RESUMEN
The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a five amino acid sequence, RPSKR, located between the DNA binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a non-canonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt two-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint, and in certain circumstances exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.
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The innate immune cGAS-STING pathway is activated by cytosolic double-stranded DNA (dsDNA), a ubiquitous danger signal, to produce interferon, a potent anti-viral and anti-cancer cytokine. However, STING activation must be tightly controlled because aberrant interferon production leads to debilitating interferonopathies. Here, we discover PELI2 as a crucial negative regulator of STING. Mechanistically, PELI2 inhibits the transcription factor IRF3 by binding to phosphorylated Thr354 and Thr356 on the C-terminal tail of STING, leading to ubiquitination and inhibition of the kinase TBK1. PELI2 sets a threshold for STING activation that tolerates low levels of cytosolic dsDNA, such as that caused by silenced TREX1, RNASEH2B, BRCA1, or SETX. When this threshold is reached, such as during viral infection, STING-induced interferon production temporarily downregulates PELI2, creating a positive feedback loop allowing a robust immune response. Lupus patients have insufficient PELI2 levels and high basal interferon production, suggesting that PELI2 dysregulation may drive the onset of lupus and other interferonopathies.
Asunto(s)
Factor 3 Regulador del Interferón , Proteínas de la Membrana , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Ubiquitinación , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Fosforilación , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Animales , Células HEK293 , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/virología , Inmunidad Innata , Interacciones Huésped-Patógeno , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ratones , Interferones/metabolismo , Interferones/inmunología , Interferones/genética , Retroalimentación Fisiológica , Ratones Endogámicos C57BL , Exodesoxirribonucleasas , FosfoproteínasRESUMEN
The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at the double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a five amino acid sequence, RPSKR, located between the DNA binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a non-canonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt two-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint, and in certain circumstances exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.
RESUMEN
Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.
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Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that ΔSisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in ΔSisarnA. The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro. ΔSisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro. These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo. Interestingly, ΔSisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.
Asunto(s)
Archaea , Factores de Transcripción Forkhead , Archaea/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Daño del ADN , FosforilaciónRESUMEN
The forkhead-associated (FHA) domain, a well-characterized small protein module that mediates protein-protein interactions by targeting motifs containing phosphothreonine, is present in many regulatory molecules like protein kinase, phosphatases, transcription factors, and other functional proteins. FHA-domain containing proteins in yeast and human are involved in a large variety of cellular processes such as DNA repair, cell cycle arrest, or pre-mRNA processing. Since the first FHA-domain protein, kinase-associated protein phosphatase (KAPP) was found in plants, the interest in plant FHA-containing proteins has increased dramatically, mainly due to the important role of FHA domain-containing proteins in plant growth and development. In this review, we provide a comprehensive overview of the fundamental properties of FHA domain-containing proteins in plants, and systematically summarized and analyzed the research progress of proteins containing the FHA domain in plants. We also emphasized that AT5G47790 and its homologs may play an important role as the regulatory subunit of protein phosphatase 1 (PP1) in plants.
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Fosfoproteínas Fosfatasas , Factores de Transcripción , Humanos , Estructura Terciaria de Proteína , Factores de Transcripción/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Saccharomyces cerevisiae/metabolismo , BiologíaRESUMEN
The objective of the research was to investigate action mechanism of oxidative stress and cerebral injuries after subarachnoid hemorrhage (SAH) by Ghrelin and angiogenic factor G-patch and FHA domain 1 (Aggf1) and offer new research ideas to SAH clinical treatment and SAH-induced early cerebral injuries. SAH rat models were prepared by prechiasmatic anterior cistern injection. Specific Ghrelin and Aggf1 small interfering ribonucleic acid (siRNA) were designed and injected into silence Ghrelin or Aggf1 in rat left lateral ventricles. Rats were divided randomly into sham-operated (sham), SAH model, negative control siRNA, Ghrelin silence (Ghrelin(-/-)), and Aggf1 silence groups. Changes of rat neurological impairment, encephaledema, cerebral tissue phosphorylated protein kinase (p-Akt), and content changes of caspase-3 protein and oxidative stress indexes were observed, including glutathione (GSH) and oxidized glutathione (GSSG). Results showed scores of neurological impairment and water content in SAH model group were reduced compared with sham group, while p-Akt protein and GSH contents were enhanced. However, caspase-3 protein and GSSG contents were declined, showing statistically meaningful difference (P < 0.05). Compared with SAH model group, scores of neurological impairment, cerebral tissue water content, and caspase-3 protein and GSSG contents in silence Ghrelin and Aggf1 groups were increased, while p-Akt protein and GSH contents were decreased, demonstrating statistically meaningful difference (P < 0.05). To conclude, silence Ghrelin and Aggf1 aggravated early cerebral injuries after SAH, revealing that Ghrelin and Aggf1 could protect brains to some degree.
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Hemorragia Subaracnoidea , Inductores de la Angiogénesis/uso terapéutico , Animales , Apoptosis , Caspasa 3/genética , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Ghrelina/genética , Ghrelina/farmacología , Ghrelina/uso terapéutico , Disulfuro de Glutatión , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Hemorragia Subaracnoidea/complicaciones , AguaRESUMEN
DNA repair scaffolds XRCC1 and XRCC4 utilize a phosphopeptide FHA domain binding motif (FBM) of the form Y-x-x-pS-pT-D-E that supports recruitment of three identified FHA domain-containing DNA repair proteins: polynucleotide kinase/phosphatase (PNKP), aprataxin (APTX), and a third protein, APLF, that functions as a scaffold in support of non-homologous end joining (NHEJ). Mammalian dimeric XRCC4 is able to interact with two of these proteins at any given time, while monomeric XRCC1 binds only one. However, sequence analysis indicates that amphibian and teleost XRCC1 generally contain two FHA binding motifs. X1-FBM1, is similar to the single mammalian XRCC1 FBM and probably functions similarly. X1-FBM2, is more similar to mammalian XRCC4 FBM; it is located closer to the XRCC1 BRCT1 domain and probably is less discriminating among its three likely binding partners. Availability of an additional PNKP or APTX recruitment motif may alleviate the bottleneck that results from using a single FBM motif for recruitment of multiple repair factors. Alternatively, recruitment of APLF by X1-FBM2 may function to rescue a misdirected or unsuccessful SSB repair response by redirecting the damaged DNA to the NHEJ pathway, - a need that results from the ambiguity of the PARP1 signal regarding the nature of the damage. Evaluation of XRCC4 FBMs in acanthomorphs, which account for a majority of the reported teleost sequences, reveals the presence of an additional XRCC4-like paralog, distinct from other previously described members of the XRCC4 superfamily. The FBM is typically absent in acanthomorph XRCC4, but present in the XRCC4-like paralog. Modeling suggests that XRCC4 and its paralog may form homodimers or XRCC4-XRCC4-like heterodimers.
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Reparación del ADN por Unión de Extremidades , Reparación del ADN , Animales , Enzimas Reparadoras del ADN/metabolismo , Mamíferos/metabolismo , Unión Proteica , Dominios Proteicos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
FIKK-9.1 is essential for parasite survival, but its structural and biochemical characterization will enable us to understand its role in the parasite life cycle. The recombinant FIKK9.1 kinase is monomeric with a native molecular weight of 60 ± 1.6 kDa. Structural characterization of FIKK9.1 kinase reveals that it consists of two domains: N-terminal FHA like domain and C-terminal kinase domain. The C-terminal domain has a well-defined pocket, but it displayed RMSD deviation of 1.38-3.2 Å from host kinases. ITC analysis indicates that ATP binds to the protein with a Kd of 45.6 ± 2.4 µM. Mutational studies confirm the role of Val-244, Met-245, Lys-320, 324, and Glu-366 for ATP binding. Co-localization studies revealed FIKK9.1 in the parasite cytosol with a component trafficked to the apicoplast and also to IRBC. FIKK9.1 has 23 pockets to serve as potential docking sites for substrates. Correlation analysis of peptides from the combinatorial library concluded that peptide P277 (MFDFHYTLGPMWGTL) was fitting nicely into the binding pocket. The peptide P277 picked up candidates from parasite and key players from RBC cytoskeleton. Interestingly, FIKK9.1 is phosphorylating spectrin, ankyrin, and band-3 from RBC cytoskeleton. Our study highlights the structural and biochemical features of FIKK9.1 to exploit it as a drug target.
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Plasmodium falciparum/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Secundaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Angiogenic factor with G-patch and FHA domains 1 (AGGF1) exhibits a dynamic distribution from the nucleus to the cytoplasm in endothelial cells during angiogenesis, but the biological significance and underlying mechanism of this nucleocytoplasmic transport remains unknown. Here, we demonstrate that the dynamic distribution is essential for AGGF1 to execute its angiogenic function. To search the structural bases for this nucleocytoplasmic transport, we characterized three potential nuclear localization regions, one potential nuclear export region, forkhead-associated (FHA), and G-patch domains to determine their effects on nucleocytoplasmic transport and angiogenesis, and we show that AGGF1 remains intact during the dynamic subcellular distribution and the region from 260 to 288 amino acids acts as a signal for its nuclear localization. The distribution of AGGF1 in cytoplasm needs both FHA domain and 14-3-3α/ß. Binding of AGGF1 via FHA domain to 14-3-3α/ß is required to complete the transport. Thus, we for the first time established structural bases for the nucleocytoplasmic transport of AGGF1 and revealed that the FHA domain of AGGF1 is essential for its nucleocytoplasmic transport and angiogenesis.
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Proteínas 14-3-3/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Angiogénicas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Neovascularización Fisiológica/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Dominios y Motivos de Interacción de ProteínasRESUMEN
Angiogenesis factors are widely known to promote tumor growth by increasing tumor angiogenesis in the tumor microenvironment, however, little is known whether their intracellular function is involved in tumorigenesis. Here we show that AGGF1 acts as a tumor suppressor by regulating p53 when acting inside tumor cells. AGGF1 antagonizes MDM2 function to inhibit p53 ubiquitination, increases the acetylation, phosphorylation, stability and expression levels of p53, activates transcription of p53 target genes, and regulates cell proliferation, cell cycle, and apoptosis. AGGF1 also interacts with p53 through the FHA domain. Somatic AGGF1 variants in the FHA domain in human tumors, including p.Q467H, p.Y469 N, and p.N483T, inhibit AGGF1 activity on tumor suppression. These results identify a key role for AGGF1 in an AGGF1-MDM2-p53 signaling axis with important functions in tumor suppression, and uncover a novel trans-tumor-suppression mechanism dependent on p53. This study has potential implications in diagnosis and therapies of cancer.
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Proteínas Angiogénicas/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Procesamiento Postranscripcional del ARN , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Angiogénicas/genética , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Pronóstico , Proteínas Proto-Oncogénicas c-mdm2/genética , Tasa de Supervivencia , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: To determine the role of angiogenic factor with G-patch and FHA domain 1 (AGGF1) in inflammatory response of human dental pulp cells (DPCs) and the underneath mechanism and to explore its role in angiogenesis. MATERIALS AND METHODS: The expression of AGGF-1 in human healthy and inflammatory pulp tissues was detected by immunohistochemistry. RT-qPCR and Western blot were used to evaluate the expression of AGGF1 in DPCs stimulated by lipopolysaccharide (LPS). After AGGF1 was knocked down, the expression of LPS-induced inflammatory cytokines in DPCs was quantified by RT-qPCR and ELISA. Immunofluorescence and Western blot were used to assess the activation of NF-κB signaling. Inflammatory cytokines were detected by RT-qPCR and ELISA in DPCs pretreated with NF-κB pathway inhibitors before LPS stimulation, and then the effect of AGGF1 on angiogenesis was also evaluated. RESULTS: AGGF1 expression increased in inflammatory dental pulp tissues. In DPCs stimulated by LPS, AGGF1 was upregulated in a dose-dependent manner (P < 0.05). In AGGF1 knockdown cells, the expression of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1/CCL-2) increased by LPS stimulation (P < 0.001). Nuclear translocation of p65 was promoted, and the addition of NF-κB inhibitors inhibited the expression of inflammatory factors. Meanwhile, knockdown of AGGF1 inhibited vascularization. CONCLUSIONS: AGGF1 inhibited the synthesis of inflammatory cytokines through NF-κB signaling pathway and promoted the angiogenesis of DPCs. CLINICAL RELEVANCE: This study might shed light in the treatment of pulpitis and regeneration of dental pulp tissues; however, more clinical trials are required to validate these findings.
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Pulpa Dental , Mediadores de Inflamación , Proteínas Angiogénicas , Pulpa Dental/metabolismo , Humanos , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Polynucleotide kinase phosphatase (PNKP) has dual enzymatic activities as kinase and phosphatase for DNA ends, which are the prerequisite for the ligation, and thus is involved in base excision repair, single-strand break repair and non-homologous end joining for double-strand break (DSB) repair. In this study, we examined mechanisms for the recruitment of PNKP to DNA damage sites by laser micro-irradiation and live-cell imaging analysis using confocal microscope. We show that the forkhead-associated (FHA) domain of PNKP is essential for the recruitment of PNKP to DNA damage sites. Arg35 and Arg48 within the FHA domain are required for interactions with XRCC1 and XRCC4. PNKP R35A/R48A mutant failed to accumulate on the laser track and siRNA-mediated depletion of XRCC1 and/or XRCC4 reduced PNKP accumulation on the laser track, indicating that PNKP is recruited to DNA damage sites via the interactions between its FHA domain and XRCC1 or XRCC4. Furthermore, cells expressing PNKP R35A/R48A mutant exhibited increased sensitivity toward ionizing radiation in association with delayed SSB and DSB repair and genome instability, represented by micronuclei and chromosome bridges. Taken together, these findings revealed the importance of PNKP recruitment to DNA damage sites via its FHA domain for DNA repair and maintenance of genome stability.
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Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/metabolismo , Inestabilidad Genómica , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Sustitución de Aminoácidos , Arginina , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HCT116 , Células HEK293 , Humanos , Mutación Missense , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Dominios Proteicos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.
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Transportadoras de Casetes de Unión a ATP/genética , Proteínas de la Membrana/genética , Mycobacterium tuberculosis/genética , Tuberculosis/genética , Transportadoras de Casetes de Unión a ATP/química , Citosol/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/ultraestructura , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/ultraestructura , Proteínas Nucleares/genética , Fosforilación/genética , Transducción de Señal/genética , Imagen Individual de Molécula , Tuberculosis/microbiologíaRESUMEN
Pellino1 is an E3 ubiquitin ligase that plays a key role in positive regulation of innate immunity signaling, specifically required for the production of interferon when induced by viral double-stranded RNA. We report the identification of the tumor suppressor protein, p53, as a binding partner of Pellino1. Their interaction has a Kd of 42⯱â¯2⯵M and requires phosphorylation of Thr18 within p53 and association with the forkhead-associated (FHA) domain of Pellino1. We employed laser micro-irradiation and live cell microscopy to show that Pellino1 is recruited to newly occurring DNA damage sites, via its FHA domain. Mutation of a hitherto unidentified nuclear localization signal within the N-terminus of Pellino1 led to its exclusion from the nucleus. This study provides evidence that Pellino1 translocates to damaged DNA in the nucleus and has a functional role in p53 signaling and the DNA damage response.
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Daño del ADN , Proteínas Nucleares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Nucleares/análisis , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína p53 Supresora de Tumor/análisis , Ubiquitina-Proteína Ligasas/análisisRESUMEN
Kawasaki disease (KD) is a self-limiting systemic vasculitis of unknown etiology. KD is often complicated by coronary artery aneurysms (CAAs), which develop in about 20-25% of untreated children and 3-5% of children treated with intravenous immunoglobulin therapy. To identify the risk loci for CAA susceptibility in patients with KD, we performed a genome-wide association study (GWAS) using our previous Illumina HumanOmni1-Quad BeadChip data (296 KD patients) and a new replication study in an independent sample set (713 KD patients) by grouping KD patients without CAA (control) versus KD patients with extremely large aneurysms (diameter ≥ 5 mm) (case). Among 44 candidate single -nucleotide polymorphisms (SNPs) selected from the initial GWAS data (33 cases vs. 215 controls), a SNP (rs899162) located 7 kb upstream of the TIFAB gene on chromosome five was replicated in an independent sample (12 cases vs. 532 controls). In the combined analysis (45 cases vs. 747 controls), the SNP (rs899162) showed a highly significant association with CAA formation (diameter ≥ 5 mm) in patients with KD (odds ratio = 3.20, 95% confidence interval = 2.02-5.05, Pcombined = 1.95 × 10-7). These results indicate that the TIFAB gene may act as a CAA susceptibility locus in patients with KD.
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Aneurisma Coronario/genética , Síndrome Mucocutáneo Linfonodular/complicaciones , Factor 6 Asociado a Receptor de TNF/genética , Estudios de Casos y Controles , Aneurisma Coronario/etiología , Vasos Coronarios/patología , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Síndrome Mucocutáneo Linfonodular/genética , Polimorfismo de Nucleótido SimpleRESUMEN
The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide. A phage-display library of variants of the Forkhead-associated 1 (FHA1) domain of yeast Rad53p was screened by affinity selection to the phosphopeptide, 301-KDGATMKpTFCGTPEY-315, and yielded 12 binding clones. The strongest binders have equilibrium dissociation constants of 160â»180 nanomolar and are phosphothreonine-specific in binding. The specificity of one Akt1-pTBD was compared to commercially available polyclonal antibodies (pAbs) generated against the same phosphopeptide. The Akt1-pTBD was either equal to or better than three pAbs in detecting the Akt1 pT308 phosphopeptide in ELISAs.
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Epítopos/inmunología , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anticuerpos/inmunología , Sitios de Unión , Proteínas de Ciclo Celular/química , Quinasa de Punto de Control 2/química , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The FHA domain-containing protein Mek1 is a meiosis-specific kinase that is involved in the regulation of interhomolog recombination in meiosis in Saccharomyces cerevisiae. The recruitment and activation of Mek1 require the phosphorylation of the chromosome axis protein Hop1 at Thr318 (pT318), which is necessary for recognition by the Mek1 FHA domain. Here, crystal structures of the Mek1 FHA domain in the apo state and in complex with the Hop1 pT318 peptide are presented, demonstrating that the hydrophobic residues Phe320 and Val321 at the pT+2 and pT+3 positions in the ligand contribute to the preferential recognition. It was further found that in Schizosaccharomyces pombe Mek1 FHA binds both pT15 in its N-terminal SQ/TQ cluster domain (SCD) and pT270 in the Hop1 SCD. The results revealed the structural basis for the preferential recognition of phosphorylated Hop1 by Mek1 in S. cerevisiae and facilitate the understanding of the interaction between the S. pombe Mek1 FHA domain and its binding targets.
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Proteínas de Unión al ADN/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/química , MAP Quinasa Quinasa 1/química , Meiosis , Fosforilación , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The ubiquitously expressed nuclear protein NIPP1 (also known as PPP1R8) recruits phosphoproteins for regulated dephosphorylation by the associated protein phosphatase PP1. To bypass the PP1 titration artifacts seen upon NIPP1 overexpression, we have engineered covalently linked fusions of PP1 and NIPP1, and demonstrate their potential to selectively explore the function of the PP1:NIPP1 holoenzyme. By using inducible stable cell lines, we show that PP1-NIPP1 fusions cause replication stress in a manner that requires both PP1 activity and substrate recruitment via the ForkHead Associated domain of NIPP1. More specifically, PP1-NIPP1 expression resulted in the build up of RNA-DNA hybrids (R-loops), enhanced chromatin compaction and a diminished repair of DNA double-strand breaks (DSBs), culminating in the accumulation of DSBs. These effects were associated with a reduced expression of DNA damage signaling and repair proteins. Our data disclose a key role for dephosphorylation of PP1:NIPP1 substrates in setting the threshold for DNA repair, and indicate that activators of this phosphatase hold therapeutic potential as sensitizers for DNA-damaging agents.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Endorribonucleasas/genética , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1/genética , Proteínas de Unión al ARN/genética , Cromatina/genética , Cromatina/metabolismo , Dimerización , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Expresión Génica , Células HEK293 , Humanos , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/metabolismo , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.