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1.
Cell ; 184(17): 4480-4494.e15, 2021 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-34320407

RESUMEN

In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.


Asunto(s)
Fagocitosis , Fosfofructoquinasa-1 Tipo Hepático/metabolismo , Estallido Respiratorio , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Proteínas de Unión a Fosfato/metabolismo , Fosfofructoquinasa-1 Tipo Hepático/antagonistas & inhibidores , Fosfofructoquinasa-1 Tipo Hepático/ultraestructura , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes/aislamiento & purificación , Estallido Respiratorio/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología
2.
Chembiochem ; 17(9): 825-35, 2016 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-26840534

RESUMEN

Emerging antibiotic resistance among human pathogens has galvanized efforts to find alternative routes to combat bacterial virulence. One new approach entails interfering with the ability of bacteria to coordinate population-wide gene expression, or quorum sensing (QS), thus inhibiting the production of virulence factors and biofilm formation. We have recently developed such a strategy by targeting LasR, the master regulator of QS in the opportunistic human pathogen Pseudomonas aeruginosa, through the rational design of covalent inhibitors closely based on the core structure of the native ligand. We now report several groups of new inhibitors, one of which, fluoro-substituted ITC-12, displayed complete covalent modification of LasR, as well as effective QS inhibition in vitro and promising in vivo results. In addition to their potential clinical relevance, this series of synthetic QS modulators can be used as a tool to further unravel the complicated QS regulation in P. aeruginosa.


Asunto(s)
Antibacterianos/farmacología , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/efectos de los fármacos , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Antibacterianos/química , Proteínas Bacterianas/agonistas , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biopelículas/efectos de los fármacos , Dominio Catalítico , Dicroismo Circular , Isotiocianatos/química , Isotiocianatos/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Estrés Oxidativo/efectos de los fármacos , Transactivadores/agonistas , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Factores de Virulencia/metabolismo
3.
Pathogens ; 11(11)2022 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-36364996

RESUMEN

Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.

4.
J Am Chem Soc ; 133(19): 7469-75, 2011 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-21513325

RESUMEN

Live cell fluorescent labeling of proteins has become a seminal tool in biology and has led to hallmark discoveries in diverse research areas such as protein trafficking, cell-to-cell interactions, and intracellular network dynamics. One of the main challenges, however, remains the ability to label intracellular proteins using fluorescent ligands with high specificity, all the while retaining viability of the targeted cells. Here, we present the first example of live cell labeling and imaging of an intracellular bacterial receptor involved in cell-to-cell communication (i.e., quorum sensing), using a novel two-step approach involving covalent attachment of a reactive mimic of the primary endogenous Pseudomonas aeruginosa quorum-sensing signal to its receptor, LasR, followed by aniline-catalyzed oxime formation between the modified receptor and a fluorescent BODIPY derivative. Our results indicate that LasR is not distributed evenly throughout the cytoplasmic membrane but is instead concentrated at the poles of the P. aeruginosa cell.


Asunto(s)
Compuestos de Anilina/química , Colorantes Fluorescentes/química , Oximas/química , Pseudomonas aeruginosa , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Datos de Secuencia Molecular , Estructura Molecular , Percepción de Quorum/fisiología , Receptores de Superficie Celular/química , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo
5.
Proc Natl Acad Sci U S A ; 105(45): 17487-92, 2008 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-18981425

RESUMEN

Obesity is a chronic, costly, and globally prevalent condition, with excess caloric intake a suspected etiologic factor. Nonsurgical treatments are modestly efficacious, and weight loss maintenance is hampered by anti-famine homeostatic mechanisms. Ghrelin, a gastric hormone linked to meal initiation, energy expenditure, and fuel partitioning, is hypothesized to facilitate weight gain and impede weight loss. Unique among known animal peptides, the serine-3 residue of ghrelin is posttranslationally acylated with an n-octanoic acid, a modification important for the peptide's active blood-brain transport and growth hormone secretagogue receptor-1 agonist activity. Pharmacological degradation of ghrelin would be hypothesized to reduce ghrelin's biological effects. To study endogenous ghrelin's role in appetite and energy expenditure, we generated antibodies that hydrolyze the octanoyl moiety of ghrelin to form des-acyl ghrelin. The most proficient antibody catalyst, GHR-11E11, was found to display a second-order rate constant of 18 M(-1) x s(-1) for the hydrolysis of ghrelin to des-acyl ghrelin. I.v. administration of GHR-11E11 (50 mg/kg) maintained a greater metabolic rate in fasting C57BL/6J mice as compared with mice receiving a control antibody and suppressed 6-h refeeding after 24 h of food deprivation. Indirect respiratory measures of metabolism after refeeding and relative fuel substrate utilization were unaffected. The results support the hypothesis that acylated ghrelin stimulates appetite and curbs energy expenditure during deficient energy intake, whereas des-acyl ghrelin does not potently share these functions. Catalytic anti-ghrelin antibodies might thereby adjunctively aid consolidation of caloric restriction-induced weight loss and might also be therapeutically relevant to Prader-Willi syndrome, characterized after infancy by hyperghrelinemia, hyperphagia, and obesity.


Asunto(s)
Anticuerpos Catalíticos/metabolismo , Apetito/fisiología , Metabolismo Energético/fisiología , Ayuno/metabolismo , Ghrelina/metabolismo , Obesidad/metabolismo , Animales , Anticuerpos Catalíticos/farmacología , Cromatografía de Afinidad , Ghrelina/farmacología , Hidrólisis , Masculino , Ratones , Ratones Endogámicos C57BL
6.
Cell Chem Biol ; 28(10): 1501-1513.e5, 2021 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-34043961

RESUMEN

The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here, we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short-chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small-molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications.


Asunto(s)
Metabolismo de los Lípidos/fisiología , Proteínas Protozoarias/metabolismo , Serina Endopeptidasas/metabolismo , Toxoplasma/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Hidrólisis , Cinética , Filogenia , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Serina Endopeptidasas/clasificación , Serina Endopeptidasas/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Especificidad por Sustrato , Toxoplasma/crecimiento & desarrollo , Toxoplasma/fisiología
7.
J Am Chem Soc ; 131(30): 10610-9, 2009 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-19585989

RESUMEN

Chemical coordination of gene expression among bacteria as a function of population density is regulated by a mechanism known as 'quorum sensing' (QS). QS in Pseudomonas aeruginosa, an opportunistic pathogen that causes disease in immunocompromised patients, is mediated by binding of the transcriptional activator, LasR, to its ligand, 3-oxo-C(12)-HSL, leading to population-wide secretion of virulence factors and biofilm formation. We have targeted QS in P. aeruginosa with a set of electrophilic probes designed to covalently bind Cys79 in the LasR binding pocket, leading to specific inhibition of QS-regulated gene expression and concomitant reduction of virulence factor secretion and biofilm formation. This first example of covalent modification of a QS receptor provides a new tool to study molecular mechanisms of bacterial group behavior and could lead to new strategies for targeting bacterial virulence.


Asunto(s)
Pseudomonas/citología , Percepción de Quorum/efectos de los fármacos , Alquilación , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cisteína/metabolismo , Isotiocianatos/química , Isotiocianatos/farmacología , Modelos Moleculares , Estructura Terciaria de Proteína , Especificidad por Sustrato , Transactivadores/química , Transactivadores/metabolismo
9.
Cell Chem Biol ; 26(1): 35-47.e7, 2019 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-30393067

RESUMEN

Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, and TgPPT1 from the parasite Toxoplasma gondii, have distinct specificities that depend on amino acid residues distal to the palmitoyl cysteine. This information informs the design of optimal and non-optimal substrates as well as isoform-selective substrates to detect the activity of a specific depalmitoylase in complex proteomes. In addition to providing tools for studying depalmitoylases, our findings identify a previously unrecognized mechanism for regulating steady-state levels of distinct palmitoylation sites by sequence-dependent control of depalmitoylation rates.


Asunto(s)
Colorantes Fluorescentes/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Tioléster Hidrolasas/metabolismo , Animales , Línea Celular , Femenino , Colorantes Fluorescentes/química , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Péptidos/química , Especificidad por Sustrato , Toxoplasma/metabolismo
11.
ACS Chem Biol ; 13(9): 2645-2654, 2018 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-30160940

RESUMEN

Matrix metalloproteases (MMPs) are a large family of zinc-dependent endopeptidases involved in a diverse set of physiological and pathological processes, most notably in cancer. Current methods for imaging and quantifying MMP activity lack sufficient selectivity and spatiotemporal resolution to allow studies of specific MMP function in vivo. Previously, we reported a strategy for selective targeting of MMPs by engineering a functionally silent cysteine mutation that enables highly specific covalent modification by a designed activity-based probe. Here, we describe the translation of that technology into a mouse model of breast cancer and subsequent demonstration of the utility of the approach for studies of MMP-14 activation in the tumor microenvironment. Using this approach, we find that MMP-14 is active in late stage tumors and is predominantly associated with stromal cell populations that have been activated by specific signaling molecules (e.g., TGFß) produced by tumor cells. Our data demonstrate the applicability of this approach for studies of MMP function in whole organisms and identify important regulatory mechanisms for MMP-14 activity in the tumor microenvironment.


Asunto(s)
Neoplasias de la Mama/enzimología , Metaloproteinasa 14 de la Matriz/metabolismo , Microambiente Tumoral , Animales , Mama/citología , Mama/enzimología , Mama/patología , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Activación Enzimática , Femenino , Humanos , Metaloproteinasa 14 de la Matriz/análisis , Ratones , Técnicas de Sonda Molecular , Células del Estroma/enzimología , Células del Estroma/patología
12.
mSphere ; 3(5)2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-30232166

RESUMEN

Hydrolase are enzymes that regulate diverse biological processes, including posttranslational protein modifications. Recent work identified four active serine hydrolases (ASHs) in Toxoplasma gondii as candidate depalmitoylases. However, only TgPPT1 (ASH1) has been confirmed to remove palmitate from proteins. ASH4 (TgME49_264290) was reported to be refractory to genetic disruption. We demonstrate that recombinant ASH4 is an esterase that processes short acyl esters but not palmitoyl thioesters. Genetic disruption of ASH4 causes defects in cell division and premature scission of parasites from residual bodies. These defects lead to the presence of vacuoles with a disordered intravacuolar architecture, with parasites arranged in pairs around multiple residual bodies. Importantly, we found that the deletion of ASH4 correlates with a defect in radial dispersion from host cells after egress. This defect in dispersion of parasites is a general phenomenon that is observed for disordered vacuoles that occur at low frequency in wild-type parasites, suggesting a possible general link between intravacuolar organization and dispersion after egress.IMPORTANCE This work defines the function of an enzyme in the obligate intracellular parasite Toxoplasma gondii We show that this previously uncharacterized enzyme is critical for aspects of cellular division by the parasite and that loss of this enzyme leads to parasites with cell division defects and which also are disorganized inside their vacuoles. This leads to defects in the ability of the parasite to disseminate from the site of an infection and may have a significant impact on the parasite's overall infectivity of a host organism.


Asunto(s)
Hidrolasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasma/crecimiento & desarrollo , Vacuolas/parasitología , División Celular , Línea Celular , Interacciones Huésped-Parásitos , Humanos , Hidrolasas/genética , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/genética , Serina/genética , Homología Estructural de Proteína , Toxoplasma/genética , Toxoplasmosis
14.
Science ; 358(6369): 1448-1453, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29146868

RESUMEN

Reversible detyrosination of α-tubulin is crucial to microtubule dynamics and functions, and defects have been implicated in cancer, brain disorganization, and cardiomyopathies. The identity of the tubulin tyrosine carboxypeptidase (TCP) responsible for detyrosination has remained unclear. We used chemical proteomics with a potent irreversible inhibitor to show that the major brain TCP is a complex of vasohibin-1 (VASH1) with the small vasohibin binding protein (SVBP). VASH1 and its homolog VASH2, when complexed with SVBP, exhibited robust and specific Tyr/Phe carboxypeptidase activity on microtubules. Knockdown of vasohibins or SVBP and/or inhibitor addition in cultured neurons reduced detyrosinated α-tubulin levels and caused severe differentiation defects. Furthermore, knockdown of vasohibins disrupted neuronal migration in developing mouse neocortex. Thus, vasohibin/SVBP complexes represent long-sought TCP enzymes.


Asunto(s)
Proteínas Angiogénicas/metabolismo , Carboxipeptidasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neurogénesis , Neuronas/citología , Tirosina/metabolismo , Proteínas Angiogénicas/genética , Animales , Carboxipeptidasas/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Movimiento Celular , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Ratones , Neocórtex/citología , Neocórtex/embriología , Neuronas/enzimología , Proteómica , Tubulina (Proteína)/metabolismo
16.
J Mol Biol ; 416(1): 21-32, 2012 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-22197379

RESUMEN

Broad specificity is believed to be a property of primordial enzymes that diverged during natural protein evolution to produce highly specific and efficient enzymes. Human estrogen sulfotransferase (SULT1E1) is a broad-specificity enzyme that detoxifies a variety of chemicals, including estrogens, by the transfer of sulfate. To study the molecular basis for the broad specificity of this enzyme and to investigate the process of SULT1E1 specialization, we have adopted a directed enzyme evolution approach. Using two iterative rounds of evolution, we generated SULT1E1 mutants with increased thermostability and narrower specificity from the broadly specific wild-type enzyme. To identify mutants with enhanced specificity, we developed an unbiased screening assay to assess sulfate transfer to three different acceptors in parallel. Such an assay enabled the isolation of SULT1E1 mutants with enhanced or wild-type activity toward an estrogen acceptor and significantly reduced activity for phenol or coumarin type of acceptors, leading to up to 3 orders of magnitude increase in specificity. We found that mutations conferring novel specificity are located in the vicinity of the active site and thus may play a direct role in reshaping the acceptor-binding site. Finally, such mutations resulted in reduced SULT1E1 thermostability, revealing a trade-off between SULT1E1 thermostability and acquisition of novel function.


Asunto(s)
Proteínas Mutantes/química , Proteínas Mutantes/genética , Sulfotransferasas/química , Sulfotransferasas/genética , Sitios de Unión , Evolución Molecular Dirigida , Estrógenos/genética , Biblioteca de Genes , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Especificidad por Sustrato , Sulfatos/metabolismo , Sulfotransferasas/metabolismo
17.
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