RESUMEN
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.
RESUMEN
Phosphorylation of histone H3 threonine 3 (H3T3) by Haspin recruits the chromosomal passenger complex to the inner centromere and ensures proper cell cycle progression through mitosis. The mechanism by which Haspin binds to nucleosomes to phosphorylate H3T3 is not known. We report here cryo-EM structures of the Haspin kinase domain bound to a nucleosome. In contrast with previous structures of histone-modifying enzymes, Haspin solely contacts the nucleosomal DNA, inserting into a supergroove formed by apposing major grooves of two DNA gyres. This unique binding mode provides a plausible mechanism by which Haspin can bind to nucleosomes in a condensed chromatin environment to phosphorylate H3T3. We identify key basic residues in the Haspin kinase domain that are essential for phosphorylation of nucleosomal histone H3 and binding to mitotic chromatin. Our structure is the first of a kinase domain bound to a nucleosome and is the first example of a histone-modifying enzyme that binds to nucleosomes solely through DNA contacts.
RESUMEN
The discovery of ubiquitin conjugation to lysines and the role of K48-linked polyubiquitin in targeting substrates for proteasomal degradation was followed by revelation of non-degradative roles of ubiquitination and, more recently, of non-canonical covalent ubiquitin linkages. Here we summarize findings of the ever-expanding array of ubiquitin signals and their biological roles.
Asunto(s)
Poliubiquitina , Ubiquitina , Ubiquitina/metabolismo , Proteolisis , Ubiquitinación , Poliubiquitina/metabolismo , Lisina/metabolismoRESUMEN
Monoubiquitination of histone H2B-K120/123 plays several roles in regulating transcription, DNA replication and the DNA damage response. The structure of a nucleosome in complex with the dimeric RING E3 ligase Bre1 reveals that one RING domain binds to the nucleosome acidic patch, where it can position the E2 ubiquitin conjugating enzyme Rad6, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggest a general mechanism of tuning histone specificity via the non-E2-binding RING domain.
Asunto(s)
Histonas , Proteínas de Saccharomyces cerevisiae , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Post-translational modification of histones plays a central role in regulating transcription. Methylation of histone H3 at lysines 4 (H3K4) and 79 (H3K79) play roles in activating transcription whereas methylation of H3K27 is a repressive mark. These modifications, in turn, depend upon prior monoubiquitination of specific histone residues in a phenomenon known as histone crosstalk. Earlier work had provided insights into the mechanism by which monoubiquitination histone H2BK120 stimulates H3K4 methylation by COMPASS/MLL1 and H3K79 methylation by DOT1L, and monoubiquitinated H2AK119 stimulates methylation of H3K27 by the PRC2 complex. Recent studies have shed new light on the role of individual subunits and paralogs in regulating the activity of PRC2 and how additional post-translational modifications regulate yeast Dot1 and human DOT1L, as well as provided new insights into the regulation of MLL1 by H2BK120ub.
Asunto(s)
Histonas , Procesamiento Proteico-Postraduccional , Humanos , Histonas/metabolismo , Ubiquitinación , Metilación , Saccharomyces cerevisiae/metabolismoRESUMEN
Monoubiquitination of histone H2BK120/123 plays multiple roles in regulating transcription, DNA replication and the DNA damage response. The structure of a nucleosome in complex with the dimeric RING E3 ligase, Bre1, reveals that one RING domain binds to the nucleosome acidic patch, where it can position the Rad6 E2, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggests a general mechanism of tuning histone specificity via the non-E2-binding RING domain.
RESUMEN
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/química , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histona Acetiltransferasas/metabolismoRESUMEN
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
The Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator contains a four-protein subcomplex called the deubiquitinating enzyme (DUB) module that removes ubiquitin from histone H2B-K120. The human DUB module contains the catalytic subunit ubiquitin-specific protease 22 (USP22), which is overexpressed in a number of cancers that are resistant to available therapies. We screened a massive combinatorial library of cyclic peptides and identified potent inhibitors of USP22. The top hit was highly specific for USP22 compared with a panel of 44 other human DUBs. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. These macrocycle inhibitors are, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module and show promise for development.
Asunto(s)
Histonas , Ubiquitina , Histonas/metabolismo , Humanos , Péptidos Cíclicos/farmacología , Factores de Transcripción/metabolismo , Ubiquitina Tiolesterasa , 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.
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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
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
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
Methylation of histone H3K4 is a hallmark of actively transcribed genes that depends on mono-ubiquitination of histone H2B (H2B-Ub). H3K4 methylation in yeast is catalyzed by Set1, the methyltransferase subunit of COMPASS. We report here the cryo-EM structure of a six-protein core COMPASS subcomplex, which can methylate H3K4 and be stimulated by H2B-Ub, bound to a ubiquitinated nucleosome. Our structure shows that COMPASS spans the face of the nucleosome, recognizing ubiquitin on one face of the nucleosome and methylating H3 on the opposing face. As compared to the structure of the isolated core complex, Set1 undergoes multiple structural rearrangements to cement interactions with the nucleosome and with ubiquitin. The critical Set1 RxxxRR motif adopts a helix that mediates bridging contacts between the nucleosome, ubiquitin and COMPASS. The structure provides a framework for understanding mechanisms of trans-histone cross-talk and the dynamic role of H2B ubiquitination in stimulating histone methylation.
Asunto(s)
Histonas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Metilación , Nucleosomas/ultraestructura , Unión Proteica , Estructura Secundaria de Proteína , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Covalent modifications of histone proteins regulate a wide variety of cellular processes. Methylation of histone H3K79 and H3K4 is associated with active transcription and is catalyzed by Dot1L and Set1, respectively. Both Dot1L and Set1 are activated by prior ubiquitination of histone H2B on K120 in a process termed 'histone crosstalk'. Recent structures of Dot1L bound to a ubiquitinated nucleosome revealed how Dot1L is activated by ubiquitin and how Dot1L distorts the nucleosome to access its substrate. Structures of Dot1L-interacting proteins have provided insight into how Dot1L is recruited to sites of active transcription. Cryo-EM and crystallographic studies of the complex of proteins associated with Set1 (COMPASS), uncovered the architecture of COMPASS and how Set1 is activated upon complex assembly.
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Histona Metiltransferasas/metabolismo , Histonas/metabolismo , Ubiquitina/metabolismo , Sitios de Unión , Activación Enzimática , Histona Metiltransferasas/química , Histonas/química , Modelos Moleculares , Conformación Molecular , Unión Proteica , Relación Estructura-Actividad , Ubiquitina/químicaRESUMEN
Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a nonhydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B.
Asunto(s)
Histonas/síntesis química , Ubiquitina/síntesis química , Proteínas de Xenopus/síntesis química , Xenopus laevis , Animales , Histonas/química , Histonas/genética , Hidrólisis , Lisina/síntesis química , Lisina/química , Lisina/genética , Modelos Moleculares , Ubiquitina/química , Ubiquitina/genética , Ubiquitinación , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
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.
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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
Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 cause different phenotypes and alter the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2B in vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Alelos , Replicación del ADN/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Hidroxiurea/farmacología , Mutación/genética , Nucleosomas/efectos de los fármacos , Fenotipo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Transcripción Genética/efectos de los fármacos , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
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
Assays of the affinity of a deubiquitinating enzyme for substrate, either through binding studies or determination of the Michaelis constant, KM, can shed light on substrate selectivity and the effects of mutations on substrate interactions. The difficulty in generating sufficient quantities of ubiquitinated substrate frequently presents a barrier to these studies. We describe here an alternative approach that can be used in cases where non-hydrolyzable, chemically ubiquitinated substrate analogs can be more readily generated. The substrate analog can be utilized as a competitive inhibitor in kinetics experiments monitoring cleavage of ubiquitin-AMC (Ub-AMC) by the deubiquitinating enzyme. The resulting inhibitory constant, Ki, provides a reliable approximation of the Kd for ubiquitinated substrate. We show how this approach can be used to assay the affinity of the yeast SAGA DUB module for nucleosomes containing monoubiquitinated H2B.