RESUMEN
Most E3 ligases use a RING domain to activate a thioester-linked E2â¼ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2â¼UBL-target intermediate representing RBX1-UBC12â¼NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2â¼UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RING-E2â¼UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.
Asunto(s)
Ubiquitinas/química , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Dominio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteína NEDD8 , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.
Asunto(s)
Péptido Hidrolasas/química , Talidomida/química , Ubiquitina-Proteína Ligasas/química , Proteínas Adaptadoras Transductoras de Señales , Cristalografía por Rayos X , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Lenalidomida , Modelos Moleculares , Complejos Multiproteicos/agonistas , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/metabolismo , Unión Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Talidomida/análogos & derivados , Talidomida/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Replication stress involving collision of replisomes with camptothecin (CPT)-stabilized DNA-Topoisomerase I adducts activates an ATR-dependent pathway to promote repair by homologous recombination. To identify human genes that protect cells from such replication stress, we performed a genome-wide CPT sensitivity screen. Among numerous candidate genes are two previously unstudied proteins: the ankyrin repeat protein NFKBIL2 and C6ORF167 (MMS22L), distantly related to yeast replication stress regulator Mms22p. MMS22L and NFKBIL2 interact with each other and with FACT (facilitator of chromatin transcription) and MCM (minichromosome maintenance) complexes. Cells depleted of NFKBIL2 or MMS22L are sensitive to DNA-damaging agents, load phosphorylated RPA onto chromatin in a CTIP-dependent manner, activate the ATR/ATRIP-CHK1 and double-strand break repair signaling pathways, and are defective in HR. This study identifies MMS22L-NFKBIL2 as components of the replication stress control pathway and provides a resource for discovery of additional components of this pathway.
Asunto(s)
Camptotecina/farmacología , Proteínas de Unión al ADN/metabolismo , Pruebas Genéticas , Genoma Humano/genética , Inestabilidad Genómica/efectos de los fármacos , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Daño del ADN , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multienzimáticos/metabolismo , FN-kappa B/deficiencia , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genética , Proteína de Replicación A/metabolismo , Reproducibilidad de los Resultados , Estrés Fisiológico/efectos de los fármacos , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Break-induced replication (BIR) is an efficient homologous recombination (HR) pathway employed to repair a DNA double-strand break (DSB) when homology is restricted to one end. All three major replicative DNA polymerases are required for BIR, including the otherwise nonessential Pol32 subunit. Here we show that BIR requires the replicative DNA helicase (Cdc45, the GINS, and Mcm2-7 proteins) as well as Cdt1. In contrast, both subunits of origin recognition complex (ORC) and Cdc6, which are required to create a prereplication complex (pre-RC), are dispensable. The Cdc7 kinase, required for both initiation of DNA replication and post-replication repair (PRR), is also required for BIR. Ubiquitination and sumoylation of the DNA processivity clamp PCNA play modest roles; in contrast, PCNA alleles that suppress pol32Delta's cold sensitivity fail to suppress its role in BIR, and are by themselves dominant inhibitors of BIR. These results suggest that origin-independent BIR involves cross-talk between normal DNA replication factors and PRR.
Asunto(s)
Roturas del ADN de Doble Cadena , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejo de Reconocimiento del Origen , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alelos , Proteínas de Ciclo Celular/metabolismo , Frío , ADN Helicasas/metabolismo , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN/genética , Mutación , Complejo de Reconocimiento del Origen/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
A DNA double-strand break (DSB) is repaired by gene conversion (GC) if both ends of the DSB share homology with an intact DNA sequence. However, if homology is limited to only one of the DSB ends, repair occurs by break-induced replication (BIR). It is not known how the homology status of the DSB ends is first assessed and what other parameters govern the choice between these repair pathways. Our data suggest that a "recombination execution checkpoint" (REC) regulates the choice of the homologous recombination pathway employed to repair a given DSB. This choice is made prior to the initiation of DNA synthesis, and is dependent on the relative position and orientation of the homologous sequences used for repair. The RecQ family helicase Sgs1 plays a key role in regulating the choice of the recombination pathway. Surprisingly, break repair and gap repair are fundamentally different processes, both kinetically and genetically, as Pol32 is required only for gap repair. We propose that the REC may have evolved to preserve genome integrity by promoting conservative repair, especially when a DSB occurs within a repeated sequence.
Asunto(s)
Reparación del ADN/genética , Reparación del ADN/fisiología , ADN de Hongos/genética , ADN de Hongos/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Roturas del ADN de Doble Cadena , Replicación del ADN , Evolución Molecular , Conversión Génica , Cinética , Modelos Biológicos , Modelos Genéticos , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cullin-RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re-sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how cullin neddylation cycles are integrated with receptor exchange.
Asunto(s)
Proteínas Cullin/metabolismo , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Complejo del Señalosoma COP9 , Proteínas Cullin/química , Humanos , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Unión Proteica , Factores de Transcripción/metabolismo , Ubiquitinas/químicaRESUMEN
Break-induced replication (BIR) is an efficient homologous recombination process to initiate DNA replication when only one end of a chromosome double-strand break shares homology with a template. BIR is thought to re-establish replication at stalled and broken replication forks and to act at eroding telomeres in cells that lack telomerase in pathways known as 'alternative lengthening of telomeres' (reviewed in refs 2, 6). Here we show that, in haploid budding yeast, Rad51-dependent BIR induced by HO endonuclease requires the lagging strand DNA Polalpha-primase complex as well as Poldelta to initiate new DNA synthesis. Polepsilon is not required for the initial primer extension step of BIR but is required to complete 30 kb of new DNA synthesis. Initiation of BIR also requires the nonessential DNA Poldelta subunit Pol32 primarily through its interaction with another Poldelta subunit, Pol31. HO-induced gene conversion, in which both ends of a double-strand break engage in homologous recombination, does not require Pol32. Pol32 is also required for the recovery of both Rad51-dependent and Rad51-independent survivors in yeast strains lacking telomerase. These results strongly suggest that both types of telomere maintenance pathways occur by recombination-dependent DNA replication. Thus Pol32, dispensable for replication and for gene conversion, is uniquely required for BIR; this finding provides an opening into understanding how DNA replication re-start mechanisms operate in eukaryotes. We also note that Pol32 homologues have been identified both in fission yeast and in metazoans where telomerase-independent survivors with alternative telomere maintenance have also been identified.
Asunto(s)
Roturas del ADN de Doble Cadena , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Telómero/genética , Telómero/metabolismo , ADN Polimerasa I/metabolismo , ADN Polimerasa II/metabolismo , ADN Polimerasa III/metabolismo , ADN Primasa/metabolismo , Reparación del ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Conversión Génica , Cinética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/citología , Telomerasa/metabolismoRESUMEN
In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5' to 3' resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Delta exo1Delta strains, where there is little 5' to 3' resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Delta, or exo1Delta cells. In sgs1Delta exo1Delta, repair by GC is severely inhibited, but cell viability remains high because of new telomere formation. These data suggest that the extensive 5' to 3' resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5' to 3' resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition.
Asunto(s)
Cromosomas Fúngicos , Exodesoxirribonucleasas/fisiología , RecQ Helicasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Telómero , Secuencia de Bases , Cartilla de ADN , Replicación del ADN , Conversión Génica , Genes Fúngicos , Reacción en Cadena de la Polimerasa , Translocación GenéticaRESUMEN
Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.
Asunto(s)
Células Madre Pluripotentes Inducidas , Pubertad Precoz , Humanos , Femenino , Ratones , Animales , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Hipotálamo/metabolismo , Pubertad , Hormona Liberadora de Gonadotropina/metabolismo , Pubertad Precoz/genética , Pubertad Precoz/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
[This corrects the article DOI: 10.1016/j.omtm.2023.101135.].
RESUMEN
Immunotherapy of acute myeloid leukemia (AML) has been challenging because the lack of tumor-specific antigens results in "on-target, off-tumor" toxicity. To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel). Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity. Here, we describe preclinical studies and process development of clinical-scale manufacturing of trem-cel. Preclinical data showed proof-of-concept with loss of CD33 surface protein and no impact on myeloid cell differentiation or function. At clinical scale, trem-cel could be manufactured reproducibly, routinely achieving >70% CD33 editing with no effect on cell viability, differentiation, and function. Trem-cel pharmacology studies using mouse xenograft models showed long-term engraftment, multilineage differentiation, and persistence of gene editing. Toxicology assessment revealed no adverse findings, and no significant or reproducible off-target editing events. Importantly, CD33-knockout myeloid cells were resistant to the CD33-targeted agent gemtuzumab ozogamicin in vitro and in vivo. These studies supported the initiation of the first-in-human, multicenter clinical trial evaluating the safety and efficacy of trem-cel in patients with AML (NCT04849910).
RESUMEN
The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to 'segregate' ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the targets and functions of UBXD proteins remain poorly understood. Through systematic proteomic analysis of UBXD proteins in human cells, we reveal a network of over 195 interacting proteins, implicating VCP in diverse cellular pathways. We have explored one such complex between an unstudied adaptor UBXN10 and the intraflagellar transport B (IFT-B) complex, which regulates anterograde transport into cilia. UBXN10 localizes to cilia in a VCP-dependent manner and both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilized the IFT-B complex and increased trafficking rates. Depletion of UBXN10 in zebrafish embryos causes defects in left-right asymmetry, which depends on functional cilia. This study provides a resource for exploring the landscape of UBXD proteins in biology and identifies an unexpected requirement for VCP-UBXN10 in ciliogenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Mapas de Interacción de Proteínas , Proteómica/métodos , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Trifosfatasas/genética , Animales , Proteínas de Ciclo Celular/genética , Cilios/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Células LLC-PK1 , Microscopía Confocal , Microscopía Fluorescente , Morfogénesis/fisiología , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Interferencia de ARN , Porcinos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína que Contiene ValosinaRESUMEN
Little is known about molecular recognition of acetylated N termini, despite prevalence of this modification among eukaryotic cytosolic proteins. We report that the family of human DCN-like (DCNL) co-E3s, which promote ligation of the ubiquitin-like protein NEDD8 to cullin targets, recognizes acetylated N termini of the E2 enzymes UBC12 and UBE2F. Systematic biochemical and biophysical analyses reveal 40- and 10-fold variations in affinities among different DCNL-cullin and DCNL-E2 complexes, contributing to varying efficiencies of different NEDD8 ligation cascades. Structures of DCNL2 and DCNL3 complexes with N-terminally acetylated peptides from UBC12 and UBE2F illuminate a common mechanism by which DCNL proteins recognize N-terminally acetylated E2s and how selectivity for interactions dependent on N-acetyl-methionine are established through side chains recognizing distal residues. Distinct preferences of UBC12 and UBE2F peptides for inhibiting different DCNLs, including the oncogenic DCNL1 protein, suggest it may be possible to develop small molecules blocking specific N-acetyl-methionine-dependent protein interactions.