RESUMEN
Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Animales , Dominio Catalítico , Cromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/ultraestructura , Histonas/química , Histonas/genética , Humanos , Metilación , Modelos Moleculares , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Receptor Cross-Talk , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinación , Xenopus laevisRESUMEN
Sirtuins are NAD(+)-dependent enzymes universally present in all organisms, where they play central roles in regulating numerous biological processes. Although early studies showed that sirtuins deacetylated lysines in a reaction that consumes NAD(+), more recent studies have revealed that these enzymes can remove a variety of acyl-lysine modifications. The specificities for varied acyl modifications may thus underlie the distinct roles of the different sirtuins within a given organism. This review summarizes the structure, chemistry, and substrate specificity of sirtuins with a focus on how different sirtuins recognize distinct substrates and thus carry out specific functions.
Asunto(s)
Histonas/química , NAD/química , Procesamiento Proteico-Postraduccional , Sirtuinas/química , Acilación , Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , Hidrólisis , Cinética , Lipoilación , Modelos Moleculares , Ácido Mirístico/química , Ácido Mirístico/metabolismo , NAD/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Estructura Secundaria de Proteína , Sirtuinas/genética , Sirtuinas/metabolismo , Especificidad por Sustrato , Ácido Succínico/química , Ácido Succínico/metabolismo , Thermotoga maritima/química , Thermotoga maritima/enzimologíaRESUMEN
Linear ubiquitin (Ub) plays a role in nuclear factor κB signaling, but the deubiquitinating enzyme that disassembles these chains was unknown. In this issue of Cell, Keusekotten et al. identify a new enzyme that disassembles linear chains with the use of a mechanism that relies on Ub itself to help catalyze peptide bond cleavage.
Asunto(s)
Endopeptidasas/química , Endopeptidasas/metabolismo , Animales , HumanosRESUMEN
Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions1. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop 'loop-seq'-a high-throughput assay to measure the propensity for DNA looping-and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors2. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a 'mechanical code' with broad functional implications.
Asunto(s)
Fenómenos Biomecánicos , ADN de Hongos/química , ADN de Hongos/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Ensamble y Desensamble de Cromatina , Codón/genética , ADN de Hongos/metabolismo , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Docilidad , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.
Asunto(s)
Microscopía por Crioelectrón , Histonas , Simulación de Dinámica Molecular , Nucleosomas , Ubiquitina , Ubiquitinación , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Nucleosomas/química , Histonas/metabolismo , Histonas/química , Ubiquitina/metabolismo , Ubiquitina/química , Ubiquitina/genética , Humanos , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
The HECT E3 ligases ubiquitinate numerous transcription factors and signaling molecules, and their activity must be tightly controlled to prevent cancer, immune disorders, and other diseases. In this study, we have found unexpectedly that peptide linkers tethering WW domains in several HECT family members are key regulatory elements of their catalytic activities. Biochemical, structural, and cellular analyses have revealed that the linkers can lock the HECT domain in an inactive conformation and block the proposed allosteric ubiquitin binding site. Such linker-mediated autoinhibition of the HECT domain can be relieved by linker post-translational modifications, but complete removal of the brake can induce hyperactive autoubiquitination and E3 self destruction. These results clarify the mechanisms of several HECT protein cancer associated mutations and provide a new framework for understanding how HECT ubiquitin ligases must be finely tuned to ensure normal cellular behavior.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regulación Alostérica , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Activación Enzimática , Estabilidad de Enzimas , Células HeLa , Humanos , Modelos Moleculares , Mutación , Ubiquitina-Proteína Ligasas Nedd4 , Fosforilación , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Represoras/química , Proteínas Represoras/genética , Relación Estructura-Actividad , Transfección , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Péptidos y Proteínas de Señalización Intracelular , Proteína de la Leucemia Mieloide-Linfoide , Nucleosomas , Ubiquitinación , Microscopía por Crioelectrón , N-Metiltransferasa de Histona-Lisina/química , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Nucleosomas/enzimología , Unión ProteicaRESUMEN
Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Reparación del ADN , Proteínas Nucleares , Poliubiquitina , Ubiquitina , Ubiquitinas , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , ADN/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Unión Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Asunto(s)
Enzimas AlkB/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN , Complejos Multiproteicos/metabolismo , Transducción de Señal , Síndromes de Tricotiodistrofia/genética , Ubiquitina/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Alquilantes/farmacología , Alquilación , Secuencia de Aminoácidos , Aductos de ADN/química , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Genes Ligados a X , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , ARN Polimerasa II/metabolismo , Empalme del ARN , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patología , UbiquitinaciónRESUMEN
The past 4 decades have seen remarkable advances in our understanding of the structural basis of gene regulation. Technological advances in protein expression, nucleic acid synthesis, and structural biology made it possible to study the proteins that regulate transcription in the context of ever larger complexes containing proteins bound to DNA. This review, written on the occasion of the 50th anniversary of the founding of the Protein Data Bank focuses on the insights gained from structural studies of protein-DNA complexes and the role the PDB has played in driving this research. I cover highlights in the field, beginning with X-ray crystal structures of the first DNA-binding domains to be studied, through recent cryo-EM structures of transcription factor binding to nucleosomal DNA.
Asunto(s)
ADN/metabolismo , Bases de Datos de Proteínas/historia , Regulación de la Expresión Génica , Biología Molecular/historia , Transcripción Genética , Animales , ADN/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Unión Proteica , Conformación ProteicaRESUMEN
OTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another's activity. OTUB1 can noncatalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2â¼Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. To assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits the polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the auto-monoubiquitination and autopolyubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3. Our quantitative analysis provides a basis for further exploring the biological roles of OTUB1:E2 complexes in cells.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Humanos , Cinética , Poliubiquitina/metabolismo , Unión Proteica , Multimerización de Proteína , Saccharomyces cerevisiae/enzimología , Termodinámica , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitinación/efectos de los fármacosRESUMEN
A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.
Asunto(s)
Enzimas Desubicuitinizantes/genética , Endopeptidasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genética , Proteasas Ubiquitina-Específicas/genética , Alanina/genética , Sustitución de Aminoácidos/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Catálisis , Cisteína/genética , Enzimas Desubicuitinizantes/química , Endopeptidasas/química , Humanos , Mutación/genética , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Transactivadores/química , Ubiquitina/química , Ubiquitina/genética , Proteasas Ubiquitina-Específicas/química , Ubiquitinación/genéticaRESUMEN
OTUB1 is a deubiquitinating enzyme that cleaves Lys-48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2â¼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1-/- knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencias de Aminoácidos , Animales , Cisteína Endopeptidasas/genética , Enzimas Desubicuitinizantes , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Estabilidad Proteica , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , UbiquitinaciónRESUMEN
A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module's molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.
Asunto(s)
Histonas/química , Hidrazinas/química , Ubiquitina/química , Alquilación , Animales , Anticuerpos/inmunología , Biomimética/métodos , Cisteína/química , Cisteína Endopeptidasas/química , Enzimas Desubicuitinizantes , Endopeptidasas/química , Escherichia coli/genética , Histonas/síntesis química , Histonas/inmunología , Histonas/aislamiento & purificación , Humanos , Hidrazinas/síntesis química , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleosomas/química , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/síntesis química , Ubiquitina/inmunología , Ubiquitina/aislamiento & purificación , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/genética , Xenopus laevisRESUMEN
Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13â¼Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13â¼Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13â¼Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13â¼Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13â¼Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13â¼Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non-catalytic manner.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Cisteína Endopeptidasas/metabolismo , Ubiquitina/antagonistas & inhibidores , Ubiquitinación , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Daño del ADN , Enzimas Desubicuitinizantes , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMEN
The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Modelos Biológicos , Nucleosomas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Acetilación , Western Blotting , Histona Acetiltransferasas/genética , Histonas/metabolismo , Cinética , Lisina/metabolismo , Metilación , Nucleosomas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/enzimología , Macrófagos/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Anciano , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Linfocitos B/citología , Donantes de Sangre , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interleucina-10/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/citología , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Microscopía Confocal , Monocitos/citología , Mutación , FN-kappa B/metabolismo , Fagocitosis , Receptores de Superficie Celular/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6â¼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/química , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Mutación , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN's C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN's cellular stability.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Fosfohidrolasa PTEN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Cromatografía Liquida , Humanos , Inmunoprecipitación , Ubiquitina-Proteína Ligasas Nedd4 , Fosforilación , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem , UbiquitinaciónRESUMEN
In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code." MS data are available via ProteomeXchange with identifier PXD001167.