RESUMEN
Poly(ADP-ribose) (PAR) is an RNA-like polymer that regulates an increasing number of biological processes. Dysregulation of PAR is implicated in neurodegenerative diseases characterized by abnormal protein aggregation, including amyotrophic lateral sclerosis (ALS). PAR forms condensates with FUS, an RNA-binding protein linked with ALS, through an unknown mechanism. Here, we demonstrate that a strikingly low concentration of PAR (1 nM) is sufficient to trigger condensation of FUS near its physiological concentration (1 µM), which is three orders of magnitude lower than the concentration at which RNA induces condensation (1 µM). Unlike RNA, which associates with FUS stably, PAR interacts with FUS transiently, triggering FUS to oligomerize into condensates. Moreover, inhibition of a major PAR-synthesizing enzyme, PARP5a, diminishes FUS condensation in cells. Despite their structural similarity, PAR and RNA co-condense with FUS, driven by disparate modes of interaction with FUS. Thus, we uncover a mechanism by which PAR potently seeds FUS condensation.
Asunto(s)
Esclerosis Amiotrófica Lateral , Poli Adenosina Difosfato Ribosa , Esclerosis Amiotrófica Lateral/genética , Humanos , Poli Adenosina Difosfato Ribosa/metabolismo , ARN/genética , Proteína FUS de Unión a ARN/metabolismoRESUMEN
Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a posttranslational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na+, Mg2+, Ca2+, and spermine4+). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR.
Asunto(s)
Adenosina Difosfato Ribosa , Poli Adenosina Difosfato Ribosa , Poli Adenosina Difosfato Ribosa/química , Poli Adenosina Difosfato Ribosa/metabolismo , Adenosina Difosfato Ribosa/química , Procesamiento Proteico-Postraduccional , Unión Proteica , Fenómenos Fisiológicos CelularesRESUMEN
Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here, we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs, but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) center with resting-state Fe-His bond lengths of â¼2.15 Å. This ligand set generates an iron environment more electropositive than those of other non-heme iron dioxygenases as observed by Mössbauer isomer shifts. Dioxygen (O2) does not coordinate iron in the absence of substrate. Substrates bind away (â¼4.7 Å) from the iron and have little impact on its electronic structure, thus excluding coordination-triggered O2 binding. However, substrate binding does perturb the spectral properties of CCO Fe-NO derivatives, indicating proximate organic substrate and O2-binding sites, which might influence Fe-O2 interactions. Together, these data provide a robust description of the CCO iron center and its interactions with substrates and substrate mimetics that illuminates commonalities as well as subtle and profound structural differences within the CCO family.
Asunto(s)
Alquenos/química , Dioxigenasas/química , Hemo/química , Conformación ProteicaRESUMEN
Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce formation of toxic retinal conjugates. Of these agents, certain ones that contain primary amine groups can also reversibly form retinaldehyde Schiff base adducts, which contributes to their retinal protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse effects resulting from slowed chromophore regeneration, whereas effective retinal sequestration can require high drug doses with potential off-target effects. We hypothesized that the RPE65-emixustat crystal structure could help guide the design of retinaldehyde-sequestering agents with varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less potently than compounds with a high affinity for the RPE65 active site. These agents were distributed into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound [3-amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol (MB-007)], these agents conferred protection against retinal phototoxicity, suggesting that both direct RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.
Asunto(s)
Visión Ocular/efectos de los fármacos , cis-trans-Isomerasas/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Sitios de Unión , Bovinos , Dermatitis Fototóxica/prevención & control , Femenino , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas/enzimología , Modelos Moleculares , Epitelio Pigmentado Ocular/efectos de los fármacos , Regeneración/efectos de los fármacos , Enfermedades de la Retina/prevención & control , Epitelio Pigmentado de la Retina/efectos de los fármacos , Bases de Schiff/química , cis-trans-Isomerasas/química , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/aislamiento & purificación , cis-trans-Isomerasas/metabolismoRESUMEN
Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.
Asunto(s)
Epitelio Pigmentado de la Retina/enzimología , Retinoides/farmacología , Visión Ocular/efectos de los fármacos , cis-trans-Isomerasas/antagonistas & inhibidores , cis-trans-Isomerasas/química , Animales , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Diterpenos/síntesis química , Diterpenos/química , Diterpenos/farmacología , Ligandos , Luz , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Palmitatos , Éteres Fenílicos/síntesis química , Éteres Fenílicos/química , Éteres Fenílicos/farmacología , Propanolaminas/síntesis química , Propanolaminas/química , Propanolaminas/farmacología , Unión Proteica , Conformación Proteica , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de la radiación , Retinoides/síntesis química , Retinoides/química , Estereoisomerismo , Visión Ocular/fisiología , Visión Ocular/efectos de la radiaciónRESUMEN
Poly(ADP-ribose) (PAR) is a homopolymer made of two or more adenosine diphosphate ribose (ADP-ribose) units. The polymer is usually conjugated to protein as a posttranslational modification playing key roles in cellular processes, such as DNA repair, RNA metabolism, and biomolecular condensate formation. Emergent data revealed that PAR length is highly regulated and determines the selection of and affinity towards protein binders. Here, we describe several fluorescence-based methods that quantify PAR length distributions. Briefly, we use the bioconjugation technique ELTA (enzymatic labeling of terminal ADP-ribose) to fluorescently label PAR, which can be isolated from in vitro and cellular samples. We describe a novel capillary electrophoresis method to separate and quantify PAR length and compare the profile to gel electrophoresis- and high-performance liquid chromatography-based methods. The capillary electrophoresis method is rapid and automatable, enabling accurate determination of the length profiles from subfemtomole quantities of PAR.
Asunto(s)
Adenosina Difosfato Ribosa , Poli Adenosina Difosfato Ribosa , Poli Adenosina Difosfato Ribosa/metabolismo , Cromatografía Líquida de Alta Presión , Adenosina Difosfato Ribosa/metabolismo , Reparación del ADN , Electroforesis CapilarRESUMEN
Poly(ADP-ribose) (PAR), as part of a post-translational modification, serves as a flexible scaffold for noncovalent protein binding. Such binding is influenced by PAR chain length through a mechanism yet to be elucidated. Structural insights have been elusive, partly due to the difficulties associated with synthesizing PAR chains of defined lengths. Here, we employ an integrated approach combining molecular dynamics (MD) simulations with small-angle X-ray scattering (SAXS) experiments, enabling us to identify highly heterogeneous ensembles of PAR conformers at two different, physiologically relevant lengths: PAR 15 and PAR 22 . Our findings reveal that numerous factors including backbone conformation, base stacking, and chain length contribute to determining the structural ensembles. We also observe length-dependent compaction of PAR upon the addition of small amounts of Mg 2+ ions, with the 22-mer exhibiting ADP-ribose bundles formed through local intramolecular coil-to-globule transitions. This study illuminates how such bundling could be instrumental in deciphering the length-dependent action of PAR.
RESUMEN
Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a post-translational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na + , Mg 2+ , Ca 2+ , and spermine). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR. Significance: Poly(ADP-ribose) (PAR) is an RNA-like homopolymer that regulates DNA repair, RNA metabolism, and biomolecular condensate formation. Dysregulation of PAR results in cancer and neurodegeneration. Although discovered in 1963, fundamental properties of this therapeutically important polymer remain largely unknown. Biophysical and structural analyses of PAR have been exceptionally challenging due to the dynamic and repetitive nature. Here, we present the first single-molecule biophysical characterization of PAR. We show that PAR is stiffer than DNA and RNA per unit length. Unlike DNA and RNA which undergoes gradual compaction, PAR exhibits an abrupt switch-like bending as a function of salt concentration and by protein binding. Our findings points to unique physical properties of PAR that may drive recognition specificity for its function.
RESUMEN
In the eye, the isomerization of all-trans-retinal to 11-cis-retinal is accomplished by a metabolic pathway termed the visual cycle that is critical for vision. RPE65 is the essential trans-cis isomerase of this pathway. Emixustat, a retinoid-mimetic RPE65 inhibitor, was developed as a therapeutic visual cycle modulator and used for the treatment of retinopathies. However, pharmacokinetic liabilities limit its further development including: (1) metabolic deamination of the γ-amino-α-aryl alcohol, which mediates targeted RPE65 inhibition, and (2) unwanted long-lasting RPE65 inhibition. We sought to address these issues by more broadly defining the structure-activity relationships of the RPE65 recognition motif via the synthesis of a family of novel derivatives, which were tested in vitro and in vivo for RPE65 inhibition. We identified a potent secondary amine derivative with resistance to deamination and preserved RPE65 inhibitory activity. Our data provide insights into activity-preserving modifications of the emixustat molecule that can be employed to tune its pharmacological properties.
Asunto(s)
Propanolaminas , Retinoides , Retinoides/farmacología , Retinoides/metabolismo , Éteres Fenílicos/farmacología , Visión Ocular , Retinaldehído/metabolismo , Proteínas del OjoRESUMEN
Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, bacteriophages. To identify such TA systems, we searched bioinformatically for those frequently encoded near known phage defence genes in bacterial genomes. This search identified homologues of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. Representatives from two different subfamilies, DarTG1 and DarTG2, strongly protected E. coli MG1655 against different phages. We demonstrate that for each system, infection with either RB69 or T5 phage, respectively, triggers release of the DarT toxin, a DNA ADP-ribosyltransferase, that then modifies viral DNA and prevents replication, thereby blocking the production of mature virions. Further, we isolated phages that have evolved to overcome DarTG defence either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defence may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.
Asunto(s)
Bacteriófagos , Sistemas Toxina-Antitoxina , Adenosina Difosfato , Bacteriófagos/genética , ADN Viral/genética , Escherichia coli/genética , Sistemas Toxina-Antitoxina/genéticaRESUMEN
Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADP-ribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-κB-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice.IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADP-ribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Hidrolasas/metabolismo , Virus Sindbis/genética , Virus Sindbis/patogenicidad , Proteínas no Estructurales Virales/metabolismo , Adenosina Difosfato Ribosa/genética , Animales , Encéfalo/virología , Línea Celular , Encefalomielitis/virología , Femenino , Hidrolasas/genética , Ratones , Mutación , Neuronas/virología , Transducción de Señal , Proteínas no Estructurales Virales/genética , Virulencia , Replicación Viral/genéticaRESUMEN
Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms.
Asunto(s)
Células Fotorreceptoras/efectos de los fármacos , Visión Ocular , cis-trans-Isomerasas/antagonistas & inhibidores , Adaptación Fisiológica , Animales , Diterpenos/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Oxidorreductasas/metabolismo , Éteres Fenílicos/farmacología , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/fisiología , Propanolaminas/farmacología , Sciuridae , Transducina/genética , cis-trans-Isomerasas/metabolismoRESUMEN
Ileal bile acid binding protein (I-BABP, gene name FABP6) is a component of the bile acid recycling system, expressed in the ileal enterocyte. The physiological role of I-BABP has been hypothesized to be either an intracellular buffering agent to protect against excess intracellular bile acids or separately as a modulator of bile acid controlled transcription. We investigated mouse I-BABP (mI-BABP) to understand the function of this protein family. Here, we studied energetics and site selectivity of binding with physiological bile acids using a combination of isothermal calorimetric analysis and NMR spectroscopy. We found that the most abundant bile acid in the mouse (ß-muricholic acid) binds with weak affinity individually and in combination with other bile acids. Further analysis showed that mI-BABP like human I-BABP (hI-BABP) specifically recognizes the conjugated form of cholic acid:chenodeoxycholic acid (CA:CDCA) in a site-selective manner, displaying the highest affinity of any bile acid combination tested. These results indicate that I-BABP specifically recognizes the ligand combination of CDCA and CA, even in a species such as the mouse where CDCA only represents a trace component of the physiological pool. Specific and conserved recognition of the CDCA and CA ligand combination suggests that I-BABP may play a critical role in the regulation of bile acid signaling in addition to its proposed role as a buffering agent.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Secuencia de Aminoácidos , Animales , Ácidos y Sales Biliares/química , Clonación Molecular , Escherichia coli , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/genética , Ratones , Modelos Moleculares , Plásmidos/genética , Unión Proteica , Conformación ProteicaRESUMEN
Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.