RESUMEN
Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure-activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indoles/farmacología , Succinimidas/farmacología , Animales , Línea Celular , Cristalografía por Rayos X , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Indoles/química , Indoles/farmacocinética , Macaca fascicularis , Masculino , Ratones , Simulación del Acoplamiento Molecular , Ratas , Relación Estructura-Actividad , Succinimidas/química , Succinimidas/farmacocinéticaRESUMEN
S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.
Asunto(s)
Metionina Adenosiltransferasa/antagonistas & inhibidores , Quinolinas/farmacología , S-Adenosilmetionina/metabolismo , Triazoles/farmacología , Sitio Alostérico/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Metionina Adenosiltransferasa/aislamiento & purificación , Metionina Adenosiltransferasa/metabolismo , Quinolinas/química , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.
Asunto(s)
Sondas Moleculares/síntesis química , Ácidos Sulfínicos/síntesis química , Química Clic , Modelos Moleculares , Sondas Moleculares/química , Estructura Molecular , Ácidos Sulfínicos/químicaRESUMEN
Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Respuesta de Proteína Desplegada/genética , eIF-2 Quinasa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Supervivencia Celular , Drosophila/genética , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Fosforilación , Biosíntesis de Proteínas/genética , Saccharomyces cerevisiae/genética , UbiquitinaciónRESUMEN
Ubiquitin-like proteins (UBLs) are activated, transferred and conjugated by E1-E2-E3 enzyme cascades. E2 enzymes for canonical UBLs such as ubiquitin, SUMO, and NEDD8 typically use common surfaces to bind to E1 and E3 enzymes. Thus, canonical E2s are required to disengage from E1 prior to E3-mediated UBL ligation. However, E1, E2, and E3 enzymes in the autophagy pathway are structurally and functionally distinct from canonical enzymes, and it has not been possible to predict whether autophagy UBL cascades are organized according to the same principles. Here, we address this question for the pathway mediating lipidation of the human autophagy UBL, LC3. We utilized bioinformatic and experimental approaches to identify a distinctive region in the autophagy E2, Atg3, that binds to the autophagy E3, Atg12â¼Atg5-Atg16. Short peptides corresponding to this Atg3 sequence inhibit LC3 lipidation in vitro. Notably, the E3-binding site on Atg3 overlaps with the binding site for the E1, Atg7. Accordingly, the E3 competes with Atg7 for binding to Atg3, implying that Atg3 likely cycles back and forth between binding to Atg7 for loading with the UBL LC3 and binding to E3 to promote LC3 lipidation. The results show that common organizational principles underlie canonical and noncanonical UBL transfer cascades, but are established through distinct structural features.
Asunto(s)
Autofagia , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Relacionadas con la Autofagia , HumanosRESUMEN
Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.
Asunto(s)
Autofagia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Modelos Biológicos , Unión Proteica , Ubiquitinas/metabolismoRESUMEN
The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub. Sequence analysis identified two potential Ub binding sites on a single α-helical surface within the coiled-coil region of the V domain. Mutation of these putative Ub binding sites inhibited polyUb binding to the isolated V domain in vitro and impaired budding of lentiviruses. These data reveal an important role for K63 polyUb binding by ALIX in retroviral release.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , VIH-1/fisiología , Virus de la Anemia Infecciosa Equina/fisiología , Retroviridae/fisiología , Ubiquitina/metabolismo , Liberación del Virus , Sitios de Unión/genética , Proteínas de Unión al Calcio/química , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/química , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Células HEK293 , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Transducción de Señal , Ubiquitina/químicaRESUMEN
Core functions of autophagy are mediated by ubiquitin-like protein (UBL) cascades, in which a homodimeric E1 enzyme, Atg7, directs the UBLs Atg8 and Atg12 to their respective E2 enzymes, Atg3 and Atg10. Crystallographic and mutational analyses of yeast (Atg7-Atg3)(2) and (Atg7-Atg10)(2) complexes reveal noncanonical, multisite E1-E2 recognition in autophagy. Atg7's unique N-terminal domain recruits distinctive elements from the Atg3 and Atg10 'backsides'. This, along with E1 and E2 conformational variability, allows presentation of 'frontside' Atg3 and Atg10 active sites to the catalytic cysteine in the C-terminal domain from the opposite Atg7 protomer in the homodimer. Despite different modes of binding, the data suggest that common principles underlie conjugation in both noncanonical and canonical UBL cascades, whereby flexibly tethered E1 domains recruit E2s through surfaces remote from their active sites to juxtapose the E1 and E2 catalytic cysteines.
Asunto(s)
Autofagia , Enzimas Activadoras de Ubiquitina/metabolismo , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Modelos Moleculares , Conformación Proteica , Enzimas Activadoras de Ubiquitina/químicaRESUMEN
UBLs (ubiquitin-like proteins) are a major class of eukaryotic post-translational modifiers. UBLs are attached to numerous cellular proteins and to other macromolecules, thereby regulating a wide array of cellular processes. In this chapter we highlight a subset of UBLs and describe their regulatory roles in the cell.
Asunto(s)
Ubiquitinas/metabolismo , Animales , Humanos , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Ubiquitina/metabolismo , Ubiquitinación/genética , Ubiquitinación/fisiología , Ubiquitinas/química , Ubiquitinas/genéticaAsunto(s)
Autofagia/fisiología , Ubiquitinas/metabolismo , Animales , Autofagia/genética , Humanos , Modelos Biológicos , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/genéticaRESUMEN
Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt-whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network.
Asunto(s)
Enfermedad de Huntington/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Ubiquitina/metabolismo , Animales , Línea Celular , Estabilidad de Enzimas , Genes Reporteros , Células HEK293 , Humanos , Proteína Huntingtina , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ubiquitina/genética , UbiquitinaciónRESUMEN
The linear ubiquitin chain assembly complex (LUBAC) is a key nuclear factor-κB (NF-κB) pathway component that produces linear polyubiquitin chains. The HOIL-1L subunit of LUBAC has been shown to bind linear chains; however, detailed structural and functional analyses on the binding between LUBAC and linear chains have not been performed. In this study, we found that the Npl4 zinc finger (NZF) domain of HOIL-1L specifically binds linear polyubiquitin chains and determined the crystal structure of the HOIL-1L NZF domain in complex with linear diubiquitin at 1.7-Å resolution. The HOIL-1L NZF domain consists of a zinc-coordinating "NZF core" region and an additional α-helical "NZF tail" region. The HOIL-1L NZF core binds both the canonical Ile44-centered hydrophobic surface on the distal ubiquitin and a Phe4-centered hydrophobic patch on the proximal ubiquitin, representing a mechanism for the specific recognition of linear chains. The NZF tail binds the proximal ubiquitin to enhance the binding affinity. These recognition mechanisms were supported by the accompanying in vitro and in vivo structure-based mutagenesis experiments.
Asunto(s)
Proteínas Portadoras/química , Proteínas Nucleares/química , Ubiquitina-Proteína Ligasas/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X/métodos , Humanos , Ratones , Conformación Molecular , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie/métodos , Factores de TranscripciónRESUMEN
Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7(NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7~Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1~UBL-E2 architecture for enzymes mediating autophagy.
Asunto(s)
Autofagia , Proteínas Portadoras/química , Fibroblastos/enzimología , Proteínas Asociadas a Microtúbulos/química , Enzimas Ubiquitina-Conjugadoras/química , Secuencia de Aminoácidos , Animales , Proteína 7 Relacionada con la Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Cristalografía por Rayos X , Fibroblastos/patología , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos , Mutación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Transfección , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.
Asunto(s)
Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Fracciones Subcelulares/metabolismo , Ubiquitina/metabolismo , Animales , Humanos , Estados UnidosRESUMEN
Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub-Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7(-/-) liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways.
Asunto(s)
Factor 2 Relacionado con NF-E2/metabolismo , Estrés Fisiológico/fisiología , Ubiquitina/metabolismo , Animales , Autofagia , Proteína 5 Relacionada con la Autofagia , Proteína 7 Relacionada con la Autofagia , Células Cultivadas , Ratones , Ratones Mutantes , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Especificidad por SustratoRESUMEN
The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.
Asunto(s)
Pliegue de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Guanidina/farmacología , Cinética , Datos de Secuencia Molecular , Proteínas de Unión al ARN/química , TermodinámicaRESUMEN
SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7A resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.
Asunto(s)
Membrana Celular/fisiología , Proteínas R-SNARE/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiología , Animales , Sitios de Unión , Cristalografía por Rayos X , Endosomas/fisiología , Exocitosis , Modelos Moleculares , Mutagénesis , Neuronas/fisiología , Conformación Proteica , Proteínas R-SNARE/genética , Proteínas R-SNARE/fisiología , Proteínas Recombinantes/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , AguaRESUMEN
The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain. We have solved crystal structures of the rat VAP-A MSP homology domain alone and in complex with an FFAT motif. The co-crystal structure was used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant also blocked function of the VAP homolog Scs2p in yeast. Finally, overexpression of the FFAT binding-defective VAP in COS7 cells dramatically altered ER morphology. Our data establish the structural basis of FFAT-mediated ER targeting and suggest that FFAT-targeted proteins play an important role in determining ER morphology.