RESUMEN
More than 200 proteins copurify with spliceosomes, the compositionally dynamic RNPs catalyzing pre-mRNA splicing. To better understand protein - protein interactions governing splicing, we systematically investigated interactions between human spliceosomal proteins. A comprehensive Y2H interaction matrix screen generated a protein interaction map comprising 632 interactions between 196 proteins. Among these, 242 interactions were found between spliceosomal core proteins and largely validated by coimmunoprecipitation. To reveal dynamic changes in protein interactions, we integrated spliceosomal complex purification information with our interaction data and performed link clustering. These data, together with interaction competition experiments, suggest that during step 1 of splicing, hPRP8 interactions with SF3b proteins are replaced by hSLU7, positioning this second step factor close to the active site, and that the DEAH-box helicases hPRP2 and hPRP16 cooperate through ordered interactions with GPKOW. Our data provide extensive information about the spliceosomal protein interaction network and its dynamics.
Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , Unión Competitiva , Proteínas Portadoras/metabolismo , Análisis por Conglomerados , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/fisiología , Humanos , Mapas de Interacción de Proteínas , Proteómica , ARN Helicasas/metabolismo , ARN Helicasas/fisiología , Factores de Empalme de ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
ADP-ribosylation is a dynamic post-translation modification that regulates the early phase of various DNA repair pathways by recruiting repair factors to chromatin. ADP-ribosylation levels are defined by the activities of specific transferases and hydrolases. However, except for the transferase PARP1/ARDT1 little is known about regulation of these enzymes. We found that MacroD2, a mono-ADP-ribosylhydrolase, is exported from the nucleus upon DNA damage, and that this nuclear export is induced by ATM activity. We show that the export is dependent on the phosphorylation of two SQ/TQ motifs, suggesting a novel direct interaction between ATM and ADP-ribosylation. Lastly, we show that MacroD2 nuclear export temporally restricts its recruitment to DNA lesions, which may decrease the net ADP-ribosylhydrolase activity at the site of DNA damage. Together, our results identify a novel feedback regulation between two crucial DNA damage-induced signaling pathways: ADP-ribosylation and ATM activation.
Asunto(s)
Adenosina Difosfato/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Daño del ADN , Enzimas Reparadoras del ADN/genética , Hidrolasas/genética , Poli(ADP-Ribosa) Polimerasas/genética , Procesamiento Proteico-Postraduccional , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Retroalimentación Fisiológica , Células HeLa , Humanos , Hidrolasas/metabolismo , Osteoblastos , Fosforilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de SeñalRESUMEN
Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein-protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes.
Asunto(s)
Fosfoproteínas/metabolismo , Mapas de Interacción de Proteínas , Tirosina/metabolismo , Humanos , Fosforilación , Unión Proteica , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Técnicas del Sistema de Dos HíbridosRESUMEN
Interleukin-11 (IL-11) is a member of the IL-6 family of cytokines and is an important factor for bone homeostasis. IL-11 binds to and signals via the membrane-bound IL-11 receptor (IL-11R, classic signaling) or soluble forms of the IL-11R (sIL-11R, trans-signaling). Mutations in the IL11RA gene, which encodes the IL-11R, are associated with craniosynostosis, a human condition in which one or several of the sutures close prematurely, resulting in malformation of the skull. The biological mechanisms of how mutations within the IL-11R are linked to craniosynostosis are mostly unexplored. In this study, we analyze two variants of the IL-11R described in craniosynostosis patients: p.T306_S308dup, which results in a duplication of three amino-acid residues within the membrane-proximal fibronectin type III domain, and p.E364_V368del, which results in a deletion of five amino-acid residues in the so-called stalk region adjacent to the plasma membrane. The stalk region connects the three extracellular domains to the transmembrane and intracellular region of the IL-11R and contains cleavage sites for different proteases that generate sIL-11R variants. Using a combination of bioinformatics and different biochemical, molecular, and cell biology methods, we show that the IL-11R-T306_S308dup variant does not mature correctly, is intracellularly retained, and does not reach the cell surface. In contrast, the IL-11R-E364_V368del variant is fully biologically active and processed normally by proteases, thus allowing classic and trans-signaling of IL-11. Our results provide evidence that mutations within the IL11RA gene may not be causative for craniosynostosis and suggest that other regulatory mechanism(s) are involved but remain to be identified.
Asunto(s)
Craneosinostosis , Interleucina-11 , Humanos , Receptores de Interleucina-11/genética , Receptores de Interleucina-11/química , Receptores de Interleucina-11/metabolismo , Interleucina-11/genética , Interleucina-11/metabolismo , Transducción de Señal , Craneosinostosis/genética , Péptido Hidrolasas/metabolismo , Receptores de Interleucina-6/genética , Receptor gp130 de Citocinas/genéticaRESUMEN
Information about the physical association of proteins is extensively used for studying cellular processes and disease mechanisms. However, complete experimental mapping of the human interactome will remain prohibitively difficult in the near future. Here we present a map of predicted human protein interactions that distinguishes functional association from physical binding. Our network classifies more than 5 million protein pairs predicting 94,009 new interactions with high confidence. We experimentally tested a subset of these predictions using yeast two-hybrid analysis and affinity purification followed by quantitative mass spectrometry. Thus we identified 462 new protein-protein interactions and confirmed the predictive power of the network. These independent experiments address potential issues of circular reasoning and are a distinctive feature of this work. Analysis of the physical interactome unravels subnetworks mediating between different functional and physical subunits of the cell. Finally, we demonstrate the utility of the network for the analysis of molecular mechanisms of complex diseases by applying it to genome-wide association studies of neurodegenerative diseases. This analysis provides new evidence implying TOMM40 as a factor involved in Alzheimer's disease. The network provides a high-quality resource for the analysis of genomic data sets and genetic association studies in particular. Our interactome is available via the hPRINT web server at: www.print-db.org.
Asunto(s)
Simulación por Computador , Modelos Moleculares , Mapeo de Interacción de Proteínas/métodos , Algoritmos , Animales , Teorema de Bayes , Células HeLa , Humanos , Ratones , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteoma/genética , Proteoma/metabolismo , Curva ROC , Proteínas Recombinantes/metabolismo , Estadísticas no ParamétricasRESUMEN
Chromatin relaxation is one of the earliest cellular responses to DNA damage. However, what determines these structural changes, including their ATP requirement, is not well understood. Using live-cell imaging and laser microirradiation to induce DNA lesions, we show that the local chromatin relaxation at DNA damage sites is regulated by PARP1 enzymatic activity. We also report that H1 is mobilized at DNA damage sites, but, since this mobilization is largely independent of poly(ADP-ribosyl)ation, it cannot solely explain the chromatin relaxation. Finally, we demonstrate the involvement of Alc1, a poly(ADP-ribose)- and ATP-dependent remodeler, in the chromatin-relaxation process. Deletion of Alc1 impairs chromatin relaxation after DNA damage, while its overexpression strongly enhances relaxation. Altogether our results identify Alc1 as an important player in the fast kinetics of the NAD+- and ATP-dependent chromatin relaxation upon DNA damage in vivo.
Asunto(s)
ADN Helicasas/metabolismo , ADN Helicasas/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Poli Adenosina Difosfato Ribosa/metabolismo , Técnicas de Cultivo de Célula , Cromatina/fisiología , Ensamble y Desensamble de Cromatina/fisiología , ADN , Daño del ADN , Reparación del ADN/fisiología , Histonas/metabolismo , Humanos , Nucleosomas , Imagen Óptica , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
The yeast two-hybrid (Y2H) system is currently one of the most important techniques for protein-protein interaction (PPI) discovery. Here, we describe a stringent three-step Y2H matrix interaction approach that is suitable for systematic PPI screening on a proteome scale. We start with the identification and elimination of autoactivating strains that would lead to false-positive signals and prevent the identification of interactions. Nonautoactivating strains are used for the primary PPI screen that is carried out in quadruplicate with arrayed preys. Interacting pairs of baits and preys are identified in a pairwise retest step. Only PPI pairs that pass the retest step are regarded as potentially biologically relevant interactions and are considered for further analysis.