RESUMEN
Glycation is a non-enzymatic reaction wherein sugars or dicarbonyls such as methylglyoxal (MGO) and glyoxal (GO) react with proteins, leading to protein inactivation. The hydrolysing enzyme human deglycase-1 (hDJ-1) is reported to decrease glycative stress by deglycating the modified proteins, specifically at cysteine, lysine, and arginine sites. This specificity of hDJ-1 is thought to be regulated by its active site cysteine residue (Cys106). Structural analysis of hDJ-1 by molecular docking and simulation studies, however, indicates a possible role of glutamate (Glu18) in determining its substrate specificity. To elucidate this, Glu18 present at the catalytic site of hDJ-1 was modified to aspartate (Asp18) by SDM, and the resultant mutant was termed mutant DJ-1 (mDJ-1). Both hDJ-1 and mDJ-1 were heterologously expressed in Escherichia coli BL21 (DE3) strain and purified to homogeneity. The hDJ-1 showed kcat values of 1.45 × 103 s-1, 3.6 × 102 s-1, and 3.1 × 102 s-1, and Km values 0.181 mM, 18.18 mM, and 12.5 mM for N-acetylcysteine (NacCys), N-acetyllysine (NacLys), and N-acetylarginine (NacArg), respectively. The mDJ-1 showed altered kcat values (8 × 102 s-1, 3.8 × 102 s-1, 4.9 × 102 s-1) and Km values of 0.14 mM, 6.25 mM, 5.88 mM for NacCys, NacLys and NacArg, respectively. A single amino acid change (Glu18 to Asp18) improved the substrate specificity of mDJ-1 toward NacLys and NacArg. Understanding hDJ-1's structure and enhanced functionality will facilitate further exploration of its therapeutic potential for the treatment of glycation-induced diabetic complications.
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Glioxal , Piruvaldehído , Humanos , Simulación del Acoplamiento Molecular , Especificidad por Sustrato , Glioxal/metabolismo , Piruvaldehído/metabolismo , Acetilcisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , CinéticaRESUMEN
BACKGROUND: Functional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential. RESULTS: Here, we identify a Parkinson-associated DJ-1/PARK7-like protein-glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A), with bacterial evolutionary affinities although not from alphaproteobacteria. We demonstrate that GATD3A localizes to the mitochondrial matrix and functions as a deglycase. Through its amidolysis domain, GATD3A removes non-enzymatic chemical modifications produced during the Maillard reaction between dicarbonyls and amines of nucleotides and amino acids. GATD3A interacts with factors involved in mitochondrial mRNA processing and translation, suggestive of a role in maintaining integrity of important biomolecules through its deglycase activity. The loss of GATD3A in mice is associated with accumulation of advanced glycation end products (AGEs) and altered mitochondrial dynamics. CONCLUSIONS: An evolutionary perspective helped us prioritize a previously uncharacterized but predicted mitochondrial protein GATD3A, which mediates the removal of early glycation intermediates. GATD3A restricts the formation of AGEs in mitochondria and is a relevant target for diseases where AGE deposition is a pathological hallmark.
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Gammaproteobacteria , Productos Finales de Glicación Avanzada , Animales , Gammaproteobacteria/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Mamíferos , Ratones , Proteínas Mitocondriales/genética , Proteína Desglicasa DJ-1/metabolismoRESUMEN
Human DJ-1 is a cytoprotective protein whose absence causes Parkinson's disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.
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Estrés Oxidativo , Piruvaldehído , Animales , Glicosilación , Guanina , Humanos , Ratones , Neuronas/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Piruvaldehído/química , Piruvaldehído/metabolismoRESUMEN
Protein deglycase DJ-1 (DJ-1) is a multifunctional protein involved in various biological processes. However, it is unclear whether DJ-1 influences atherosclerosis development and plaque stability. Accordingly, we evaluated the influence of DJ-1 deletion on the progression of atherosclerosis and elucidate the underlying mechanisms. We examine the expression of DJ-1 in atherosclerotic plaques of human and mouse models which showed that DJ-1 expression was significantly decreased in human plaques compared with that in healthy vessels. Consistent with this, the DJ-1 levels were persistently reduced in atherosclerotic lesions of ApoE-/- mice with the increasing time fed by western diet. Furthermore, exposure of vascular smooth muscle cells (VSMCs) to oxidized low-density lipoprotein down-regulated DJ-1 in vitro. The canonical markers of plaque stability and VSMC phenotypes were evaluated in vivo and in vitro. DJ-1 deficiency in Apoe-/- mice promoted the progression of atherosclerosis and exaggerated plaque instability. Moreover, isolated VSMCs from Apoe-/- DJ-1-/- mice showed lower expression of contractile markers (α-smooth muscle actin and calponin) and higher expression of synthetic indicators (osteopontin, vimentin and tropoelastin) and Kruppel-like factor 4 (KLF4) by comparison with Apoe-/- DJ-1+/+ mice. Furthermore, genetic inhibition of KLF4 counteracted the adverse effects of DJ-1 deletion. Therefore, our results showed that DJ-1 deletion caused phenotype switching of VSMCs and exacerbated atherosclerotic plaque instability in a KLF4-dependent manner.
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Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Placa Aterosclerótica/etiología , Placa Aterosclerótica/metabolismo , Proteína Desglicasa DJ-1/deficiencia , Animales , Apolipoproteínas E/deficiencia , Biomarcadores , Biopsia , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Lipoproteínas LDL/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Fenotipo , Placa Aterosclerótica/patología , Transducción de SeñalRESUMEN
Loss-of-function mutations in the gene encoding human protein DJ-1 cause early onset of Parkinson's disease, suggesting that DJ-1 protects dopaminergic neurons. The molecular mechanisms underlying this neuroprotection are unclear; however, DJ-1 has been suggested to be a GSH-independent glyoxalase that detoxifies methylglyoxal (MGO) by converting it into lactate. It has also been suggested that DJ-1 serves as a deglycase that catalyzes hydrolysis of hemithioacetals and hemiaminals formed by reactions of MGO with the thiol and amino groups of proteins. In this report, we demonstrate that the equilibrium constant of reaction of MGO with thiols is â¼500 m-1 at 37 °C and that the half-life of the resulting hemithioacetal is only 12 s. These thermodynamic parameters would dictate that a significant fraction of free MGO will be present in a fast equilibrium with hemithioacetals in solution. We found that removal of free MGO by DJ-1's glyoxalase activity forces immediate spontaneous decomposition of hemithioacetals due to the shift in equilibrium position. This spontaneous decomposition of hemithioacetals could be mistaken for deglycase activity of DJ-1. Furthermore, we demonstrate that higher initial concentrations of hemithioacetals are associated with lower rates of DJ-1-mediated conversion of MGO, ruling out the possibility that hemithioacetals are DJ-1 substrates. Experiments with CRISPR/Cas-generated DJ-1-knockout HEK293 cells revealed that DJ-1 does not protect against acute MGO toxicity or carboxymethylation of lysine residues in cells. Combined, our results suggest that DJ-1 does not possess protein deglycase activity.
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Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Humanos , Piruvaldehído/metabolismo , Albúmina Sérica Bovina/metabolismoRESUMEN
DJ-1 is a conserved, ubiquitous protein associated to a large number of intracellular processes. Human DJ-1 has been linked to several pathologies, including hereditary forms of Parkinson's disease, cancer, and amyotrophic lateral sclerosis. Several cytoprotective functions of DJ-1 have been reported, however, its actual mechanisms of action remain elusive. In vitro, DJ-1 has been shown to bind zinc and copper(II) at its active site, which contains a conserved cysteine (C106), and copper(I) at a different binding site. C106 is essential to DJ-1 function, and is easily oxidized upon oxidative stress. Here, we investigated the metal-binding- and redox properties of DJ-1 in living human cells by in-cell NMR. Intracellular DJ-1 is surprisingly free from interactions with any other cellular components and as such is clearly detectable by NMR. Metal-bound forms of DJ-1 were not observed upon treating the cells with excess zinc or copper. No copper binding was observed when co-expressing DJ-1 with the copper chaperone for superoxide dismutase 1 (SOD1). Co-expression of DJ-1 with SOD1 itself did not promote copper binding to SOD1, excluding a previously suggested function of DJ-1 as a copper chaperone. Overall, our data do not support the role of DJ-1 as a metalloprotein. Conversely, oxidative treatment to the cells caused the complete and selective oxidation of C106 to sulfinic acid, consistent with the reported role of DJ-1 as a redox sensor.
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Calcio/química , Proteína Desglicasa DJ-1/química , Zinc/química , Dominio Catalítico , Cisteína/química , Células HEK293 , Humanos , Oxidación-Reducción , Superóxido Dismutasa-1/químicaRESUMEN
Amadori rearrangement products are stable sugar-amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose-lysine (F-Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F-Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6-phosphofructose-lysine (6-P-F-Lys). FrlB, a deglycase, then converts 6-P-F-Lys to L-lysine and glucose-6-phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8-Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6-P-F-Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine-6-phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB-6-P-F-Lys on GlmS-fructose-6-phosphate structures revealed parallels in their active-site arrangement and guided our selection of seven putative active-site residues in FrlB for site-directed mutagenesis. Activity assays with eight recombinant single-substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface-induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x-ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure-function and mechanistic studies of enzymes.
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Aminoácidos , Lisina , Animales , Bacterias , Escherichia coli/genética , Azúcares , FructosaRESUMEN
Despite the documented bi-enzymatic mode of methylglyoxal detoxification, the single-step catalysis of methylglyoxal by DJ-1/Pfp-I domain containing proteins has been in the limelight. Prasad et al. recently discovered another functional facet of these moonlighting proteins: the deglycase potential of DJ-1D to repair the glycated DNA, RNA, and proteins in plants.
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Piruvaldehído , Piruvaldehído/metabolismoRESUMEN
DJ-1, a protein encoded by PARK7 plays a protective role against neurodegeneration. Since its glyoxalase III activity catalyzing methylglyoxal (MG) to lactate was discovered, DJ-1 has been re-established as a deglycase decomposing the MG-intermediates with amino acids and nucleotides (hemithioacetal and hemiaminal) rather than MG itself, but it is still debatable. Here, we have clarified that human DJ-1 directly recognizes MG, and not MG-intermediates, by monitoring the detailed catalytic processes and enantiomeric lactate products. The hemithioacetal intermediate between C106 of 15 N-labeled DJ-1 (15N DJ-1) and MG was also monitored by NMR. TRIS molecule formed stable diastereotopic complexes with MG (Kd , 1.57 ± 0.27 mM) by utilizing its three OH groups, which likely disturbed the assay of deglycase activity. The low kcat of DJ-1 for MG and its MG-induced structural perturbation may suggest that DJ-1 has a regulatory function as an in vivo sensor of reactive carbonyl stress.
Asunto(s)
Enfermedad de Parkinson , Humanos , Aldehído Oxidorreductasas , Ácido Láctico/metabolismo , Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1/genética , Proteína Desglicasa DJ-1/metabolismo , Piruvaldehído/química , Piruvaldehído/metabolismoRESUMEN
Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 µM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.
RESUMEN
Parkinsonism-associated deglycase-PARK7/DJ-1 (PARK7) is a multifunctional protein having significant roles in inflammatory and immune disorders and cell protection against oxidative stress. Mutations in PARK7 may result in the onset and progression of a few neurodegenerative disorders such as Parkinson's disease. This study has analyzed the non-synonymous single nucleotide polymorphisms (nsSNPs) resulting in single amino acid substitutions in PARK7 to explore its disease-causing variants and their structural dysfunctions. Initially, we retrieved the mutational dataset of PARK7 from the Ensembl database and performed detailed analyses using sequence-based and structure-based approaches. The pathogenicity of the PARK7 was then performed to distinguish the destabilizing/deleterious variants. Aggregation propensity, noncovalent interactions, packing density, and solvent accessible surface area analyses were carried out on the selected pathogenic mutations. The SODA study suggested that mutations in PARK7 result in aggregation, inducing disordered helix and altering the strand propensity. The effect of mutations alters the number of hydrogen bonds and hydrophobic interactions in PARK7, as calculated from the Arpeggio server. The study indicated that the alteration in the hydrophobic contacts and frustration of the protein could alter the stability of the missense variants of the PARK7, which might result in disease progression. This study provides a detailed understanding of the destabilizing effects of single amino acid substitutions in PARK7.
RESUMEN
Glycation, the term for non-enzymatic covalent reactions between aldehyde metabolites and nucleophiles on biopolymers, results in deleterious cellular damage and diseases. Since Parkinsonism-associated protein DJ-1 was proposed as a novel deglycase that directly repairs glycated adducts, it has been considered a major contributor to glycation damage repair. Recently, an interesting debate over the mechanism of glycation repair by DJ-1 has emerged, focusing on whether the substrate of DJ-1 is glycated adducts or the free small aldehydes. The physiological significance of DJ-1 on glycation defense also remains in question. This debate is complicated by the fact that glycated biomolecular adducts are in rapid equilibrium with free aldehydes. Here, we summarize experimental evidence for the two possibilities, highlighting both consistencies and conflicts. We discuss the experimental complexities from a mechanistic perspective, and suggest classes of experiments that should help clarify this debate.
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Productos Finales de Glicación Avanzada/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Piruvaldehído/metabolismo , Cisteína/metabolismo , Productos Finales de Glicación Avanzada/química , Humanos , Cinética , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Proteína Desglicasa DJ-1/química , Proteína Desglicasa DJ-1/genética , Proteínas/química , Proteínas/metabolismo , Piruvaldehído/química , Especificidad por SustratoRESUMEN
Salmonellais a foodborne pathogen that causes annually millions of cases of salmonellosis globally, yet Salmonella-specific antibacterials are not available. During inflammation, Salmonella utilizes the Amadori compound fructose-asparagine (F-Asn) as a nutrient through the successive action of three enzymes, including the terminal FraB deglycase. Salmonella mutants lacking FraB are highly attenuated in mouse models of inflammation due to the toxic build-up of the substrate 6-phosphofructose-aspartate (6-P-F-Asp). This toxicity makes Salmonella FraB an appealing drug target, but there is currently little experimental information about its catalytic mechanism. Therefore, we sought to test our postulated mechanism for the FraB-catalyzed deglycation of 6-P-F-Asp (via an enaminol intermediate) to glucose-6-phosphate and aspartate. A FraB homodimer model generated by RosettaCM was used to build substrate-docked structures that, coupled with sequence alignment of FraB homologs, helped map a putative active site. Five candidate active-site residues-including three expected to participate in substrate binding-were mutated individually and characterized. Native mass spectrometry and ion mobility were used to assess collision cross sections and confirm that the quaternary structure of the mutants mirrored the wild type, and that there are two active sites/homodimer. Our biochemical studies revealed that FraB Glu214Ala, Glu214Asp, and His230Ala were inactive in vitro, consistent with deprotonated-Glu214 and protonated-His230 serving as a general base and a general acid, respectively. Glu214Ala or His230Ala introduced into the Salmonella chromosome by CRISPR/Cas9-mediated genome editing abolished growth on F-Asn. Results from our computational and experimental approaches shed light on the catalytic mechanism of Salmonella FraB and of phosphosugar deglycases in general.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hidrolasas/química , Hidrolasas/metabolismo , Salmonella/enzimología , Proteínas Bacterianas/genética , Edición Génica , Hidrolasas/genética , Espectrometría de Masas , Mutación/genética , Especificidad por SustratoRESUMEN
DJ-1 is a highly conserved soluble protein that is associated to several cellular pathways. In humans, DJ-1 has been implicated in several pathologies such as cancer, Parkinson's disease and amyotrophic lateral sclerosis. Several roles have been attributed to DJ-1, including defense against oxidative stress, chaperone activity and proteasome regulation. The recent finding that DJ-1 acts as a protein and DNA deglycase further confirms the protective function of DJ-1 and suggests a common mechanism of action in the various pathways in which DJ-1 is involved. Cysteine 106, located in the putative active site of DJ-1, is critical for the biological activity of DJ-1 and is easily oxidized to cysteine-sulfinate. While such oxidation modulates DJ-1 activity, the underlying molecular mechanism has not yet been elucidated. Cysteine oxidation does not perturb the protein structure, therefore changes in protein dynamics in solution could modulate its function. Here, we report a revised and completed (98%) backbone assignment of reduced DJ-1, together with the backbone assignment of oxidized DJ-1. Chemical shift perturbation is observed in several regions across the sequence, while no changes in secondary structure are observed. These data will provide the starting point for further characterization of the changes in the backbone dynamics of DJ-1 upon oxidation in solution at physiological temperature.
Asunto(s)
Cisteína/análogos & derivados , Resonancia Magnética Nuclear Biomolecular , Proteína Desglicasa DJ-1/química , Proteína Desglicasa DJ-1/metabolismo , Cisteína/metabolismo , Humanos , Oxidación-ReducciónRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with the degeneration of dopamine neurons of the substantia nigra pars compacta (SNpc) and the presence of intra-neuronal aggregates of α-synuclein and its post-translational products. Based on emerging reports on the association between glycated α-synuclein and PD; and the newly identified deglycase activity of DJ-1, we sought to find the relevance of deglycase activity of DJ-1 on glycation of α-synuclein and its plausible role in PD. Our results demonstrate that DJ-1 has a higher affinity towards the substrate methylglyoxal (MGO) (Kmâ¯=â¯900â¯mM) as compared to its familial mutant, L166P (Kmâ¯=â¯1900â¯mM). Also, CML α-synuclein (CML-syn) served as a substrate for the deglycase activity of DJ-1. Treatment of cells with Parkinsonian mimetic, 1-methyl-4-phenylpyridinium ion (MPP+); oxidants, such as H2O2 and methylglyoxal (MGO) lead to a dose-dependent decrease in the levels of DJ-1 with a concomitant increase in CML-syn. Also, MGO induced cytosolic α-synuclein aggregates in cells which stained positive with the anti-CML antibody. Further, unilateral stereotaxic administration of MGO into the SNpc of mice induced α-synuclein aggregates and CML-syn with a concomitant reduction in the number of TH positive neurons, protein levels of TH and DJ-1 at the site of injection. Interestingly, overexpression of DJ-1 enhanced the clearance of preformed CML-syn in cells, mitigated MGO induced CML-syn and intracellular α-synuclein aggregates. Overall, the findings of our present study demonstrate that DJ-1 plays a pivotal role in the glycation and aggregation of α-synuclein. Reduced DJ-1 activity due to mutations or oxidative stress may lead to the accumulation of glycated α-synuclein and its aggregates.
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Peróxido de Hidrógeno/metabolismo , Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1/genética , alfa-Sinucleína/genética , 1-Metil-4-fenilpiridinio/farmacología , Animales , Línea Celular , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Productos Finales de Glicación Avanzada/genética , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Ratones , Estrés Oxidativo/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Porción Compacta de la Sustancia Negra/metabolismo , Porción Compacta de la Sustancia Negra/patología , Agregado de Proteínas/genética , Proteína Desglicasa DJ-1/metabolismo , Piruvaldehído/metabolismo , Especificidad por Sustrato , alfa-Sinucleína/metabolismoRESUMEN
Plasmodium falciparum DJ1 (PfDJ1) belongs to the DJ-1/ThiJ/PfpI superfamily whose members are present in all the kingdoms of life and exhibit diverse cellular functions and biochemical activities. The common feature of the superfamily is the class I glutamine amidotransferase domain with a conserved redox-active cysteine residue, which mediates various activities of the superfamily members, including anti-oxidative activity in PfDJ1 and human DJ1 (hDJ1). As the superfamily members represent diverse functional classes, to investigate if there is any sequence feature unique to hDJ1-like proteins, sequences of the representative proteins of different functional classes were compared and analysed. A novel motif unique to PfDJ1 and several other hDJ1-like proteins, with the consensus sequence of TSXGPX5FXLX5L, was identified that we designated as the hDJ1-subfamily motif (DJSM). Several mutations that have been associated with Parkinson's disease are also present in DJSM, suggesting its functional importance in hDJ1-like proteins. Mutations of the conserved residues of DJSM of PfDJ1 did not significantly affect overall secondary structure, but caused both a significant loss (S151A and P154A) and gain (L168A) of anti-oxidative activity. We also report that PfDJ1 has deglycase activity, which was significantly decreased in its mutants of the catalytic cysteine (C106A) and DJSM (S151A and P154A). Episomal expression of the catalytic cysteine (C106A) or DJSM (P154A) mutant decreased growth rates of parasites as compared to that of wild type parasites or parasites expressing wild type PfDJ1. S151 appears to properly position the nucleophilic elbow containing C106 and P154 forms a hydrogen bond with C106, which could be a reason for the loss of activities of PfDJ1 upon their mutations. Taken together, DJSM delineates PfDJ1 and other hDJ1-subfamily proteins from the remaining superfamily, and is critical for anti-oxidative and deglycase activities of PfDJ1.
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Estrés Oxidativo , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Proteína Desglicasa DJ-1/química , Proteína Desglicasa DJ-1/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Catálisis , Secuencia Conservada , Humanos , Plasmodium falciparum/química , Plasmodium falciparum/genética , Proteína Desglicasa DJ-1/genética , Proteínas Protozoarias/genética , Alineación de SecuenciaRESUMEN
The present study examined the effect of diallyl disulfide (DADS) on the invasion and migration ability of HL-60 cells with a high expression of parkinsonism associated deglycase (DJ-1) in the nucleus (HHDN), and its molecular mechanism. A western blot assay was used to measure the effects of DADS and an Src inhibitor on the expression of DJ-1 and the Src signal pathway in HHDN. The effects of DADS and Src inhibitors on the invasion and migration ability of HHDN was detected using Transwell migration and invasion chamber experiments. The experiments were divided into three groups: A control group (HL-60 cells), an empty vector group and a high expression group (HHDN cells). Western blot assays revealed that the expression of DJ-1 in HHDN was inhibited in a time-dependent manner following treatment with DADS for 24, 48 and 72 h. Following DADS treatment, the expression of phosphorylated Src (p-Src) and phosphorylated Fak (p-Fak) were significantly decreased in all groups compared with the untreated groups, however the expression level of Src, Fak and integrin did not change significantly. Western blot analysis results revealed that following treatment with DADS and Src inhibitor, the expression levels of p-Src and p-Fak significantly decreased in all three groups compared with untreated groups, whereas the expression levels of Src, Fak and integrin did not change significantly. The expression of DJ-1 in HHND was inhibited in time-dependent manner following treatment with DADS and Src inhibitor for 24, 48 and 72 h. Transwell migration and invasion assay results revealed that DADS and Src inhibitors may suppress migration and invasion in leukemic cells, and a combination of the two treatments may result in more efficient suppression. DADS may downregulate DJ-1-mediated invasion and migration in leukemic cells through suppressing the Src-Fak-Integrin signaling pathway, and the Src inhibitor may enhance the antitumor effect of DADS.
RESUMEN
Cerebellar damage and granular and Purkinje cell loss in sporadic Creutzfeldt-Jakob disease (sCJD) highlight a critical involvement of the cerebellum during symptomatic progression of the disease. In this project, global proteomic alterations in the cerebellum of brain from the two most prevalent subtypes (MM1 and VV2) of sCJD were studied. Two-dimensional gel electrophoresis (2DE) coupled mass spectrometric identification revealed 40 proteins in MM1 and 43 proteins in VV2 subtype to be differentially expressed. Of those, 12 proteins showed common differential expression in their expression between two subtypes. Differentially expressed proteins mainly belonged to (i) cell cycle, gene expression and cell death; (ii) cellular stress response/oxidative stress (OS) and (iii) signal transduction and synaptic functions, related molecular functions. We verified 10 differentially expressed proteins at transcriptional and translational level as well. Interestingly, protein deglycase DJ-1 (an antioxidative protein) showed an increase in its messenger RNA (mRNA) expression in both MM1 and VV2 subtypes but protein expression only in VV2 subtype in cerebellum of sCJD patients. Nuclear translocalization of DJ-1 confirmed its expressional alteration due to OS in sCJD. Downstream experiments showed the activation of nuclear factor erythroid-2 related factor 2 (Nrf2)/antioxidative response element (ARE) pathway. DJ-1 protein concentration was significantly increased during the clinical phase in cerebrospinal fluid of sCJD patients and also at presymptomatic and symptomatic stages in cerebellum of humanized PrP transgenic mice inoculated with sCJD (MM1 and VV2) brain. These results suggest the implication of oxidative stress during the pathophysiology of sCJD.
Asunto(s)
Cerebelo/metabolismo , Cerebelo/patología , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Estrés Oxidativo , Proteína Desglicasa DJ-1/metabolismo , Animales , Síndrome de Creutzfeldt-Jakob/fisiopatología , Progresión de la Enfermedad , Humanos , Ratones Noqueados , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Programas Informáticos , Espectrometría de Masas en TándemRESUMEN
Reactive species play an important role in physiological functions. Overproduction of reactive species, notably reactive oxygen (ROS) and nitrogen (RNS) species along with the failure of balance by the body's antioxidant enzyme systems results in destruction of cellular structures, lipids, proteins, and genetic materials such as DNA and RNA. Moreover, the effects of reactive species on mitochondria and their metabolic processes eventually cause a rise in ROS/RNS levels, leading to oxidation of mitochondrial proteins, lipids, and DNA. Oxidative stress has been considered to be linked to the etiology of many diseases, including neurodegenerative diseases (NDDs) such as Alzheimer diseases, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Multiple sclerosis, and Parkinson's diseases. In addition, oxidative stress causing protein misfold may turn to other NDDs include Creutzfeldt-Jakob disease, Bovine Spongiform Encephalopathy, Kuru, Gerstmann-Straussler-Scheinker syndrome, and Fatal Familial Insomnia. An overview of the oxidative stress and mitochondrial dysfunction-linked NDDs has been summarized in this review.