Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 40
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
bioRxiv ; 2024 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-39026748

RESUMO

Targeted protein degradation (TPD) modulates protein function beyond inhibition of enzyme activity or protein-protein interactions. Most degraders function by proximity induction, and directly bridge an E3 ligase with the target to be degraded. However, many proteins might not be addressable via proximity-based degraders, and other challenges, such as resistance acquisition, exist. Here, we identified pseudo-natural products derived from (-)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs induce a unique conformational change and, thereby, boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2 KLHDC3 , which we identified to also mediate native IDO1 degradation. Therefore, iDegs supercharge the native proteolytic pathway of IDO1, rendering this mechanism of action distinct from traditional degrader approaches involving proteolysis-targeting chimeras (PROTACs) or molecular-glue degraders (MGDs). In contrast to clinically explored IDO1 inhibitors, iDegs reduce formation of kynurenine by both inhibition and induced degradation of the enzyme and should also modulate non-enzymatic functions of IDO1. This unique mechanism of action may open up new therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

2.
Nat Struct Mol Biol ; 31(2): 378-389, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38326650

RESUMO

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL-E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.


Assuntos
Proteínas Culina , Enzimas de Conjugação de Ubiquitina , Proteínas Culina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Poliubiquitina/metabolismo
3.
Mol Cell ; 84(7): 1304-1320.e16, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38382526

RESUMO

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.


Assuntos
Proteínas Culina , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Poliubiquitina/metabolismo , Proteínas de Transporte/metabolismo
4.
Biochem J ; 480(22): 1817-1831, 2023 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-37870100

RESUMO

Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation. Given the homology between HOIL-1 and ARIH1, an RBR E3 that functions with the large superfamily of cullin-RING E3 ligases (CRLs), a biochemical investigation was undertaken, showing ARIH1 catalyzes Ser ubiquitylation to CRL-bound substrates. However, the efficiency of ubiquitylation was exquisitely dependent on the location and chemical environment of the Ser residue within the primary structure of the substrate. Comprehensive mutagenesis of the ARIH1 Rcat domain identified residues whose mutation severely impacted both oxyester and isopeptide bond formation at the preferred site for Ser ubiquitylation while only modestly affecting Lys ubiquitylation at the physiological site. The results reveal dual isopeptide and oxyester protein ubiquitylation activities of ARIH1 and set the stage for physiological investigations into this function of emerging importance.


Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Proteínas/metabolismo , Catálise
6.
Cell ; 186(9): 1895-1911.e21, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37028429

RESUMO

Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of ∼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.


Assuntos
Proteínas Culina , Proteínas F-Box , Proteínas Ligases SKP Culina F-Box , Fatores de Transcrição , Humanos , Proteínas Culina/química , Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Conformação Molecular , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
7.
Mol Cell ; 83(5): 770-786.e9, 2023 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-36805027

RESUMO

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.


Assuntos
Proteínas , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Transporte , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
8.
Mol Cell ; 83(1): 57-73.e9, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608670

RESUMO

The TFE3 and MITF master transcription factors maintain metabolic homeostasis by regulating lysosomal, melanocytic, and autophagy genes. Previous studies posited that their cytosolic retention by 14-3-3, mediated by the Rag GTPases-mTORC1, was key for suppressing transcriptional activity in the presence of nutrients. Here, we demonstrate using mammalian cells that regulated protein stability plays a fundamental role in their control. Amino acids promote the recruitment of TFE3 and MITF to the lysosomal surface via the Rag GTPases, activating an evolutionarily conserved phospho-degron and leading to ubiquitination by CUL1ß-TrCP and degradation. Elucidation of the minimal functional degron revealed a conserved alpha-helix required for interaction with RagA, illuminating the molecular basis for a severe neurodevelopmental syndrome caused by missense mutations in TFE3 within the RagA-TFE3 interface. Additionally, the phospho-degron is recurrently lost in TFE3 genomic translocations that cause kidney cancer. Therefore, two divergent pathologies converge on the loss of protein stability regulation by nutrients.


Assuntos
Aminoácidos , Fator de Transcrição Associado à Microftalmia , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Aminoácidos/metabolismo , Nutrientes , Estabilidade Proteica , Lisossomos/genética , Lisossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Mamíferos/metabolismo
9.
J Med Chem ; 64(9): 5850-5862, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33945681

RESUMO

The cullin-RING ubiquitin ligases (CRLs) are ubiquitin E3 enzymes that play a key role in controlling proteasomal degradation and are activated by neddylation. We previously reported inhibitors that target CRL activation by disrupting the interaction of defective in cullin neddylation 1 (DCN1), a CRL neddylation co-E3, and UBE2M, a neddylation E2. Our first-generation inhibitors possessed poor oral bioavailability and fairly rapid clearance that hindered the study of acute inhibition of DCN-controlled CRL activity in vivo. Herein, we report studies to improve the pharmacokinetic performance of the pyrazolo-pyridone inhibitors. The current best inhibitor, 40, inhibits the interaction of DCN1 and UBE2M, blocks NEDD8 transfer in biochemical assays, thermally stabilizes cellular DCN1, and inhibits anchorage-independent growth in a DCN1 amplified squamous cell carcinoma cell line. Additionally, we demonstrate that a single oral 50 mg/kg dose sustains plasma exposures above the biochemical IC90 for 24 h in mice.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pirazóis/química , Piridinas/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Administração Oral , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Estabilidade de Medicamentos , Meia-Vida , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Camundongos , Simulação de Dinâmica Molecular , Pirazóis/metabolismo , Pirazóis/farmacologia , Piridinas/metabolismo , Piridinas/farmacologia , Relação Estrutura-Atividade , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores
10.
Nature ; 590(7847): 671-676, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33536622

RESUMO

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies3-7. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.


Assuntos
Proteínas F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Regulação Alostérica , Biocatálise , Microscopia Crioeletrônica , Ciclina E/metabolismo , Humanos , Fosforilação , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
11.
Blood ; 137(2): 155-167, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33156908

RESUMO

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain-containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks at their promoters. In FBXO11-/- erythroblasts, these gene promoters bind BAHD1 and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2, and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at many developmentally poised bivalent genes during erythropoiesis.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Eritropoese/fisiologia , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexo Repressor Polycomb 2/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Linhagem Celular , Eritroblastos/metabolismo , Humanos , Proteólise
12.
Curr Opin Struct Biol ; 67: 101-109, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33160249

RESUMO

RING E3s comprise the largest family of ubiquitin (UB) and ubiquitin-like protein (UBL) ligases. RING E3s typically promote UB or UBL transfer from the active site of an associated E2 enzyme to a distally-recruited substrate. Many RING E3s - including the cullin-RING ligase family - are multifunctional, interacting with various E2s (or other E3s) to target distinct proteins, transfer different UBLs, or to initially modify substrates with UB or subsequently elongate UB chains. Here we consider recent structures of cullin-RING ligases, and their partner E2 enzymes, representing ligation reactions. The studies collectively reveal multimodal mechanisms - interactions between ancillary E2 or E3 domains, post-translational modifications, or auxiliary binding partners - directing cullin-RING E3-E2 enzyme active sites to modify their specific targets.


Assuntos
Proteínas Culina , Ubiquitina , Domínio Catalítico , Proteínas Culina/genética , Proteínas Culina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo
14.
J Biol Chem ; 295(15): 4974-4984, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32098871

RESUMO

The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). We genetically introduced pairs of Cys residues in different regions of the FepA tertiary structure, with the potential to form disulfide bonds. These included Cys pairs on adjacent ß-strands of the N-domain (intra-N) and Cys pairs that bridged the external surface of the N-domain to the interior of the C-terminal transmembrane ß-barrel (inter-N-C). We characterized FeEnt uptake by these mutants with siderophore nutrition tests, [59Fe]Ent binding and uptake experiments, and fluorescence decoy sensor assays. The three methods consistently showed that the intra-N disulfide bonds, which restrict conformational motion within the N-domain, prevented FeEnt uptake, whereas most inter-N-C disulfide bonds did not prevent FeEnt uptake. These outcomes indicate that conformational rearrangements must occur in the N terminus of FepA during FeEnt transport. They also argue against disengagement of the N-domain out of the channel as a rigid body and suggest instead that it remains within the transmembrane pore as FeEnt enters the periplasm.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Enterobactina/metabolismo , Escherichia coli/metabolismo , Mutação , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Proteínas de Transporte/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios Proteicos , Receptores de Superfície Celular/genética
15.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868589

RESUMO

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.


Assuntos
Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Genoma Humano/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Espectrometria de Massas , Poliubiquitina/genética , Transdução de Sinais/genética , Ubiquitinação/genética
16.
J Med Chem ; 62(18): 8429-8442, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31465221

RESUMO

Chemical control of cullin neddylation is attracting increased attention based largely on the successes of the NEDD8-activating enzyme (E1) inhibitor pevonedistat. Recently reported chemical probes enable selective and time-dependent inhibition of downstream members of the neddylation trienzymatic cascade including the co-E3, DCN1. In this work, we report the optimization of a novel class of small molecule inhibitors of the DCN1-UBE2M interaction. Rational X-ray co-structure enabled optimization afforded a 25-fold improvement in potency relative to the initial screening hit. The potency gains are largely attributed to additional hydrophobic interactions mimicking the N-terminal acetyl group that drives binding of UBE2M to DCN1. The compounds inhibit the protein-protein interaction, block NEDD8 transfer in biochemical assays, engage DCN1 in cells, and selectively reduce the steady-state neddylation of Cul1 and Cul3 in two squamous carcinoma cell lines harboring DCN1 amplification.


Assuntos
Proteínas Culina/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteína NEDD8/química , Pirazóis/química , Piridonas/química , Amidas/química , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Ciclopentanos/farmacologia , Desenho de Fármacos , Fibroblastos/metabolismo , Glicina/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Domínios Proteicos , Mapeamento de Interação de Proteínas , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/química , Relação Estrutura-Atividade , Enzimas de Conjugação de Ubiquitina/química
17.
Methods Enzymol ; 618: 29-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850057

RESUMO

Many fundamental discoveries in ubiquitin-proteasome research have relied on reconstitution of activities from purified or recombinantly expressed components. These include landmark discoveries of E1-E2-E3 mechanisms, in which ubiquitin (UB) is initially activated and then covalently shuttled between enzyme active sites and ultimately ligated to substrate or substrate-linked UBs during polyubiquitination. However, recent studies have unearthed enormous variations on the E1-E2-E3 theme; for example, one E3 may employ two distinct E2s, or two different E3s may act in a single assembly or in series, to prime substrates directly with UB and subsequently decorate them with myriad types of polyubiquitin chains. To dissect this complexity, it can be helpful to monitor specific UB transfer reactions in isolation, rather than the end-point products formed upon mixing all enzymes in a cascade. Pulse-chase assays enable observation of a single reaction step and also allow one to differentially label UBs carried by different enzymes within the same tube. In such assays, the "pulse" reaction generates a thioester-linked enzyme~UB intermediate, while the "chase" monitors UB transfer to downstream components over time. Here, we describe pulse-chase assays for detecting fluorescent-UB in E2~UB intermediates. These assays enable direct assessment of particular ligation reactions, alone and in combination, to explore roles of multiple enzymatic cascades in the same tube. We anticipate this technique can be adapted to many different E2s, as well as thioester-forming E3s, to dissect ubiquitination by many distinct enzyme cascades.


Assuntos
Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Animais , Ensaios Enzimáticos/métodos , Fluorescência , Humanos , Especificidade por Substrato , Proteínas Ubiquitinadas/metabolismo
18.
Mol Cell ; 72(1): 19-36.e8, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30244836

RESUMO

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.


Assuntos
Compartimento Celular/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteostase/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Humanos , Mutação , Neoplasias/patologia , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
19.
PLoS One ; 13(6): e0199197, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29958295

RESUMO

The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKß. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated.


Assuntos
Quinase I-kappa B/imunologia , Imunidade Inata , Proteínas Oncogênicas/imunologia , Peptídeo Sintases/imunologia , Transdução de Sinais/imunologia , Animais , Células HEK293 , Humanos , Quinase I-kappa B/genética , Camundongos , Proteína NEDD8/genética , Proteína NEDD8/imunologia , Proteínas Oncogênicas/genética , Peptídeo Sintases/genética , Fosforilação/genética , Fosforilação/imunologia , Células RAW 264.7 , Transdução de Sinais/genética
20.
J Med Chem ; 61(7): 2694-2706, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29547693

RESUMO

We previously reported the discovery, validation, and structure-activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1-UBE2M interaction. We demonstrated that compound 7 inhibits both the DCN1-UBE2M protein-protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound 7 exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CLint = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (67). Compound 67 retains the favorable biochemical and cellular activity of compound 7 but is significantly more stable both in vitro and in vivo. Compound 67 is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1-UBE2M interaction on the NEDD8/CUL pathway.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas Culina/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Proteína NEDD8/antagonistas & inibidores , Proteína NEDD8/efeitos dos fármacos , Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-Atividade , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...