RESUMO
The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.
Assuntos
Proteína Centromérica A , Centrômero , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/química , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Ligação Proteica , Ciclo Celular/genética , Modelos Moleculares , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleossomos/metabolismo , Nucleossomos/química , Proteínas Adaptadoras de Transdução de SinalRESUMO
Mammalian FNDC5 encodes a protein precursor of Irisin, which is important for exercise-dependent regulation of whole-body metabolism. In a genetic screen in Drosophila, we identified Iditarod (Idit), which shows substantial protein homology to mouse and human FNDC5, as a regulator of autophagy acting downstream of Atg1/Atg13. Physiologically, Idit-deficient flies showed reduced exercise performance and defective cold resistance, which were rescued by exogenous expression of Idit. Exercise training increased endurance in wild-type flies, but not in Idit-deficient flies. Conversely, Idit is induced upon exercise training, and transgenic expression of Idit in wild-type flies increased endurance to the level of exercise trained flies. Finally, Idit deficiency prevented both exercise-induced increase in cardiac Atg8 and exercise-induced cardiac stress resistance, suggesting that cardiac autophagy may be an additional mechanism by which Idit is involved in the adaptive response to exercise. Our work suggests an ancient role of an Iditarod/Irisin/FNDC5 family of proteins in autophagy, exercise physiology, and cold adaptation, conserved throughout metazoan species.
Assuntos
Proteínas de Drosophila , Fibronectinas , Animais , Humanos , Camundongos , Animais Geneticamente Modificados , Autofagia , Drosophila , Fibronectinas/metabolismo , Mamíferos , Fatores de Transcrição , Proteínas de Drosophila/metabolismoRESUMO
Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity.
Assuntos
Histona-Lisina N-Metiltransferase , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Nucleossomos/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Histonas/química , Histonas/genética , Microscopia Crioeletrônica/métodosRESUMO
Atmospheric-pressure plasmas have been widely applied for surface modification and biomedical treatment because of their ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used to generate hydrophilic surfaces and eliminate surface contaminants to embed specimens onto grids. In addition, plasma treatment is a prerequisite for negative-stain and Quantifoil grids, whose surfaces are coated with hydrophobic amorphous carbon. Although the conventional glow discharge system has been used successfully in this purpose, there has been no further effort to take an advantage from the recent progress in the plasma field. Here, we developed a nonthermal atmospheric plasma jet system as an alternative tool for treatment of surfaces. The low-temperature plasma is a nonequilibrium system that has been widely used in biomedical area. Unlike conventional glow discharge systems, the plasma jet system successfully cleans and introduces hydrophilicity on the grid surface in the ambient environment without a vacuum. Therefore, we anticipate that the plasma jet system will have numerous benefits, such as convenience and versatility, as well as having potential applications in surface modification for both negative-stain and cryo-EM grid treatment.
Assuntos
Microscopia Crioeletrônica , Temperatura Baixa , Microscopia Crioeletrônica/instrumentação , VácuoRESUMO
Cryo-EM structures of the KMT2A/MLL1 core complex bound on nucleosome core particles (NCPs) suggest unusual rotational dynamics of the MLL1 complex approaching its physiological substrate. However, the functional implication of such dynamics remains unclear. Here, we show that the MLL1 core complex also shows high rotational dynamics bound on the NCP carrying the catalytically inert histone H3 lysine 4 to methionine (K4M) mutation. There are two major binding modes of the MLL1 complex on the NCPK4M. Importantly, disruption of only one of the binding modes compromised the overall MLL1 activity in an NCP-specific manner. We propose that the MLL1 core complex probably exists in an equilibrium of poised and active binding modes. The high rotational dynamics of the MLL1 complex on the NCP is a feature that can be exploited for loci-specific regulation of H3K4 methylation in higher eukaryotes.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nucleossomos/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/ultraestrutura , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/ultraestrutura , Ligação Proteica , Conformação ProteicaRESUMO
Excessive accumulation of reactive oxygen species (ROS) and chronic activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are well-characterized promoters of aging and age-associated degenerative pathologies. Sestrins, a family of highly conserved stress-inducible proteins, are important negative regulators of both ROS and mTORC1 signaling pathways; however, the mechanistic basis of how Sestrins suppress these pathways remains elusive. In the past couple of years, breakthrough discoveries about Sestrin signaling and its molecular nature have markedly increased our biochemical understanding of Sestrin function. These discoveries have also uncovered new potential therapeutic strategies that may eventually enable us to attenuate aging and age-associated diseases.
Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas Nucleares/metabolismo , Fenômenos Bioquímicos , Proteínas de Choque Térmico/química , Humanos , Proteínas Nucleares/químicaRESUMO
Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 µm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine ß-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.
Assuntos
Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Alcinos/metabolismo , Animais , Linhagem Celular , Cistationina gama-Liase/fisiologia , Cisteína/farmacologia , Glicina/análogos & derivados , Glicina/metabolismo , Humanos , Sulfeto de Hidrogênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfetos/farmacologiaRESUMO
The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle. CENP-C (centromere protein C), a conserved inner kinetochore component, has been broadly characterized as a scaffolding protein and is required for the recruitment of multiple kinetochore proteins to the centromere. At its C terminus, CENP-C harbors a conserved cupin domain that has an established role in protein dimerization. Although the crystal structure of the Saccharomyces cerevisiae Mif2CENP-C cupin domain has been determined, centromeric organization and kinetochore composition vary greatly between S. cerevisiae (point centromere) and other eukaryotes (regional centromere). Therefore, whether the structural and functional role of the cupin domain is conserved throughout evolution requires investigation. Here, we report the crystal structures of the Schizosaccharomyces pombe and Drosophila melanogaster CENP-C cupin domains at 2.52 and 1.81 Å resolutions, respectively. Although the central jelly roll architecture is conserved among the three determined CENP-C cupin domain structures, the cupin domains from organisms with regional centromeres contain additional structural features that aid in dimerization. Moreover, we found that the S. pombe Cnp3CENP-C jelly roll fold harbors an inner binding pocket that is used to recruit the meiosis-specific protein Moa1. In summary, our results unveil the evolutionarily conserved and unique features of the CENP-C cupin domain and uncover the mechanism by which it functions as a recruitment factor.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/ultraestrutura , Animais , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Cristalografia por Raios X/métodos , Proteínas de Ligação a DNA/metabolismo , Dimerização , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in sulfide clearance, coupling H2S oxidation to coenzyme Q reduction. Recent structures of human SQOR revealed a sulfur atom bridging the SQOR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic, and computational modeling approaches. Cyanolysis of SQOR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. We captured a disulfanyl-methanimido thioate intermediate in the SQOR crystal structure, revealing how cyanolysis leads to reversible loss of SQOR activity that is restored in the presence of sulfide. Computational modeling and MD simulations revealed an â¼105-fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQOR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Sulfetos/metabolismo , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Sulfetos/químicaRESUMO
Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH-MMOB (hereafter termed H-B) complex. Here we remedy this deficiency by providing a crystal structure of H-B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α(2)ß(2) interface of α(2)ß(2)γ(2) MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.
Assuntos
Methylococcus capsulatus/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Ferro/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Oxirredutases/química , Oxirredutases/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal.
Assuntos
Hemoglobinas/química , Hemoglobinas/metabolismo , Sulfeto de Hidrogênio/metabolismo , Sulfetos/metabolismo , Catálise , Cristalografia por Raios X , Humanos , Conformação Molecular , Oxirredução , Conformação Proteica , Sulfetos/química , Tiossulfatos/metabolismoRESUMO
Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to dephosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB'C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled coil that docks simultaneously onto PP2A's C and B' subunits. Sgo1 homodimerization is a prerequisite for PP2A binding. While hSgo1 interacts only with the AB'C holoenzymes, its relative, Sgo2, interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, we provide evidence that PP2A's recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/enzimologia , Meiose/fisiologia , Proteína Fosfatase 2/metabolismo , Fuso Acromático/enzimologia , Animais , Sítios de Ligação , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Endopeptidases/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutação , Proteínas Nucleares/metabolismo , Oócitos/enzimologia , Conformação Proteica , Multimerização Proteica , Proteína Fosfatase 2/química , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Separase , CoesinasRESUMO
A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4 : H4)(2) heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.
Assuntos
Centrômero/metabolismo , Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Kluyveromyces/metabolismo , Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalização , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Histonas/química , Histonas/genética , Kluyveromyces/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Protein phosphatase 2A (PP2A) is a principal Ser/Thr phosphatase, the deregulation of which is associated with multiple human cancers, Alzheimer's disease and increased susceptibility to pathogen infections. How PP2A is structurally organized and functionally regulated remains unclear. Here we report the crystal structure of an AB'C heterotrimeric PP2A holoenzyme. The structure reveals that the HEAT repeats of the scaffold A subunit form a horseshoe-shaped fold, holding the catalytic C and regulatory B' subunits together on the same side. The regulatory B' subunit forms pseudo-HEAT repeats and interacts with the C subunit near the active site, thereby defining substrate specificity. The methylated carboxy-terminal tail of the C subunit interacts with a highly negatively charged region at the interface between A and B' subunits, suggesting that the C-terminal carboxyl methylation of the C subunit promotes B' subunit recruitment by neutralizing charge repulsion. Together, our structural results establish a crucial foundation for understanding PP2A assembly, substrate recruitment and regulation.
Assuntos
Fosfoproteínas Fosfatases/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Metilação , Modelos Moleculares , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2 , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por SubstratoRESUMO
Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air-water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air-water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.
Assuntos
Grafite , Methylococcus capsulatus , Grafite/química , Microscopia Crioeletrônica/métodos , Água , ProteínasRESUMO
How cell-type-specific chromatin landscapes emerge and progress during metazoan ontogenesis remains an important question. Transcription factors are expressed in a cell-type-specific manner and recruit chromatin-regulatory machinery to specific genomic loci. In contrast, chromatin-regulatory proteins are expressed broadly and are assumed to exert the same intrinsic function across cell types. However, human genetics studies have revealed an unexpected vulnerability of neurodevelopment to chromatin factor mutations with unknown mechanisms. Here, we report that 14 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Of the 14 chromatin regulators, two are integral components of a histone H3K4 demethylase complex; the catalytic subunit LSD1 and an H3K4me0-reader protein PHF21A adopt neuron-specific forms. We found that canonical PHF21A (PHF21A-c) binds to DNA by AT-hook motif, and the neuronal counterpart PHF21A-n lacks this DNA-binding function yet maintains H3K4me0 recognition intact. In-vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes identified the neuronal complex as a hypomorphic H3K4 demethylating machinery with reduced nucleosome engagement. Furthermore, an autism-associated PHF21A missense mutation, 1285 G>A, at the last nucleotide of the common exon immediately upstream of the neuronal microexon led to impaired splicing of PHF21A -n. Thus, ubiquitous chromatin regulatory complexes exert unique intrinsic functions in neurons via alternative splicing of their subunits and potentially contribute to faithful human brain development.
RESUMO
Epigenetic dysregulation is a prominent feature in cancer, as exemplified by frequent mutations in chromatin regulators, including the MLL/KMT2 family of histone methyltransferases. Although MLL1/KMT2A activity on H3K4 methylation is well documented, their non-canonical activities remain mostly unexplored. Here we show that MLL1/KMT2A methylates Borealin K143 in the intrinsically disordered region essential for liquid-liquid phase separation of the chromosome passenger complex (CPC). The co-crystal structure highlights the distinct binding mode of the MLL1 SET domain with Borealin K143. Inhibiting MLL1 activity or mutating Borealin K143 to arginine perturbs CPC phase separation, reduces Aurora kinase B activity, and impairs the resolution of erroneous kinetochore-microtubule attachments and sister-chromatid cohesion. They significantly increase chromosome instability and aneuploidy in a subset of hepatocellular carcinoma, resulting in growth inhibition. These results demonstrate a non-redundant function of MLL1 in regulating inner centromere liquid condensates and genome stability via a non-canonical enzymatic activity.
Assuntos
Proteínas Cromossômicas não Histona , Mitose , Humanos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Centrômero/genética , Centrômero/metabolismo , Proteínas de Ciclo Celular/genética , Instabilidade Genômica , Aurora Quinase B/genética , Aurora Quinase B/metabolismoRESUMO
Recent cryo-EM structures show the highly dynamic nature of the MLL1-NCP (nucleosome core particle) interaction. Functional implication and regulation of such dynamics remain unclear. Here we show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together in restricting the rotational dynamics of the MLL1 complex on the NCP. We show that DPY30 binding to ASH2L leads to stabilization and integration of ASH2L IDRs into the MLL1 complex and establishes new ASH2L-NCP contacts. The significance of ASH2L-DPY30 interactions is demonstrated by requirement of both ASH2L IDRs and DPY30 for dramatic increase of processivity and activity of the MLL1 complex. This DPY30 and ASH2L-IDR dependent regulation is NCP-specific and applies to all members of the MLL/SET1 family of enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in ESCs. Our study provides a paradigm of how H3K4me3 is regulated on chromatin and how H3K4me3 heterogeneity can be modulated by ASH2L IDR interacting proteins.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Técnicas In Vitro , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Espalhamento a Baixo Ângulo , Fatores de Transcrição/genética , Difração de Raios XRESUMO
The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 A resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3-7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST.
Assuntos
Antígenos Virais de Tumores/metabolismo , Conformação Proteica , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/química , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Vírus 40 dos Símios/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais de Tumores/química , Antígenos Virais de Tumores/genética , Sítios de Ligação , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/metabolismo , Alinhamento de SequênciaRESUMO
In eukaryotes, histone H3K4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The level of H3K4 methylation is highly correlated with transcription activation and is one of the most frequently used histone post-translational modifications to predict transcriptional outcome. Recently, it has been shown that rearrangement of the cellular landscape of H3K4 mono-methylation at distal enhancers precedes cell fate transition and is used for identification of novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions have also been used to predict intrinsic tumor suppression properties of regulator regions in a variety of cellular models. Understanding the regulation for how H3K4 methylation is deposited and regulated is of paramount importance. In this review, we will discuss new findings on how the MLL/SET1 family enzymes are regulated on chromatin and their potential functional and regulatory implications. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.