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1.
Biochemistry ; 60(38): 2902-2914, 2021 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-34491035

RESUMEN

Citrullination is an enzyme-catalyzed post-translational modification (PTM) that is essential for a host of biological processes, including gene regulation, programmed cell death, and organ development. While this PTM is required for normal cellular functions, aberrant citrullination is a hallmark of autoimmune disorders as well as cancer. Although aberrant citrullination is linked to human pathology, the exact role of citrullination in disease remains poorly characterized, in part because of the challenges associated with identifying the specific arginine residues that are citrullinated. Tandem mass spectrometry is the most precise method for uncovering sites of citrullination; however, due to the small mass shift (+0.984 Da) that results from citrullination, current database search algorithms commonly misannotate spectra, leading to a high number of false-positive assignments. To address this challenge, we developed an automated workflow to rigorously and rapidly mine proteomic data to unambiguously identify the sites of citrullination from complex peptide mixtures. The crux of this streamlined workflow is the ionFinder software program, which classifies citrullination sites with high confidence on the basis of the presence of diagnostic fragment ions. These diagnostic ions include the neutral loss of isocyanic acid, which is a dissociative event that is unique to citrulline residues. Using the ionFinder program, we have mapped the sites of autocitrullination on purified protein arginine deiminases (PAD1-4) and mapped the global citrullinome in a PAD2-overexpressing cell line. The ionFinder algorithm is a highly versatile, user-friendly, and open-source program that is agnostic to the type of instrument and mode of fragmentation that are used.


Asunto(s)
Citrulinación/fisiología , Minería de Datos/métodos , Proteómica/métodos , Algoritmos , Arginina/metabolismo , Citrulinación/genética , Citrulina/química , Citrulina/genética , Citrulina/metabolismo , Análisis de Datos , Manejo de Datos/métodos , Humanos , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Espectrometría de Masas en Tándem/métodos
2.
Circ Res ; 125(5): 507-519, 2019 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-31248335

RESUMEN

RATIONALE: PAD4 (peptidylarginine deiminase type IV), an enzyme essential for neutrophil extracellular trap formation (NETosis), is released together with neutrophil extracellular traps into the extracellular milieu. It citrullinates histones and holds the potential to citrullinate other protein targets. While NETosis is implicated in thrombosis, the impact of the released PAD4 is unknown. OBJECTIVE: This study tests the hypothesis that extracellular PAD4, released during inflammatory responses, citrullinates plasma proteins, thus affecting thrombus formation. METHODS AND RESULTS: Here, we show that injection of r-huPAD4 in vivo induces the formation of VWF (von Willebrand factor)-platelet strings in mesenteric venules and that this is dependent on PAD4 enzymatic activity. VWF-platelet strings are naturally cleaved by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motif-13). We detected a reduction of endogenous ADAMTS13 activity in the plasma of wild-type mice injected with r-huPAD4. Using mass spectrometry and in vitro studies, we found that r-huPAD4 citrullinates ADAMTS13 on specific arginine residues and that this modification dramatically inhibits ADAMTS13 enzymatic activity. Elevated citrullination of ADAMTS13 was observed in plasma samples of patients with sepsis or noninfected patients who were elderly (eg, age >65 years) and had underlying comorbidities (eg, diabetes mellitus and hypertension) as compared with healthy donors. This shows that ADAMTS13 is citrullinated in vivo. VWF-platelet strings that form on venules of Adamts13-/- mice were immediately cleared after injection of r-huADAMTS13, while they persisted in vessels of mice injected with citrullinated r-huADAMTS13. Next, we assessed the effect of extracellular PAD4 on platelet-plug formation after ferric chloride-induced injury of mesenteric venules. Administration of r-huPAD4 decreased time to vessel occlusion and significantly reduced thrombus embolization. CONCLUSIONS: Our data indicate that PAD4 in circulation reduces VWF-platelet string clearance and accelerates the formation of a stable platelet plug after vessel injury. We propose that this effect is, at least in part, due to ADAMTS13 inhibition.


Asunto(s)
Plaquetas/metabolismo , Arginina Deiminasa Proteína-Tipo 4/sangre , Trombosis/sangre , Lesiones del Sistema Vascular/sangre , Factor de von Willebrand/metabolismo , Anciano , Animales , Plaquetas/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Arginina Deiminasa Proteína-Tipo 4/toxicidad , Trombosis/inducido químicamente , Lesiones del Sistema Vascular/inducido químicamente , Adulto Joven
3.
Angew Chem Int Ed Engl ; 58(36): 12476-12480, 2019 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-31276611

RESUMEN

Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.


Asunto(s)
Citrulinación/efectos de los fármacos , Citrulina/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Arginina Deiminasa Proteína-Tipo 1/antagonistas & inhibidores , Animales , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/enzimología , Células HEK293 , Histonas/química , Humanos , Isoenzimas , Ratones , Arginina Deiminasa Proteína-Tipo 1/metabolismo
4.
Biochemistry ; 55(32): 4509-18, 2016 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-27404806

RESUMEN

Enzymes belonging to the GNAT superfamily are widely distributed in nature where they play key roles in the transfer of acyl groups from acyl-CoAs to primary amine acceptors. The amine acceptors run the gamut from histones to aminoglycoside antibiotics to small molecules such as serotonin. Whereas those family members that function on histones have been extensively studied, the GNAT enzymes that employ nucleotide-linked sugars as their substrates have not been well characterized. Indeed, though the structures of two of these "amino sugar" GNAT enzymes have been determined within the past 10 years, details concerning their active site architectures have been limited because of a lack of bound nucleotide-linked sugar substrates. Here we describe a combined structural and biochemical analysis of FdhC from Acinetobacter nosocomialis O2. On the basis of bioinformatics, it was postulated that FdhC catalyzes the transfer of a 3-hydroxybutanoyl group from 3-hydroxylbutanoyl-CoA to dTDP-3-amino-3,6-dideoxy-d-galactose, to yield an unusual sugar that is ultimately incorporated into the surface polysaccharides of the bacterium. We present data confirming this activity. In addition, the structures of two ternary complexes of FdhC, in the presence of CoA and either 3-hydroxybutanoylamino-3,6-dideoxy-d-galactose or 3-hydroxybutanoylamino-3,6-dideoxy-d-glucose, were solved by X-ray crystallographic analyses to high resolution. Kinetic parameters were determined, and activity assays demonstrated that FdhC can also utilize acetyl-CoA, 3-methylcrotonyl-CoA, or hexanoyl-CoA as acyl donors, albeit at reduced rates. Site-directed mutagenesis experiments were conducted to probe the catalytic mechanism of FdhC. Taken together, the data presented herein provide significantly new molecular insight into those GNAT superfamily members that function on nucleotide-linked amino sugars.


Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , Acinetobacter/enzimología , Biocatálisis , Cinética , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta
5.
Biochemistry ; 54(29): 4495-506, 2015 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-26125548

RESUMEN

3-Acetamido-3,6-dideoxy-d-galactose (Fuc3NAc) and 3-acetamido-3,6-dideoxy-d-glucose (Qui3NAc) are unusual sugars found on the lipopolysaccharides of Gram-negative bacteria and on the S-layers of Gram-positive bacteria. The 3,4-ketoisomerases, referred to as FdtA and QdtA, catalyze the third steps in the respective biosynthetic pathways for these sugars. Whereas both enzymes utilize the same substrate, the stereochemistries of their products are different. Specifically, the hydroxyl groups at the hexose C-4' positions assume the "galactose" and "glucose" configurations in the FdtA and QdtA products, respectively. In 2007 we reported the structure of the apoform of FdtA from Aneurinibacillus thermoaerophilus, which was followed in 2014 by the X-ray analysis of QdtA from Thermoanaerobacterium thermosaccharolyticum as a binary complex. Both of these enzymes belong to the cupin superfamily. Here we report a combined structural and enzymological study to explore the manner in which these enzymes control the stereochemistry of their products. Various site-directed mutant proteins of each enzyme were constructed, and their dTDP-sugar products were analyzed by NMR spectroscopy. In addition, the kinetic parameters for these protein variants were measured, and the structure of one, namely, the QdtA Y17R/R97H double mutant form, was determined to 2.3-Å resolution. Finally, in an attempt to obtain a model of FdtA with a bound dTDP-linked sugar, the 3,4-ketoisomerase domain of a bifunctional enzyme from Shewanella denitrificans was cloned, purified, and crystallized in the presence of a dTDP-linked sugar analogue. Taken together, the results from this investigation demonstrate that it is possible to convert a "galacto" enzyme into a "gluco" enzyme and vice versa.


Asunto(s)
Isomerasas Aldosa-Cetosa/química , Proteínas Bacterianas/química , Secuencia de Aminoácidos , Bacillales/enzimología , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Shewanella/enzimología , Estereoisomerismo
6.
Sci Adv ; 9(51): eadj1397, 2023 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-38117877

RESUMEN

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosis-induced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils.


Asunto(s)
Trampas Extracelulares , Neutrófilos , Neutrófilos/metabolismo , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Arginina Deiminasa Proteína-Tipo 4/genética , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Trampas Extracelulares/genética , Trampas Extracelulares/metabolismo , Histonas/metabolismo , Epigénesis Genética
7.
Cell Chem Biol ; 28(12): 1728-1739.e5, 2021 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-34352225

RESUMEN

Aberrant protein citrullination is associated with many pathologies; however, the specific effects of this modification remain unknown. We have previously demonstrated that serine protease inhibitors (SERPINs) are highly citrullinated in rheumatoid arthritis (RA) patients. These citrullinated SERPINs include antithrombin, antiplasmin, and t-PAI, which regulate the coagulation and fibrinolysis cascades. Notably, citrullination eliminates their inhibitory activity. Here, we demonstrate that citrullination of antithrombin and t-PAI impairs their binding to their cognate proteases. By contrast, citrullination converts antiplasmin into a substrate. We recapitulate the effects of SERPIN citrullination using in vitro plasma clotting and fibrinolysis assays. Moreover, we show that citrullinated antithrombin and antiplasmin are increased and decreased in a deep vein thrombosis (DVT) model, accounting for how SERPIN citrullination shifts the equilibrium toward thrombus formation. These data provide a direct link between increased citrullination and the risk of thrombosis in autoimmunity and indicate that aberrant SERPIN citrullination promotes pathological thrombus formation.


Asunto(s)
Antifibrinolíticos/farmacología , Antitrombinas/farmacología , Inactivadores Plasminogénicos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Trombosis de la Vena/tratamiento farmacológico , Animales , Antifibrinolíticos/química , Antitrombinas/química , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Péptido Hidrolasas/metabolismo , Inactivadores Plasminogénicos/química , Inhibidores de Serina Proteinasa/química , Trombosis de la Vena/metabolismo
8.
Nat Commun ; 12(1): 3910, 2021 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-34162877

RESUMEN

Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.


Asunto(s)
Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Procesamiento Proteico-Postraduccional , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Chlorocebus aethiops , Citrulinación , Citomegalovirus/fisiología , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/virología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Proteínas de Resistencia a Mixovirus/metabolismo , Desiminasas de la Arginina Proteica/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Vero , Proteínas Virales/metabolismo
9.
ACS Chem Biol ; 14(4): 613-618, 2019 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-30933557

RESUMEN

Nicotinamide N-methyltransferase (NNMT) catalyzes the S-adenosyl-l-methionine-dependent methylation of nicotinamide to form N-methylnicotinamide. This enzyme detoxifies xenobiotics and regulates NAD+ biosynthesis. Additionally, NNMT is overexpressed in various cancers. Herein, we describe the first NNMT-targeted suicide substrates. These compounds, which include 4-chloropyridine and 4-chloronicotinamide, exploit the broad substrate scope of NNMT; methylation of the pyridine nitrogen enhances the electrophilicity of the C4 position, thereby promoting an aromatic nucleophilic substitution by C159, a noncatalytic cysteine. On the basis of this activity, we developed a suicide inhibition-based protein labeling strategy using an alkyne-substituted 4-chloropyridine that selectively labels NNMT in vitro and in cells. In total, this study describes the first NNMT-directed activity-based probes.


Asunto(s)
Nicotinamida N-Metiltransferasa/metabolismo , Proteínas/metabolismo , Catálisis , Células HEK293 , Humanos , Cinética , Metilación , Nicotinamida N-Metiltransferasa/antagonistas & inhibidores
10.
ACS Chem Biol ; 13(9): 2663-2672, 2018 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-30044909

RESUMEN

Nicotinamide- N-methyltransferase (NNMT) catalyzes the irreversible methylation of nicotinamide (NAM) to form N-methyl nicotinamide using S-adenosyl methionine as a methyl donor. NNMT is implicated in several chronic disease conditions, including cancers, kidney disease, cardiovascular disease, and Parkinson's disease. Although phosphorylation of NNMT in gastric tumors is reported, the functional effects of this post-translational modification has not been investigated. We previously reported that citrullination of NNMT by Protein Arginine Deiminases abolished its methyltransferase activity. Herein, we investigate the mechanism of inactivation. Using tandem mass spectrometry, we identified three sites of citrullination in NNMT. With this information in hand, we used a combination of site-directed mutagenesis, kinetics, and circular dichoism experiments to demonstrate that citrullination of R132 leads to a structural perturbation that ultimately promotes NNMT inactivation.


Asunto(s)
Citrulinación , Nicotinamida N-Metiltransferasa/metabolismo , Activación Enzimática , Humanos , Cinética , Metilación , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Nicotinamida N-Metiltransferasa/química , Nicotinamida N-Metiltransferasa/genética , Conformación Proteica
11.
ACS Chem Biol ; 13(3): 712-722, 2018 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-29341591

RESUMEN

Citrullination is the post-translational hydrolysis of peptidyl-arginines to form peptidyl-citrulline, a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calcium-regulated enzymes. Aberrantly increased protein citrullination is associated with a slew of autoimmune diseases (e.g., rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis) and certain cancers. Given the clear link between increased PAD activity and human disease, the PADs are therapeutically relevant targets. Herein, we report the development of next generation cell permeable and "clickable" probes (BB-Cl-Yne and BB-F-Yne) for covalent labeling of the PADs both in vitro and in cell-based systems. Using advanced chemoproteomic technologies, we also report the off targets of both BB-Cl-Yne and BB-F-Yne. The probes are highly specific for the PADs, with relatively few off targets, especially BB-F-Yne, suggesting the preferential use of the fluoroacetamidine warhead in next generation irreversible PAD inhibitors. Notably, these compounds can be used in a variety of modalities, including the identification of off targets of the parent compounds and as activity-based protein profiling probes in target engagement assays to demonstrate the efficacy of PAD inhibitors.


Asunto(s)
Bencimidazoles/química , Sondas Moleculares/química , Desiminasas de la Arginina Proteica/análisis , Coloración y Etiquetado/métodos , Enfermedades Autoinmunes , Citrulina , Química Clic , Fluoroacetatos/farmacología , Desiminasas de la Arginina Proteica/antagonistas & inhibidores , Proteómica
12.
Cell Chem Biol ; 25(6): 691-704.e6, 2018 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-29628436

RESUMEN

Increased protein citrullination is linked to various diseases including rheumatoid arthritis (RA), lupus, and cancer. Citrullinated autoantigens, a hallmark of RA, are recognized by anti-citrullinated protein antibodies (ACPAs) which are used to diagnose RA. ACPA-recognizing citrullinated enolase, vimentin, keratin, and filaggrin are also pathogenic. Here, we used a chemoproteomic approach to define the RA-associated citrullinome. The identified proteins include numerous serine protease inhibitors (Serpins), proteases and metabolic enzymes. We demonstrate that citrullination of antiplasmin, antithrombin, t-PAI, and C1 inhibitor (P1-Arg-containing Serpins) abolishes their ability to inhibit their cognate proteases. Citrullination of nicotinamide N-methyl transferase (NNMT) also abolished its methyltransferase activity. Overall, these data advance our understanding of the roles of citrullination in RA and suggest that extracellular protein arginine deiminase (PAD) activity can modulate protease activity with consequent effects on Serpin-regulated pathways. Moreover, our data suggest that inhibition of extracellular PAD activity will be therapeutically relevant.


Asunto(s)
Artritis Reumatoide/metabolismo , Citrulina/metabolismo , Proteínas Filagrina , Humanos , Proteómica
13.
Protein Sci ; 24(10): 1633-9, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26174084

RESUMEN

Yersinia enterocolitica is a Gram-negative bacterium that causes yersiniosis, a zoonotic disease affecting the gastrointestinal tract of humans, cattle, and pigs, among others. The lipopolysaccharide of Y. enterocolitica O:8 contains an unusual sugar, 6-deoxy-d-gulose, which requires four enzymes for its biosynthesis. Here, we describe a combined structural and functional investigation of WbcA, which catalyzes the third step in the pathway, namely an epimerization about the C-3' carbon of a CDP-linked sugar. The structure of WbcA was determined to 1.75-Å resolution, and the model was refined to an overall R-factor of 19.5%. The fold of WbcA places it into the well-defined cupin superfamily of sugar epimerases. Typically, these enzymes contain both a conserved histidine and a tyrosine residue that play key roles in catalysis. On the basis of amino acid sequence alignments, it was anticipated that the "conserved" tyrosine had been replaced with a cysteine residue in WbcA (Cys 133), and indeed this was the case. However, what was not anticipated was the fact that another tyrosine residue (Tyr 50) situated on a neighboring ß-strand moved into the active site. Site-directed mutant proteins were subsequently constructed and their kinetic properties analyzed to address the roles of Cys 133 and Tyr 50 in WbcA catalysis. This study emphasizes the continuing need to experimentally verify assumptions that are based solely on bioinformatics approaches.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/metabolismo , Modelos Moleculares , Yersinia enterocolitica/enzimología , Secuencia de Carbohidratos , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Estructura Terciaria de Proteína
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