatic steatosis frequently occurring in type 2 diabetes. Targeting ChREBP might represent a new intervention strategy for fatty liver.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Lípidos/biosíntesis , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.
Asunto(s)
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/fisiología , Secuencias de Aminoácidos/genética , Factores de Restricción Antivirales , Biocatálisis , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.