RESUMEN
The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4)2 tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4)2 tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA.
Asunto(s)
Proteínas de Ciclo Celular , Microscopía por Crioelectrón , Chaperonas de Histonas , Histonas , Unión Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Histonas/química , Histonas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Multimerización de Proteína , Sitios de Unión , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Dominios y Motivos de Interacción de ProteínasRESUMEN
The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.
Asunto(s)
Inflamasomas , Sistemas de Secreción Tipo III , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Flagelina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Adaptadoras de Señalización CARD , Proteína Inhibidora de la Apoptosis Neuronal/metabolismoRESUMEN
The heme-regulated inhibitor (HRI) is a heme-sensing kinase that regulates mRNA translation in erythroid cells. In heme deficiency, HRI is activated to phosphorylate eukaryotic initiation factor 2α and halt production of globins, thus avoiding accumulation of heme-free globin chains. HRI is inhibited by heme via binding to one or two heme-binding domains within the HRI N-terminal and kinase domains. HRI has recently been found to inhibit fetal hemoglobin (HbF) production in adult erythroid cells. Depletion of HRI increases HbF production, presenting a therapeutically exploitable target for the treatment of patients with sickle cell disease or thalassemia, which benefit from elevated HbF levels. HRI is known to be an oligomeric enzyme that is activated through autophosphorylation, although the exact nature of the HRI oligomer, its relation to autophosphorylation, and its mode of heme regulation remain unclear. Here, we employ biochemical and biophysical studies to demonstrate that HRI forms a dimeric species that is not dependent on autophosphorylation, the C-terminal coiled-coil domain in HRI is essential for dimer formation, and dimer formation facilitates efficient autophosphorylation and activation of HRI. We also employ kinetic studies to demonstrate that the primary avenue by which heme inhibits HRI is through the heme-binding site within the kinase domain, and that this inhibition is relatively independent of binding of ATP and eukaryotic initiation factor 2α substrates. Together, these studies highlight the mode of heme inhibition and the importance of dimerization in human HRI heme-sensing activity.
Asunto(s)
Hemo , eIF-2 Quinasa , Humanos , Dimerización , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Hemo/metabolismo , Cinética , Fosforilación , Unión ProteicaRESUMEN
Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for new Rap1 targets at senescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392-394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1SHY ), and thus disrupts Rap1-H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1-H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1-mediated nucleosome displacement and gene activation. Remarkably, RAP1SHY does not affect the pace of senescence-related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.
Asunto(s)
Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genética , Regulación Fúngica de la Expresión Génica , Nucleosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The HIRA histone chaperone complex is composed of the proteins HIRA, UBN1, and CABIN1 and cooperates with the histone chaperone ASF1a to specifically bind and deposit H3.3/H4 into chromatin. We recently reported that the UBN1 Hpc2-related domain (HRD) specifically binds to H3.3/H4 over H3.1/H4. However, the mechanism for HIRA complex deposition of H3.3/H4 into nucleosomes remains unclear. Here, we characterize a central region of UBN1 (UBN1 middle domain) that is evolutionarily conserved and predicted to have helical secondary structure. We report that the UBN1 middle domain has dimer formation activity and binds to H3/H4 in a manner that does not discriminate between H3.1 and H3.3. We additionally identify a nearby DNA-binding domain in UBN1, located between the UBN1 HRD and middle domain, which binds DNA through electrostatic contacts involving several conserved lysine residues. Together, these observations suggest a mechanism for HIRA-mediated H3.3/H4 deposition whereby UBN1 associates with DNA and dimerizes to mediate formation of an (H3.3/H4)2 heterotetramer prior to chromatin deposition.
Asunto(s)
ADN/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Cromatina/metabolismo , Dimerización , Histonas/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Alineación de Secuencia , Electricidad Estática , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Epigenetic regulation of the chromatin landscape is often orchestrated through modulation of nucleosomes. Nucleosomes are composed of two copies each of the four core histones, H2A, H2B, H3, and H4, wrapped in ~150 bp of DNA. We focus this review on recent structural studies that further elucidate the mechanisms used by macromolecular complexes to mediate histone modification and nucleosome assembly. Nucleosome assembly, spacing, and variant histone incorporation are coordinated by chromatin remodeler and histone chaperone complexes. Several recent structural studies highlight how disparate families of histone chaperones and chromatin remodelers share similar features that underlie how they interact with their respective histone or nucleosome substrates. Post-translational modification of histone residues is mediated by enzymatic subunits within large complexes. Until recently, relatively little was known about how association with auxiliary subunits serves to modulate the activity and specificity of the enzymatic subunit. Analysis of several recent structures highlights the different modes that auxiliary subunits use to influence enzymatic activity or direct specificity toward individual histone residues.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Animales , ADN/metabolismo , HumanosRESUMEN
The HIRA histone chaperone complex deposits the histone variant H3.3 onto chromatin in a DNA synthesis-independent manner. It comprises three identified subunits, HIRA, UBN1 and CABIN1, however the functional oligomerization state of the complex has not been investigated. Here we use biochemical and crystallographic analysis to show that the HIRA subunit forms a stable homotrimer that binds two subunits of CABIN1 in vitro. A HIRA mutant that is defective in homotrimer formation interacts less efficiently with CABIN1, is not enriched at DNA damage sites upon UV irradiation and cannot rescue new H3.3 deposition in HIRA knockout cells. The structural homology with the homotrimeric replisome component Ctf4/AND-1 enables the drawing of parallels and discussion of the functional importance of the homotrimerization state of the HIRA subunit.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Ciclo Celular/química , ADN/química , Chaperonas de Histonas/química , Histonas/química , Chaperonas Moleculares/química , Proteínas Nucleares/química , Factores de Transcripción/química , Sitios de Unión , Línea Celular Tumoral , Cromatina/química , Cristalografía por Rayos X , Daño del ADN , Bases de Datos de Proteínas , Proteínas Fluorescentes Verdes/química , Células HeLa , Humanos , Plásmidos , Unión Proteica , Conformación Proteica , Rayos UltravioletaRESUMEN
The human enzyme histone acetyltransferase binding to ORC1 (HBO1) regulates DNA replication, cell proliferation, and development. HBO1 is part of a multiprotein histone acetyltransferase (HAT) complex that also contains inhibitor of growth family member (ING) 4/5, MYST/Esa1-associated factor (MEAF) 6, and the scaffolding proteins Jade family PHD finger (JADE) 1/2/3 or bromodomain and PHD finger-containing protein (BRPF) 2/3 to acetylate histone H4 H4K5/8/12 or H3K14, respectively. Within this four-protein complex, JADE1 determines histone H4 substrate specificity of the HBO1-HAT complex. However, the mechanism by which JADE1 controls the H4-specific acetyltransferase activity of HBO1 is unknown. Here we used recombinant proteins in vitro to dissect the specific regions and activities of HBO1 and JADE1 that mediate histone H3-H4 acetylation via the HBO1-HAT domain. We found that JADE1 increases the catalytic efficiency of HBO1 acetylation of an H3-H4 substrate by about 5-fold through an N-terminal, 21-residue HBO1- and histone-binding domain and a nearby second histone core-binding domain. We also demonstrate that HBO1 contains an N-terminal histone-binding domain (HBD) that makes additional contacts with H3-H4 independent of JADE1 interactions with histones and that the HBO1 HBD does not significantly contribute to HBO1's overall HAT activity. Experiments with JADE1 deletions in vivo recapitulated these in vitro interactions and their roles in HBO1 histone acetylation activity. Together, these results indicate that the N-terminal region of JADE1 functions as a platform that brings together the catalytic HBO1 subunit with its cognate H3-H4 substrate for histone acetylation.
Asunto(s)
Cromatina/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Acetilación , Secuencia de Aminoácidos , Cromatina/genética , Replicación del ADN , Células HEK293 , Histona Acetiltransferasas/genética , Histonas/genética , Proteínas de Homeodominio/genética , Humanos , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Homología de Secuencia , Especificidad por Sustrato , Proteínas Supresoras de Tumor/genéticaRESUMEN
Incorporation of variant histone sequences, in addition to post-translational modification of histones, serves to modulate the chromatin environment. Different histone chaperone proteins mediate the storage and chromatin deposition of variant histones. Although the two non-centromeric histone H3 variants, H3.1 and H3.3, differ by only 5 aa, replacement of histone H3.1 with H3.3 can modulate the transcription for highly expressed and developmentally required genes, lead to the formation of repressive heterochromatin, or aid in DNA and chromatin repair. The human histone cell cycle regulator (HIRA) complex composed of HIRA, ubinuclein-1, CABIN1, and transiently anti-silencing function 1, forms one of the two complexes that bind and deposit H3.3/H4 into chromatin. A number of recent biochemical and structural studies have revealed important details underlying how these proteins assemble and function together as a multiprotein H3.3-specific histone chaperone complex. Here, we present a review of existing data and present a new model for the assembly of the HIRA complex and for the HIRA-mediated incorporation of H3.3/H4 into chromatin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Factores de Transcripción/metabolismo , Cromatina/metabolismo , Reparación del ADN , Regulación de la Expresión Génica , Humanos , Chaperonas Moleculares , Transcripción GenéticaRESUMEN
The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.
Asunto(s)
Proteínas de Ciclo Celular/química , Histona Acetiltransferasas/química , Histonas/química , Chaperonas Moleculares/química , Complejos Multienzimáticos/química , Acetilación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , MutaciónRESUMEN
Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.
Asunto(s)
Autofagia , Lámina Nuclear/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Familia de las Proteínas 8 Relacionadas con la Autofagia , Transformación Celular Neoplásica , Células Cultivadas , Senescencia Celular , Cromatina/química , Cromatina/metabolismo , Citoplasma/metabolismo , Fibroblastos , Células HEK293 , Humanos , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Lisosomas/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Unión Proteica , ProteolisisRESUMEN
Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1-H3.3-binding specificity. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex.
Asunto(s)
Histonas/metabolismo , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Calorimetría , Proteínas de Ciclo Celular/metabolismo , Cromatina , Proteínas Co-Represoras , Cristalización , Cristalografía por Rayos X , Chaperonas de Histonas/metabolismo , Humanos , Chaperonas Moleculares , Unión Proteica , Proteínas Recombinantes , Células Sf9 , Spodoptera , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
The mammalian HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex deposits the histone H3 variant H3.3 into chromatin and is linked to gene activation, repression, and chromatin assembly in diverse cell contexts. We recently reported that a short N-terminal fragment of UBN1 containing amino acids 1-175 is necessary and sufficient for interaction with the WD repeats of HIRA and attributed this interaction to a region from residues 120-175 that is highly conserved with the yeast ortholog Hpc2 and so termed the HRD for Hpc2-related domain. In this report, through a more comprehensive and refined biochemical and mutational analysis, we identify a smaller and more moderately conserved region within residues 41-77 of UBN1, which we term the NHRD, that is essential for interaction with the HIRA WD repeats; we further demonstrate that the HRD is dispensable for this interaction. We employ analytical ultracentrifugation studies to demonstrate that the NHRD of UBN1 and the WD repeats of HIRA form a tight 1:1 complex with a dissociation constant in the nanomolar range. Mutagenesis experiments identify several key residues in the NHRD that are required for interaction with the HIRA WD repeat domain, stability of the HUCA complex in vitro and in vivo, and changes in chromatin organization in primary human cells. Together, these studies implicate the NHRD domain of UBN1 as being an essential region for HIRA interaction and chromatin organization by the HUCA complex.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Cromatina/metabolismo , Chaperonas de Histonas/química , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Mutación Puntual , Estabilidad Proteica , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Solubilidad , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino ß-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 °C, an observed half-life of 5 min at 30 °C, and a "T-50" value, the temperature at which the half-life is 30 min, of 27 °C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a "T-50" value of 34 °C (7 °C improvement) and 1.3-fold activity compared to wild-type.