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
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
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
Zinc fingers can be loosely defined as protein domains containing one or more tetrahedrally-co-ordinated zinc ions whose role is to stabilise the structure rather than to be involved in enzymatic chemistry; such zinc ions are often referred to as "structural zincs". Although structural zincs can occur in proteins of any size, they assume particular significance for very small protein domains, where they are often essential for maintaining a folded state. Such small structures, that sometimes have only marginal stability, can present particular difficulties in terms of sample preparation, handling and structure determination, and early on they gained a reputation for being resistant to crystallisation. As a result, NMR has played a more prominent role in structural studies of zinc finger proteins than it has for many other types of proteins. This review will present an overview of the particular issues that arise for structure determination of zinc fingers by NMR, and ways in which these may be addressed.
Asunto(s)
Proteínas , Dedos de Zinc , Secuencia de Aminoácidos , Zinc/química , Zinc/metabolismoRESUMEN
Single-stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 domains led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1's multi-domain organization. Here, we directly probe this process at the single-molecule level. Quantitative smFRET and structural ensemble calculations reveal how PARP-1's N-terminal zinc fingers convert DNA SSBs from a largely unperturbed conformation, via an intermediate state into the highly kinked DNA conformation. Our data suggest an induced fit mechanism via a multi-domain assembly cascade that drives SSB sensing and stimulates an interplay with the scaffold protein XRCC1 orchestrating subsequent DNA repair events. Interestingly, a clinically used PARP-1 inhibitor Niraparib shifts the equilibrium towards the unkinked DNA conformation, whereas the inhibitor EB47 stabilizes the kinked state.
Asunto(s)
Roturas del ADN de Cadena Simple , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Reparación del ADN , Daño del ADN , ADN/metabolismoRESUMEN
Cilia formation is essential for human life. One of the earliest events in the ciliogenesis program is the recruitment of tau-tubulin kinase 2 (TTBK2) by the centriole distal appendage component CEP164. Due to the lack of high-resolution structural information on this complex, it is unclear how it is affected in human ciliopathies such as nephronophthisis. Furthermore, it is poorly understood if binding to CEP164 influences TTBK2 activities. Here, we present a detailed biochemical, structural, and functional analysis of the CEP164-TTBK2 complex and demonstrate how it is compromised by two ciliopathic mutations in CEP164. Moreover, we also provide insights into how binding to CEP164 is coordinated with TTBK2 activities. Together, our data deepen our understanding of a crucial step in cilia formation and will inform future studies aimed at restoring CEP164 functionality in a debilitating human ciliopathy.
Asunto(s)
Ciliopatías/genética , Proteínas de Microtúbulos/química , Proteínas de Microtúbulos/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Sitios de Unión , Dicroismo Circular , Células HEK293 , Humanos , Proteínas de Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Estabilidad ProteicaRESUMEN
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
Attachment of ubiquitin (Ub) to proteins is one of the most abundant and versatile of all posttranslational modifications and affects outcomes in essentially all physiological processes. RING E3 ligases target E2 Ub-conjugating enzymes to the substrate, resulting in its ubiquitination. However, the mechanism by which a ubiquitin chain is formed on the substrate remains elusive. Here we demonstrate how substrate binding can induce a specific RING topology that enables self-ubiquitination. By analyzing a catalytically trapped structure showing the initiation of TRIM21 RING-anchored ubiquitin chain elongation, and in combination with a kinetic study, we illuminate the chemical mechanism of ubiquitin conjugation. Moreover, biochemical and cellular experiments show that the topology found in the structure can be induced by substrate binding. Our results provide insights into ubiquitin chain formation on a structural, biochemical and cellular level with broad implications for targeted protein degradation.
Asunto(s)
Biocatálisis , Dominios RING Finger , Ubiquitinación , Animales , Femenino , Masculino , Ratones , Modelos Moleculares , Células 3T3 NIH , Proteolisis , Ribonucleoproteínas/química , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps in coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here, we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but it decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind polyubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes, and it also shows an mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for polyubiquitin, and prevents its binding to nonspecific substrates.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Cinética , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Soluciones , Resonancia por Plasmón de Superficie , Transcripción GenéticaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Complejo 2 de Proteína Adaptadora/genética , Subunidades alfa de Complejo de Proteína Adaptadora/genética , Endocitosis/genética , Nexinas de Clasificación/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Clatrina/genética , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/genética , Vesículas Cubiertas por Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/genética , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Humanos , Fosforilación/genética , Unión Proteica/genéticaRESUMEN
The peripheral stalk of ATP synthase acts as a stator holding the alpha(3)beta(3) catalytic subcomplex and the membrane subunit a against the torque of the rotating central stalk and attached c ring. In bovine mitochondria, the N-terminal domain of the oligomycin sensitivity conferral protein (OSCP-NT; residues 1-120) anchors one end of the peripheral stalk to the N-terminal tails of one or more alpha subunits of the F(1) subcomplex. Here, we present an NMR characterisation of the interaction between OSCP-NT and a peptide corresponding to residues 1-25 of the alpha-subunit of bovine F(1)-ATPase. The interaction site contains adjoining hydrophobic surfaces of helices 1 and 5 of OSCP-NT binding to hydrophobic side-chains of the alpha-peptide.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , TermodinámicaRESUMEN
Cell surface Fc receptors activate inflammation and are tightly controlled to prevent autoimmunity. Antibodies also simulate potent immune signalling from inside the cell via the cytosolic antibody receptor TRIM21, but how this is regulated is unknown. Here we show that TRIM21 signalling is constitutively repressed by its B-Box domain and activated by phosphorylation. The B-Box occupies an E2 binding site on the catalytic RING domain by mimicking E2-E3 interactions, inhibiting TRIM21 ubiquitination and preventing immune activation. TRIM21 is derepressed by IKKß and TBK1 phosphorylation of an LxxIS motif in the RING domain, at the interface with the B-Box. Incorporation of phosphoserine or a phosphomimetic within this motif relieves B-Box inhibition, promoting E2 binding, RING catalysis, NF-κB activation and cytokine transcription upon infection with DNA or RNA viruses. These data explain how intracellular antibody signalling is regulated and reveal that the B-Box is a critical regulator of RING E3 ligase activity.
Asunto(s)
Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , Receptores Fc/metabolismo , Ribonucleoproteínas/metabolismo , Transducción de Señal , Animales , Línea Celular , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes - the pstA cells - as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Resorcinoles/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dictyostelium/citología , Dictyostelium/efectos de los fármacos , Espectrometría de Masas , Resorcinoles/química , Resorcinoles/aislamiento & purificaciónRESUMEN
Homeodomain proteins control the developmental transition between the haploid and diploid phases in several eukaryotic lineages, but it is not known whether this regulatory mechanism reflects the ancestral condition or, instead, convergent evolution. We have characterized the mating-type locus of the amoebozoan Dictyostelium discoideum, which encodes two pairs of small proteins that determine the three mating types of this species; none of these proteins display recognizable homology to known families. We report that the nuclear magnetic resonance structures of two of them, MatA and MatB, contain helix-turn-helix folds flanked by largely disordered amino- and carboxyl-terminal tails. This fold closely resembles that of homeodomain transcription factors, and, like those proteins, MatA and MatB each bind DNA characteristically using the third helix of their folded domains. By constructing chimeric versions containing parts of MatA and MatB, we demonstrate that the carboxyl-terminal tail, not the central DNA binding motif, confers mating specificity, providing mechanistic insight into how a third mating type might have originated. Finally, we show that these homeodomain-like proteins specify zygote function: Hemizygous diploids, formed in crosses between a wild-type strain and a mat null mutant, grow and differentiate identically to haploids. We propose that Dictyostelium MatA and MatB are divergent homeodomain proteins with a conserved function in triggering the haploid-to-diploid transition that can be traced back to the last common ancestor of eukaryotes.