RESUMEN
Fast synaptic neurotransmission in the vertebrate central nervous system relies primarily on ionotropic glutamate receptors (iGluRs), which drive neuronal excitation, and type A γ-aminobutyric acid receptors (GABAARs), which are responsible for neuronal inhibition. However, the GluD1 receptor, an iGluR family member, is present at both excitatory and inhibitory synapses. Whether and how GluD1 activation may affect inhibitory neurotransmission is unknown. In this work, by using a combination of biochemical, structural, and functional analyses, we demonstrate that GluD1 binds GABA, a previously unknown feature of iGluRs. GluD1 activation produces long-lasting enhancement of GABAergic synaptic currents in the adult mouse hippocampus through a non-ionotropic mechanism that is dependent on trans-synaptic anchoring. The identification of GluD1 as a GABA receptor that controls inhibitory synaptic plasticity challenges the classical dichotomy between glutamatergic and GABAergic receptors.
Asunto(s)
Inhibición Neural , Plasticidad Neuronal , Receptores de GABA , Transmisión Sináptica , Ácido gamma-Aminobutírico , Animales , Ratones , Ácido gamma-Aminobutírico/metabolismo , Glutamato Deshidrogenasa/metabolismo , Hipocampo/metabolismo , Receptores de GABA/metabolismo , Sinapsis/fisiología , Ratones Noqueados , Racemasas y Epimerasas/genéticaRESUMEN
NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like or endosomal vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Tomografía con Microscopio Electrónico , Gasderminas , Caspasa 1/metabolismo , Caspasas/metabolismo , Piroptosis , Orgánulos/metabolismoRESUMEN
Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2-PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast.
Asunto(s)
Ribonucleoproteína Nuclear Pequeña U5 , Proteínas de Saccharomyces cerevisiae , Humanos , Ribonucleoproteína Nuclear Pequeña U5/química , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Saccharomyces cerevisiae/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Ribonucleasa H/metabolismo , Proteínas de Unión al ARN/químicaRESUMEN
The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni2+-NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.
Asunto(s)
Histidina/química , Indicadores y Reactivos/química , Sondas Moleculares/química , Proteínas/química , Coloración y Etiquetado , Sulfonamidas/química , Células HEK293 , Histidina/metabolismo , Humanos , Indicadores y Reactivos/metabolismo , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Sondas Moleculares/metabolismo , Estructura Molecular , Níquel/química , Níquel/metabolismo , Ácido Nitrilotriacético/química , Ácido Nitrilotriacético/metabolismo , Proteínas/metabolismo , Sulfonamidas/metabolismoRESUMEN
Prolonged drug residence times may result in longer-lasting drug efficacy, improved pharmacodynamic properties, and "kinetic selectivity" over off-targets with high drug dissociation rates. However, few strategies have been elaborated to rationally modulate drug residence time and thereby to integrate this key property into the drug development process. Herein, we show that the interaction between a halogen moiety on an inhibitor and an aromatic residue in the target protein can significantly increase inhibitor residence time. By using the interaction of the serine/threonine kinase haspin with 5-iodotubercidin (5-iTU) derivatives as a model for an archetypal active-state (typeâ I) kinase-inhibitor binding mode, we demonstrate that inhibitor residence times markedly increase with the size and polarizability of the halogen atom. The halogen-aromatic πâ interactions in the haspin-inhibitor complexes were characterized by means of kinetic, thermodynamic, and structural measurements along with binding-energy calculations.
RESUMEN
Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation.
Asunto(s)
Compuestos de Dansilo/metabolismo , Galactosamina/análogos & derivados , Neurturina/metabolismo , Transducción de Señal/fisiología , Sinapsis/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Células COS , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Pollos , Técnicas de Cocultivo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Galactosamina/genética , Galactosamina/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Neurturina/genética , Mapas de Interacción de Proteínas , Receptores de N-Metil-D-Aspartato/metabolismo , Alineación de SecuenciaRESUMEN
Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/genética , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Animales , Sitios de Unión , Biomarcadores , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/química , Línea Celular , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Sustrato del Receptor de Insulina/química , Ligandos , Ratones , Modelos Biológicos , Desarrollo de Músculos , Mioblastos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-akt/química , Ratas , Proteínas Smad/química , UbiquitinaciónRESUMEN
Haspin is a mitotic protein kinase that is responsible for the phosphorylation of Thr3 of histone H3, thereby creating a recognition motif for docking of the chromosomal passenger complex that is crucial for the progression of cell division. Here, two high-resolution models of haspin with previously reported inhibitors consisting of an ATP analogue and a histone H3(1-7) peptide analogue are presented. The structures of the complexes confirm the bisubstrate character of the inhibitors by revealing the signature binding modes of the moieties targeting the ATP-binding site and the protein substrate-binding site of the kinase. This is the first structural model of a bisubstrate inhibitor targeting haspin. The presented structural data represent a model for the future development of more specific haspin inhibitors.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Conformación Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Especificidad por SustratoRESUMEN
Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson's disease).