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Cdc14 phosphatases are related structurally and mechanistically to protein tyrosine phosphatases (PTP) but evolved a unique specificity for phosphoSer-Pro-X-Lys/Arg sites primarily deposited by cyclin-dependent kinases. This specialization is widely conserved in eukaryotes. The evolutionary reconfiguration of the Cdc14 active site to selectively accommodate phosphoSer-Pro likely required modification to the canonical PTP catalytic cycle. While studying Saccharomyces cerevisiae Cdc14 we discovered a short sequence in the disordered C-terminus, distal to the catalytic domain, that mimics an optimal substrate. Kinetic analyses demonstrated this pseudosubstrate binds the active site and strongly stimulates rate-limiting phosphoenzyme hydrolysis, and we named it "substrate-like catalytic enhancer" (SLiCE). The SLiCE motif is found in all Dikarya fungal Cdc14 orthologs and contains an invariant glutamine, which we propose is positioned via substrate-like contacts to assist orientation of the hydrolytic water, similar to a conserved active site glutamine in other PTPs that Cdc14 lacks. AlphaFold2 predictions revealed vertebrate Cdc14 orthologs contain a conserved C-terminal alpha helix bound to the active site. Although apparently unrelated to the fungal sequence, this motif also makes substrate-like contacts and has an invariant glutamine in the catalytic pocket. Altering these residues in human Cdc14A and Cdc14B demonstrated that it functions by the same mechanism as the fungal motif. However, the fungal and vertebrate SLiCE motifs were not functionally interchangeable, illuminating potential active site differences during catalysis. Finally, we show that the fungal SLiCE motif is a target for phosphoregulation of Cdc14 activity. Our study uncovered evolution of an unusual stimulatory pseudosubstrate motif in Cdc14 phosphatases.
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Together with protein tyrosine kinases, protein tyrosine phosphatases (PTPs) control protein tyrosine phosphorylation and regulate numerous cellular functions. Dysregulated PTP activity is associated with the onset of multiple human diseases. Nevertheless, understanding of the physiological function and disease biology of most PTPs remains limited, largely due to the lack of PTP-specific chemical probes. In this study, starting from a well-known nonhydrolyzable phosphotyrosine (pTyr) mimetic, phosphonodifluoromethyl phenylalanine (F2Pmp), we synthesized 7 novel phosphonodifluoromethyl-containing bicyclic/tricyclic aryl derivatives with improved cell permeability and potency toward various PTPs. Furthermore, with fragment- and structure-based design strategies, we advanced compound 9 to compound 15, a first-in-class, potent, selective, and bioavailable inhibitor of human CDC14A and B phosphatases. This study demonstrates the applicability of the fragment-based design strategy in creating potent, selective, and bioavailable PTP inhibitors and provides a valuable probe for interrogating the biological roles of hCDC14 phosphatases and assessing their potential for therapeutic interventions.
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Inhibidores Enzimáticos , Fosfotirosina , Humanos , Fosfotirosina/metabolismo , Fosfotirosina/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Relación Estructura-Actividad , Proteínas Tirosina Fosfatasas no Receptoras/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Estructura Molecular , Disponibilidad BiológicaRESUMEN
Reversible protein phosphorylation is an abundant post-translational modification dynamically regulated by opposing kinases and phosphatases. Protein phosphorylation has been extensively studied in cell division, where waves of cyclin-dependent kinase activity, peaking in mitosis, drive the sequential stages of the cell cycle. Here we developed and employed a strategy to specifically probe kinase or phosphatase substrates at desired times or experimental conditions in the model organism Saccharomyces cerevisiae. We combined auxin-inducible degradation (AID) with mass spectrometry-based phosphoproteomics, which allowed us to arrest physiologically normal cultures in mitosis prior to rapid phosphatase degradation and phosphoproteome analysis. Our results revealed that protein phosphatase 2A coupled with its B56 regulatory subunit, Rts1 (PP2ARts1), is involved in dephosphorylation of numerous proteins in mitosis, highlighting the need for phosphatases to selectively maintain certain proteins in a hypophosphorylated state in the face of high mitotic kinase activity. Unexpectedly, we observed elevated phosphorylation at many sites on several subunits of the fungal eisosome complex following rapid Rts1 degradation. Eisosomes are dynamic polymeric assemblies that create furrows in the plasma membrane important in regulating nutrient import, lipid metabolism, and stress responses, among other things. We found that PP2ARts1-mediated dephosphorylation of eisosomes promotes their plasma membrane association and we provide evidence that this regulation impacts eisosome roles in metabolic homeostasis. The combination of rapid, inducible protein degradation with proteomic profiling offers several advantages over common protein disruption methods for characterizing substrates of regulatory enzymes involved in dynamic biological processes.
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A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation, AID2, systems work efficiently and rapidly in the human pathogenic yeasts, Candida albicans and Candida glabrata. We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid. Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens. IMPORTANCE Life-threatening fungal infections are an escalating human health problem, complicated by limited treatment options and the evolution of drug resistant pathogen strains. Identification of new targets for therapeutics to combat invasive fungal infections, including those caused by Candida species, is an urgent need. In this report, we establish and validate an inducible protein degradation methodology in Candida albicans and Candida glabrata that provides a new tool for protein functional characterization in these, and likely other, fungal pathogen species. We expect this tool will ultimately be useful for the identification and characterization of promising drug targets and factors involved in virulence and drug resistance.
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Candida , Micosis , Humanos , Proteolisis , Candida albicans/genética , Micosis/tratamiento farmacológico , Candida glabrata/genéticaRESUMEN
This research examined the differential motivational effects of a pre-college science enrichment program delivered in both online and in-person learning formats. Using self-determination theory as a guiding framework, we hypothesized that (a) students would exhibit growth in their perceived satisfaction of needs for autonomy, competence, and relatedness, (b) online learning would be associated with greater growth in autonomy, and (c) in-person learning would be associated with greater growth in both competence and relatedness. Using a sample of 598 adolescent participants, results of latent growth curve modeling indicated that satisfaction of the three needs grew unconditionally over the course of the program. However, format type was unrelated to growth in need satisfaction. Rather, this effect was found to be conditional upon the type of science project undertaken by students: astrophysics students exhibited significantly greater autonomy growth when receiving online instruction than did biochemistry students. Our findings suggest that online science learning can be just as effective in motivating students as in-person learning provided that the learning tasks are conducive to remote instruction.
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Embryonic expression of DNMT3B is critical for establishing de novo DNA methylation. This study uncovers the mechanism through which the promoter-associated long non-coding RNA (lncRNA) Dnmt3bas controls the induction and alternative splicing of Dnmt3b during embryonic stem cell (ESC) differentiation. Dnmt3bas recruits the PRC2 (polycomb repressive complex 2) at cis-regulatory elements of the Dnmt3b gene expressed at a basal level. Correspondingly, Dnmt3bas knockdown enhances Dnmt3b transcriptional induction, whereas overexpression of Dnmt3bas dampens it. Dnmt3b induction coincides with exon inclusion, switching the predominant isoform from the inactive Dnmt3b6 to the active Dnmt3b1. Intriguingly, overexpressing Dnmt3bas further enhances the Dnmt3b1:Dnmt3b6 ratio, attributed to its interaction with hnRNPL (heterogeneous nuclear ribonucleoprotein L), a splicing factor that promotes exon inclusion. Our data suggest that Dnmt3bas coordinates alternative splicing and transcriptional induction of Dnmt3b by facilitating the hnRNPL and RNA polymerase II (RNA Pol II) interaction at the Dnmt3b promoter. This dual mechanism precisely regulates the expression of catalytically active DNMT3B, ensuring fidelity and specificity of de novo DNA methylation.
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ADN (Citosina-5-)-Metiltransferasas , Metilación de ADN , Diferenciación Celular , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Células Madre Embrionarias/metabolismo , Exones/genética , Complejo Represivo Polycomb 2/metabolismo , Ratones , ADN Metiltransferasa 3B , AnimalesRESUMEN
A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation AID2 systems work efficiently and rapidly in the human pathogenic yeasts Candida albicans and Candida glabrata . We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid (5-Ad-IAA). Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens.
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Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here, we survey common LC-MS/MS-based phosphoproteomic workflows for identifying protein kinase and phosphatase substrates, noting key advantages and limitations of each. We conclude by discussing the value of inducible degradation technologies coupled with phosphoproteomics as a new approach that overcomes some limitations of current methods for substrate identification of kinases, phosphatases, and other regulatory enzymes.
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Monoéster Fosfórico Hidrolasas , Espectrometría de Masas en Tándem , Monoéster Fosfórico Hidrolasas/metabolismo , Cromatografía Liquida , Fosforilación , Proteínas Quinasas/metabolismo , Fosfoproteínas/metabolismoRESUMEN
The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm ) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.
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Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer's disease and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, we apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry, to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, we detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using in vitro phosphorylation, among 15 candidate kinases we attempted to purify and test, four candidate kinases, OXSR1 (oxidative-stress responsive gene 1), DAPK2 (death-associated protein kinase 2), CSK (C-terminal SRC kinase), and ZAP70 (zeta chain of T-cell receptor-associated protein kinase 70), displayed the ability to phosphorylate Tau in time-course experiments. Furthermore, coexpression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. We demonstrate that fluorescence complementation mass spectrometry is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing Alzheimer's disease therapeutic strategies.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/metabolismo , Proteómica , Proteínas tau/genética , Fosforilación , Espectrometría de Masas , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Accurate segregation of chromosomes during mitosis depends on the correct assembly of the mitotic spindle, a bipolar structure composed mainly of microtubules. The augmin complex, or homologous to augmin subunits (HAUS) complex, is an eight-subunit protein complex required for building robust mitotic spindles in metazoa. Augmin increases microtubule density within the spindle by recruiting the γ-tubulin ring complex (γ-TuRC) to pre-existing microtubules and nucleating branching microtubules. Here, we elucidate the molecular architecture of augmin by single particle cryo-electron microscopy (cryo-EM), computational methods, and crosslinking mass spectrometry (CLMS). Augmin's highly flexible structure contains a V-shaped head and a filamentous tail, with the head existing in either extended or contracted conformational states. Our work highlights how cryo-EM, complemented by computational advances and CLMS, can elucidate the structure of a challenging protein complex and provides insights into the function of augmin in mediating microtubule branching nucleation.
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Proteínas Asociadas a Microtúbulos , Tubulina (Proteína) , Microscopía por Crioelectrón , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND: Catheter ablation of ventricular tachycardia (VT) is associated with potential major complications, including mortality. The risk of acute complications in patients with ischaemic cardiomyopathy (ICM) and non-ischaemic cardiomyopathy (NICM) has not been systematically evaluated. METHODS: PubMed was searched for studies of catheter ablation of VT published between September 2009 and September 2019. Pre-specified primary outcomes were (1) rate of major acute complications, including death, and (2) mortality rate. RESULTS: A total of 7395 references were evaluated for relevance. From this, 50 studies with a total of 3833 patients undergoing 4319 VT ablation procedures fulfilled the inclusion criteria (mean age 59 years; male 82%; 2363 [62%] ICM; 1470 [38%] NICM). The overall major complication rate in ICM cohorts was 9.4% (95% CI, 8.1-10.7) and NICM cohorts was 7.1% (95% CI, 6.0-8.3). Reported complication rates were highly variable between studies (ICM I2 = 90%; NICM I2 = 89%). Vascular complications (ICM 2.5% [95% CI, 1.9-3.1]; NICM 1.2% [95% CI, 0.7-1.7]) and cerebrovascular events (ICM 0.5% [95% CI, 0.2-0.7]; NICM, 0.1% [95% CI, 0-0.2]) were significantly higher in ICM cohorts. Acute mortality rates in the ICM and NICM cohorts were low (ICM 0.9% [95% CI, 0.5-1.3]; NICM 0.6% [95% CI, 0.3-1.0]) with the majority of overall deaths (ICM 75%; NICM 80%) due to either recurrent VT or cardiogenic shock. CONCLUSION: Overall acute complication rates of VT ablation are comparable between ICM and NICM patients. However, the pattern and predictors of complications vary depending on the underlying cardiomyopathy.
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Cardiomiopatías , Ablación por Catéter , Isquemia Miocárdica , Taquicardia Ventricular , Cardiomiopatías/cirugía , Humanos , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/cirugía , Taquicardia Ventricular/cirugíaRESUMEN
Protein α-N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free α-amino group at protein N-termini. To systematically explore the extent of α-N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the α-N-terminal methylome. This repurposing approach found evidence of α-N-methylation of established and novel protein substrates with canonical N-terminal motifs of established α-N-terminal methyltransferases, including human NTMT1/2 and yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but have unclear and context-dependent roles. Moreover, α-N-methylation of noncanonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of α-N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif. We delineated the distribution of the two types of prevalent N-terminal modifications, acetylation and methylation, on amino acids at the first position. We tested three potentially methylated proteins and confirmed the α-N-terminal methylation of Hsp31 by additional proteomic analysis and immunoblotting. The other two proteins, Vma1 and Ssa3, were found to be predominantly acetylated, indicating that proteomic searching for α-N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global α-N-terminal methylome investigations.
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Proteómica , Proteínas de Saccharomyces cerevisiae , Epigenoma , Proteínas HSP70 de Choque Térmico , Proteínas de Choque Térmico , Humanos , Metilación , Procesamiento Proteico-Postraduccional , ATPasas de Translocación de Protón , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Reversible phosphorylation is a pervasive regulatory event in cellular physiology controlled by reciprocal actions of protein kinases and phosphatases. Determining the inherent substrate specificity of kinases and phosphatases is essential for understanding their cellular roles. Synthetic peptides have long served as substrate proxies for defining intrinsic kinase and phosphatase specificities. Here, we describe a high throughput protocol to simultaneously measure specificity constants (kcat/KM) of many synthetic peptide substrates in a single pool using label-free quantitative mass spectrometry. The generation of specificity constants from a single pooled reaction provides a rigorous and rapid comparison of substrate variants to help define an enzyme's specificity. Equally applicable to kinases and phosphatases, as well as other enzyme classes, the protocol consists of three general steps: (1) reaction of enzyme with pooled peptide substrates, each ideally with a unique mass and at concentrations well below KM, (2) analysis of reaction products using liquid chromatography-coupled mass spectrometry (LC-MS), and (3) automated extraction and integration of elution peaks for each substrate/product pair. We incorporate an ionization correction strategy allowing direct calculation of reaction progress, and subsequently kcat/KM, from substrate and product peak areas in a single sample, obviating the need for stable isotope labeling. Peptide consumption is minimal, and high peptide purity and accurate concentrations are not required. Access to a high-resolution LC-MS system is the only nonstandard equipment need. We present an analysis pipeline consisting entirely of established open-source software tools, and demonstrate proof of principle with the highly selective cell cycle phosphatase Cdc14 from Saccharomyces cerevisiae.
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Péptidos/análisis , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Proteómica/métodos , Cromatografía Liquida , Biología Computacional , Ensayos Analíticos de Alto Rendimiento , Biblioteca de Péptidos , Péptidos/metabolismo , Fosforilación , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
The identification of a pathogenic SCN5A variant confers an increased risk of conduction defects and ventricular arrhythmias (VA) in Brugada syndrome (BrS). However, specific aspects of sodium channel function that influence clinical phenotype have not been defined. A systematic literature search identified SCN5A variants associated with BrS. Sodium current (INa ) functional parameters (peak current, decay, steady-state activation and inactivation, and recovery from inactivation) and clinical features (conduction abnormalities [CA], spontaneous VA or family history of sudden cardiac death [SCD], and spontaneous BrS electrocardiogram [ECG]) were extracted. A total of 561 SCN5A variants associated with BrS were identified, for which data on channel function and clinical phenotype were available in 142. In the primary analysis, no relationship was found between any aspect of channel function and CA, VA/SCD, or spontaneous BrS ECG pattern. Sensitivity analyses including only variants graded pathogenic or likely pathogenic suggested that reduction in peak current and positive shift in steady-state activation were weakly associated with CA and VA/SCD, although sensitivity and specificity remained low. The relationship between in vitro assessment of channel function and BrS clinical phenotype is weak. The assessment of channel function does not enhance risk stratification. Caution is needed when extrapolating functional testing to the likelihood of variant pathogenicity.
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Síndrome de Brugada/genética , Síndrome de Brugada/patología , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Arritmias Cardíacas/genética , Síndrome de Brugada/diagnóstico por imagen , Electrocardiografía , Sistema de Conducción Cardíaco/patología , Humanos , FenotipoRESUMEN
Cdc14 protein phosphatases play an important role in plant infection by several fungal pathogens. This and other properties of Cdc14 enzymes make them an intriguing target for development of new antifungal crop treatments. Active site architecture and substrate specificity of Cdc14 from the model fungus Saccharomyces cerevisiae (ScCdc14) are well-defined and unique among characterized phosphatases. Cdc14 appears absent from some model plants. However, the extent of conservation of Cdc14 sequence, structure, and specificity in fungal plant pathogens is unknown. We addressed this by performing a comprehensive phylogenetic analysis of the Cdc14 family and comparing the conservation of active site structure and specificity among a sampling of plant pathogen Cdc14 homologs. We show that Cdc14 was lost in the common ancestor of angiosperm plants but is ubiquitous in ascomycete and basidiomycete fungi. The unique substrate specificity of ScCdc14 was invariant in homologs from eight diverse species of dikarya, suggesting it is conserved across the lineage. A synthetic substrate mimetic inhibited diverse fungal Cdc14 homologs with similar low µM Ki values, but had little effect on related phosphatases. Our results justify future exploration of Cdc14 as a broad spectrum antifungal target for plant protection.
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Evolución Biológica , Resistencia a la Enfermedad , Interacciones Huésped-Patógeno , Plantas/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Hongos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Filogenia , Plantas/clasificación , Plantas/genética , Plantas/microbiología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif-dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.
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Subunidad Apc10 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Ciclo Celular , Proteínas de Ciclo Celular/química , Mapas de Interacción de Proteínas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/química , Especificidad por Sustrato , UbiquitinaciónRESUMEN
Background Identification and elimination of nonpulmonary vein targets may improve clinical outcomes in patients with persistent atrial fibrillation (AF). We report on the use of a novel, noncontact imaging and mapping system that uses ultrasound to reconstruct atrial chamber anatomy and measures timing and density of dipolar, ionic activation (ie, charge density) across the myocardium to guide ablation of atrial arrhythmias. Methods The prospective, nonrandomized UNCOVER AF trial (Utilizing Novel Dipole Density Capabilities to Objectively Visualize the Etiology of Rhythms in Atrial Fibrillation) was conducted at 13 centers across Europe and Canada. Patients with persistent AF (>7 days, <1 year) aged 18 to 80 years, scheduled for de novo catheter ablation, were eligible. Before pulmonary vein isolation, AF was mapped and then iteratively remapped to guide each subsequent ablation of charge density-identified targets. AF recurrence was evaluated at 3, 6, 9, and 12 months using continuous 24-hour ECG monitors. The primary effectiveness outcome was freedom from AF >30 seconds at 12 months for a single procedure with a secondary outcome being acute procedural efficacy. The primary safety outcome was freedom from device/procedure-related major adverse events. Results Between October 2016 and April 2017, 129 patients were enrolled, and 127 underwent mapping and catheter ablation. Acute procedural efficacy was demonstrated in 125 patients (98%). At 12 months, single procedure freedom from AF on or off antiarrhythmic drugs was 72.5% (95% CI, 63.9%-80.3%). After 1 or 2 procedures, freedom from AF was 93.2% (95% CI, 87.1%-97.0%). A total of 29 (23%) retreatments because of arrhythmia recurrence were performed with average time from index procedure to first retreatment being 7 months. The primary safety outcome was 98% with no device-related major adverse events reported. Conclusions This novel ultrasound imaging and charge density mapping system safely guided ablation of nonpulmonary vein targets in persistent AF patients with 73% single procedure and 93% second procedure freedom from AF at 12 months. Clinical Trial Registration URL: https://www.clinicaltrials.gov . Unique identifier: NCT02825992 EU/NCT02462980 CN.
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Potenciales de Acción , Fibrilación Atrial/diagnóstico por imagen , Técnicas Electrofisiológicas Cardíacas , Sistema de Conducción Cardíaco/diagnóstico por imagen , Frecuencia Cardíaca , Ultrasonografía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/cirugía , Canadá , Ablación por Catéter , Electrocardiografía Ambulatoria , Europa (Continente) , Femenino , Sistema de Conducción Cardíaco/fisiopatología , Sistema de Conducción Cardíaco/cirugía , Humanos , Masculino , Persona de Mediana Edad , Ensayos Clínicos Controlados no Aleatorios como Asunto , Valor Predictivo de las Pruebas , Supervivencia sin Progresión , Estudios Prospectivos , Venas Pulmonares/fisiopatología , Recurrencia , Factores de Tiempo , Adulto JovenRESUMEN
AIMS: Video-assisted thoracoscopic surgery (VATS) ablation has been advocated as a treatment option for non-paroxysmal atrial fibrillation (AF) in recent guidelines. Real-life data on its safety and efficacy during a centre's early experience are sparse. METHODS AND RESULTS: Thirty patients (28 persistent/longstanding persistent AF) underwent standalone VATS ablation for AF by an experienced thoracoscopic surgeon, with the first 20 cases proctored by external surgeons. Procedural and follow-up outcomes were collected prospectively, and compared with 90 propensity-matched patients undergoing contemporaneous catheter ablation (CA). Six (20.0%) patients undergoing VATS ablation experienced ≥1 major complication (death n = 1, stroke n = 2, conversion to sternotomy n = 3, and phrenic nerve injury n = 2). This was significantly higher than the 1.1% major complication rate (tamponade requiring drainage n = 1) seen with CA (P < 0.001). Twelve-month single procedure arrhythmia-free survival rates without antiarrhythmic drugs were 56% in the VATS and 57% in the CA cohorts (P = 0.22), and 78% and 80%, respectively given an additional CA and antiarrhythmic drugs (P = 0.32). CONCLUSION: During a centre's early experience, VATS ablation may have similar success rates to those from an established CA service, but carry a greater risk of major complications. Those embarking on a programme of VATS AF ablation should be aware that complication and success rates may differ from those reported by selected high-volume centres.