RESUMEN
Monoubiquitination of the Fanconi anemia complementation group D2 (FANCD2) protein by the FA core ubiquitin ligase complex is the central event in the FA pathway. FANCA and FANCG play major roles in the nuclear localization of the FA core complex. Mutations of these two genes are the most frequently observed genetic alterations in FA patients, and most point mutations in FANCA are clustered in the C-terminal domain (CTD). To understand the basis of the FA-associated FANCA mutations, we determined the cryo-electron microscopy (EM) structures of Xenopus laevis FANCA alone at 3.35 Å and 3.46 Å resolution and two distinct FANCA-FANCG complexes at 4.59 and 4.84 Å resolution, respectively. The FANCA CTD adopts an arc-shaped solenoid structure that forms a pseudo-symmetric dimer through its outer surface. FA- and cancer-associated point mutations are widely distributed over the CTD. The two different complex structures capture independent interactions of FANCG with either FANCA C-terminal HEAT repeats, or the N-terminal region. We show that mutations that disturb either of these two interactions prevent the nuclear localization of FANCA, thereby leading to an FA pathway defect. The structure provides insights into the function of FANCA CTD, and provides a framework for understanding FA- and cancer-associated mutations.
Asunto(s)
Proteína del Grupo de Complementación A de la Anemia de Fanconi/ultraestructura , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/ultraestructura , Proteína del Grupo de Complementación G de la Anemia de Fanconi/ultraestructura , Anemia de Fanconi/genética , Animales , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Anemia de Fanconi/patología , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/química , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Mutación , Unión Proteica/genética , Conformación Proteica , Xenopus laevis/genéticaRESUMEN
Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI-FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.
Asunto(s)
Exodesoxirribonucleasas/química , Modelos Moleculares , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/enzimología , Dominio Catalítico , Cristalización , Exodesoxirribonucleasas/metabolismo , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Humanos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.
Asunto(s)
ADN Cruciforme/metabolismo , ADN Cruciforme/fisiología , Resolvasas de Unión Holliday/metabolismo , Animales , Proteínas de Arabidopsis , Segregación Cromosómica/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Desoxirribonucleasa I , Endodesoxirribonucleasas , Endonucleasas , Proteínas de Escherichia coli , Transferencia Resonante de Energía de Fluorescencia/métodos , Resolvasas de Unión Holliday/fisiología , Humanos , Unión Proteica , Recombinación Genética/genética , Imagen Individual de Molécula/métodos , Especificidad por SustratoRESUMEN
ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here,Methanococcus jannaschii MR-ATPγS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Arqueales/metabolismo , ADN/metabolismo , Methanococcus/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , ADN/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
DNA interstrand cross-links (ICLs) block the progress of the replication and transcription machineries and can weaken chromosomal stability, resulting in various diseases. FANCD2-FANCI-associated nuclease (FAN1) is a conserved structure-specific nuclease that unhooks DNA ICLs independently of the Fanconi anemia pathway. Recent structural studies have proposed two different mechanistic features for ICL unhooking by human FAN1: a specific basic pocket that recognizes the terminal phosphate of a 1-nucleotide (nt) 5' flap or FAN1 dimerization. Herein, we show that despite lacking these features, Pseudomonas aeruginosa FAN1 (PaFAN1) cleaves substrates at â¼3-nt intervals and resolves ICLs. Crystal structures of PaFAN1 bound to various DNA substrates revealed that its conserved basic Arg/Lys patch comprising Arg-228 and Lys-260 recognizes phosphate groups near the 5' terminus of a DNA substrate with a 1-nt flap or a nick. Substitution of Lys-260 did not affect PaFAN1's initial endonuclease activity but significantly decreased its subsequent exonuclease activity and ICL unhooking. The Arg/Lys patch also interacted with phosphates at a 3-nt gap, and this interaction could drive movement of the scissile phosphates into the PaFAN1-active site. In human FAN1, the ICL-resolving activity was not affected by individual disruption of the Arg/Lys patch or basic pocket. However, simultaneous substitution of both FAN1 regions significantly reduced its ICL-resolving activity, suggesting that these two basic regions play a complementary role in ICL repair. On the basis of these findings, we propose a conserved role for two basic regions in FAN1 to guide ICL unhooking and to maintain genomic stability.
Asunto(s)
Proteínas Bacterianas/química , Desoxirribonucleasa I/química , Simulación de Dinámica Molecular , Pseudomonas aeruginosa/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Dominios Proteicos , Pseudomonas aeruginosa/genética , Relación Estructura-ActividadRESUMEN
Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-ß clamp complex. This complex contains two pairs of Hda dimers sandwiched between two ß clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the ß clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-ß clamp complex indicate that the interaction of the ß clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-ß clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Bacterianas/metabolismo , ADN Polimerasa III/química , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ADN Polimerasa III/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutación , Multimerización de Proteína , Alineación de SecuenciaRESUMEN
Communication between Mre11 and Rad50 in the MR complex is critical for the sensing, damage signaling, and repair of DNA double-strand breaks. To understand the basis for interregulation between Mre11 and Rad50, we determined the crystal structure of the Mre11-Rad50-ATPγS complex. Mre11 brings the two Rad50 molecules into close proximity and promotes ATPase activity by (1) holding the coiled-coil arm of Rad50 through its C-terminal domain, (2) stabilizing the signature motif and P loop of Rad50 via its capping domain, and (3) forming a dimer through the nuclease domain. ATP-bound Rad50 negatively regulates the nuclease activity of Mre11 by blocking the active site of Mre11. Hydrolysis of ATP disengages Rad50 molecules, and, concomitantly, the flexible linker that connects the C-terminal domain and the capping domain of Mre11 undergoes substantial conformational change to relocate Rad50 and unmask the active site of Mre11. Our structural and biochemical data provide insights into understanding the interplay between Mre11 and Rad50 to facilitate efficient DNA damage repair.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Proteínas Arqueales/química , Endodesoxirribonucleasas/química , Exodesoxirribonucleasas/química , Methanococcus/química , Methanococcus/metabolismo , Modelos Moleculares , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Proteínas Arqueales/metabolismo , Sitios de Unión , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Regulación de la Expresión Génica Arqueal , Hidrólisis , Methanococcus/enzimología , Methanococcus/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3' flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a "5' end binding pocket" that hosts the 5' nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3' flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5' flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3' flap DNA substrates with 5' nicked ends.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Endodesoxirribonucleasas/química , Endonucleasas/química , Región de Flanqueo 5' , Cristalografía por Rayos X , Roturas del ADN de Cadena Simple , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.
Asunto(s)
Proteínas Arqueales/química , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Endodesoxirribonucleasas/química , Exodesoxirribonucleasas/química , Methanocaldococcus/enzimología , Modelos Moleculares , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , Análisis Mutacional de ADN , ADN de Archaea/genética , ADN de Archaea/metabolismo , Dimerización , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Methanocaldococcus/química , Methanocaldococcus/genética , Modelos Estructurales , Datos de Secuencia Molecular , Unión Proteica , Alineación de SecuenciaRESUMEN
In higher eukaryotes, one of the two arginyl-tRNA synthetases (ArgRSs) has evolved to have an extended N-terminal domain that plays a crucial role in protein synthesis and cell growth and in integration into the multisynthetase complex (MSC). Here, we report a crystal structure of the MSC subcomplex comprising ArgRS, glutaminyl-tRNA synthetase (GlnRS), and the auxiliary factor aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1)/p43. In this complex, the N-terminal domain of ArgRS forms a long coiled-coil structure with the N-terminal helix of AIMP1 and anchors the C-terminal core of GlnRS, thereby playing a central role in assembly of the three components. Mutation of AIMP1 destabilized the N-terminal helix of ArgRS and abrogated its catalytic activity. Mutation of the N-terminal helix of ArgRS liberated GlnRS, which is known to control cell death. This ternary complex was further anchored to AIMP2/p38 through interaction with AIMP1. These findings demonstrate the importance of interactions between the N-terminal domains of ArgRS and AIMP1 for the catalytic and noncatalytic activities of ArgRS and for the assembly of the higher-order MSC protein complex.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Arginino-ARNt Ligasa/química , Citocinas/química , Proteínas de Neoplasias/química , Proteínas de Unión al ARN/química , Sitios de Unión , Cromatografía en Gel , Dicroismo Circular , Cristalografía por Rayos X , Escherichia coli/metabolismo , Glutatión Transferasa/química , Humanos , Modelos Moleculares , Complejos Multiproteicos , Mutagénesis , Mutación , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de RadiaciónRESUMEN
The Ssl1/p44 subunit is a core component of the yeast/mammalian general transcription factor TFIIH, which is involved in transcription and DNA repair. Ssl1/p44 binds to and stimulates the Rad3/XPD helicase activity of TFIIH. To understand the helicase stimulatory mechanism of Ssl1/p44, we determined the crystal structure of the N-terminal regulatory domain of Ssl1 from Saccharomyces cerevisiae. Ssl1 forms a von Willebrand factor A fold in which a central six-stranded ß-sheet is sandwiched between three α helices on both sides. Structural and biochemical analyses of Ssl1/p44 revealed that the ß4-α5 loop, which is frequently found at the interface between von Willebrand factor A family proteins and cellular counterparts, is critical for the stimulation of Rad3/XPD. Yeast genetics analyses showed that double mutation of Leu-239 and Ser-240 in the ß4-α5 loop of Ssl1 leads to lethality of a yeast strain, demonstrating the importance of the Rad3-Ssl1 interactions to cell viability. Here, we provide a structural model for the Rad3/XPD-Ssl1/p44 complex and insights into how the binding of Ssl1/p44 contributes to the helicase activity of Rad3/XPD and cell viability.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIH/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , ADN Helicasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/metabolismoRESUMEN
Cohesin is a multiprotein complex that establishes sister chromatid cohesion from S phase until mitosis or meiosis. In vertebrates, sister chromatid cohesion is dissolved in a stepwise manner: most cohesins are removed from the chromosome arms via a process that requires polo-like kinase 1 (Plk1), aurora B and Wapl, whereas a minor amount of cohesin, found preferentially at the centromere, is cleaved by separase following its activation by the anaphase-promoting complex/cyclosome. Here, we report that our budding yeast two-hybrid assay identified hsSsu72 phosphatase as a Rad21-binding protein. Additional experiments revealed that Ssu72 directly interacts with Rad21 and SA2 in vitro and in vivo, and associates with sister chromatids in human cells. Interestingly, depletion or mutational inactivation of Ssu72 phosphatase activity caused the premature resolution of sister chromatid arm cohesion, whereas the overexpression of Ssu72 yielded high resistance to this resolution. Interestingly, it appears that Ssu72 regulates the cohesion of chromosome arms but not centromeres, and acts by counteracting the phosphorylation of SA2. Thus, our study provides important new evidence, suggesting that Ssu72 is a novel cohesin-binding protein capable of regulating cohesion between sister chromatid arms.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos Híbridos , CohesinasRESUMEN
Generation of the 3' overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5'-3' nuclease, NurA, plays an important role in generating 3' single-stranded DNA during archaeal HR, together with Mre11-Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn(2)(+)-bound NurA from Pyrococcus furiousus (Pf NurA) to provide the basis for its cleavage mechanism. Pf NurA forms a pyramid-shaped dimer containing a large central channel on one side, which becomes narrower towards the peak of the pyramid. The structure contains a PIWI domain with high similarity to argonaute, endoV nuclease and RNase H. The two active sites, each of which contains Mn(2)(+) ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer. The 3' OH group of the ribose ring is directed toward the channel entrance, explaining the 5'-3' nuclease activity of Pf NurA. We provide a DNA binding and cleavage model for Pf NurA.
Asunto(s)
Proteínas Arqueales/química , Nucleótidos de Desoxiadenina/química , Endodesoxirribonucleasas/química , Exodesoxirribonucleasas/química , Manganeso/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía , ADN Helicasas/metabolismo , ADN de Cadena Simple/química , Dimerización , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Modelos Moleculares , Mutación , Pyrococcus furiosus/enzimología , Thermotoga maritima/enzimologíaRESUMEN
Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal antibody production from plasma cells. Despite advances in the treatment, many patients experience disease relapse or become refractory to treatment. G-protein-coupled receptor class C group 5 member D (GPRC5D), an orphan GPCR predominantly expressed in MM cells, is emerging as a promising target for MM immunotherapy. Talquetamab, a Food and Drug Administration-approved T-cell-directing bispecific antibody developed for treatment of MM, targets GPRC5D. Here, we elucidate the structure of GPRC5D complexed with the Fab fragment of talquetamab, using cryo-electron microscopy, providing the basis for recognition of GPRC5D by the bispecific antibody. GPRC5D forms a symmetric homodimer with the interface between transmembrane helix (TM)4 of one protomer and TM4/5 of the other protomer. A single talquetamab Fab interacts with the GPRC5D dimer with its orientation toward the dimer interface. All six complementarity-determining regions of talquetamab engage with extracellular loops and TM3/5/7. In particular, the side-chain of an arginine residue from the antibody penetrates into a shallow pocket on the extracellular surface of GPRC5D. The structure offers insights for optimizing antibody design against GPRC5D for relapsed or refractory MM therapy.
RESUMEN
Class C G-protein-coupled receptors (GPCRs) are activated through binding of agonists to the large extracellular domain (ECD) followed by rearrangement of the transmembrane domains (TMDs). GPR156, a class C orphan GPCR, is unique because it lacks an ECD and exhibits constitutive activity. Impaired GPR156-Gi signaling contributes to loss of hearing. Here we present the cryo-electron microscopy structures of human GPR156 in the Go-free and Go-coupled states. We found that an endogenous phospholipid molecule is located within each TMD of the GPR156 dimer. Asymmetric binding of Gα to the phospholipid-bound GPR156 dimer restructures the first and second intracellular loops and the carboxy-terminal part of the elongated transmembrane 7 (TM7) without altering dimer conformation. Our findings reveal that GPR156 is a transducer for phospholipid signaling. Constant binding of abundant phospholipid molecules and the G-protein-induced reshaping of the cytoplasmic face provide a basis for the constitutive activation of GPR156.
Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Humanos , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP/metabolismo , FosfolípidosRESUMEN
DDS, 4,4'-diaminodiphenylsulfone, is the most common drug prescribed to treat Hansen disease patients. In addition to its antibacterial activity, DDS has been reported to be involved in other cellular processes that occur in eukaryotic cells. Because DDS treatment significantly enhances the antioxidant activity in humans, we examined its effect on lifespan extension. Here we show that DDS extends organismic lifespan using Caenorhabditis elegans as a model system. DDS treatment caused a delay in aging and decreased the levels of a mitochondrial complex. The oxygen consumption rate was also significantly lowered. Consistent with these data, paraquat treatment evoked less reactive oxygen species in DDS-treated worms, and these worms were less sensitive to paraquat. Interestingly enough, all of the molecular events caused by DDS treatment were consistently reproduced in mice treated with DDS for 3 mo and in the C2C12 muscle cell line. Structural prediction identified pyruvate kinase (PK) as a protein target of DDS. Indeed, DDS bound and inhibited PK in vitro and inhibited it in vivo, and a PK mutation conferred extended lifespan of C. elegans. Supplement of pyruvate to the media protected C2C12 cells from apoptosis caused by paraquat. Our findings establish the significance of DDS in lowering reactive oxygen species generation and extending the lifespan, which renders the rationale to examining the possible effect of DDS on human lifespan extension.
Asunto(s)
Caenorhabditis elegans/fisiología , Dapsona/farmacología , Longevidad/efectos de los fármacos , Envejecimiento/efectos de los fármacos , Animales , Antioxidantes , Línea Celular , Proteínas Mitocondriales/efectos de los fármacos , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Paraquat/farmacología , Piruvato Quinasa/antagonistas & inhibidores , Piruvato Quinasa/genética , Especies Reactivas de OxígenoRESUMEN
The binding of insulin to the insulin receptor (IR) triggers a cascade of receptor conformational changes and autophosphorylation, leading to the activation of metabolic and mitogenic pathways. Recent advances in the structural and functional analyses of IR have revealed the conformations of the extracellular domains of the IR in inactive and fully activated states. However, the early activation mechanisms of this receptor remain poorly understood. The structures of partially activated IR in complex with aptamers provide clues for understanding the initial activation mechanism. In this review, we discuss the structural and functional features of IR complexed with various ligands and propose a model to explain the sequential activation mechanism. Moreover, we discuss the structures of IR complexed with biased agonists that selectively activate metabolic pathways and provide insights into the design of selective agonists and their clinical implications.
Asunto(s)
Receptor de Insulina , Transducción de Señal , Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Insulina/metabolismo , Fosforilación , Proteínas Portadoras/metabolismoRESUMEN
Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate-dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate-recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5-1.9â Å resolution using synchrotron X-rays and an X-ray free-electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate-binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate-binding and reaction process of cyanase.
Asunto(s)
Bicarbonatos , Escherichia coli , Bicarbonatos/metabolismo , Bicarbonatos/farmacología , Liasas de Carbono-Nitrógeno/química , Cianatos/metabolismo , Cianatos/farmacologíaRESUMEN
Prostate cancer is the most prevalent cancer in men worldwide and is promoted by the sex hormone androgen. Expression of androgen from the testis can be significantly reduced through castration. However, as most prostate cancer patients acquire castration resistance, additional therapeutic solutions are necessary. Although anti-androgens, such as enzalutamide, have been used to treat castration-resistant prostate cancer (CRPC), enzalutamide-resistant CRPC (Enz-resistant CRPC) has emerged. Therefore, development of novel treatments for Enz-resistant CRPC is urgent. In this study, we found a novel anti-androgen called pinostilbene through screening with a GAL4-transactivation assay. We confirmed that pinostilbene directly binds to androgen receptor (AR) and inhibits its activation and translocalization. Pinostilbene treatment also reduced the protein level and downstream gene expression of AR. Furthermore, pinostilbene reduced the protein level of AR variant 7 in the Enz-resistant prostate cancer cell line 22Rv1 and inhibited cell viability and proliferation. Our results suggest that pinostilbene has the potential to treat Enz-resistant CRPC.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Andrógenos/farmacología , Línea Celular Tumoral , Nitrilos/uso terapéutico , Antagonistas de Andrógenos/uso terapéutico , Resistencia a Antineoplásicos/genéticaRESUMEN
A complex of Drosha with DGCR8 (or its homolog Pasha) cleaves primary microRNA (pri-miRNA) substrates into precursor miRNA and initiates the microRNA maturation process. Drosha provides the catalytic site for this cleavage, whereas DGCR8 or Pasha provides a frame for anchoring substrate pri-miRNAs. To clarify the molecular basis underlying recognition of pri-miRNA by DGCR8 and Pasha, we determined the crystal structure of the human DGCR8 core (DGCR8S, residues 493-720). In the structure, the two double-stranded RNA-binding domains (dsRBDs) are arranged with pseudo two-fold symmetry and are tightly packed against the C-terminal helix. The H2 helix in each dsRBD is important for recognition of pri-miRNA substrates. This structure, together with fluorescent resonance energy transfer and mutational analyses, suggests that the DGCR8 core recognizes pri-miRNA in two possible orientations. We propose a model for DGCR8's recognition of pri-miRNA.