Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
1.
Appl Opt ; 59(25): 7490-7495, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32902446

RESUMO

Soluble, small amyloid-ß oligomers (AßO) are recognized as significant contributors to the pathology of Alzheimer's disease (AD). Although drugs for treating AD symptoms have been approved, no therapy targeting amyloid-ß (Aß) capable of modifying the course of the disease is available. In an effort to develop a label-free method for screening new anti-AD therapeutic agents, we show the use of a surface-enhanced Raman scattering (SERS) active substrate for detecting the interactions between Aß peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600cm-1 and 2900cm-1 can be used to monitor the molecular interactions between SLF and Aß. This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti-Aß therapeutic agents.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Flúor/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/prevenção & controle , Análise Espectral Raman , Peptídeos beta-Amiloides/química , Interações Medicamentosas , Flúor/química , Agregação Patológica de Proteínas/metabolismo , Marcadores de Spin
2.
Int J Mol Sci ; 19(8)2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30111691

RESUMO

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.


Assuntos
Venenos de Crotalídeos/efeitos adversos , Edema/induzido quimicamente , Proteínas de Répteis/efeitos adversos , Serina Proteases/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Crotalus/metabolismo , Edema/metabolismo , Edema/patologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Camundongos , Modelos Moleculares , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores Ativados por Proteinase/metabolismo , Proteínas de Répteis/química , Proteínas de Répteis/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Venenos de Serpentes , Fosfolipases Tipo C/metabolismo
3.
Pharmaceuticals (Basel) ; 16(4)2023 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-37111388

RESUMO

Snake venom serine protease (SVSP) interferes with the regulation and control of important biological reactions in homeostasis and can be classified as an activator of the fibrinolytic system and platelet aggregation. Our group has recently isolated a new serine protease from Crotalus durissus terrificus total venom (Cdtsp-2). This protein exhibits edematogenic capacity and myotoxic activity. A Kunitz-like EcTI inhibitor protein with a molecular mass of 20 kDa was isolated from Enterolobium contortisiliquum and showed high trypsin inhibition. Thus, the objective of this work is to verify the possible inhibition of the pharmacological activities of Cdtsp-2 by the Kutinz-type inhibitor EcTI. To isolate Cdtsp-2 from total C. d. terrificus venom, we used three-step chromatographic HPLC. Using the mice paw edema model, we observed an edematogenic effect, myotoxicity and hepatotoxicity caused by Cdtsp-2. In vitro and in vivo experiments showed that the alterations in hemostasis caused by Cdtsp-2 are crucial for the development of marked hepatotoxicity and that EcTI significantly inhibits the enzymatic and pharmacological activities of Cdtsp-2. Kunitz-like inhibitor may be a viable alternative for the development of ancillary treatments against the biological activities of venoms.

4.
J Biomed Opt ; 27(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36071559

RESUMO

SIGNIFICANCE: Follicular thyroid carcinoma carries a substantially poor prognosis due to its unique biological behavior and less favorable outcomes. In particular, fine-needle aspiration (FNA) biopsies, which play a key role in screening thyroid nodules, cannot differentiate benign from malignant follicular neoplasm. AIM: We report on the use of hyperspectral Raman microscopy in combination with chemometric analysis for identifying and classifying single cells obtained from clinical samples of human follicular thyroid neoplasms. APPROACH: We used a method intended to simulate the FNA procedure to obtain single cells from thyroid nodules. A total of 392 hyperspectral Raman images of single cells from follicular thyroid neoplasms were collected. RESULTS: Malignant cells were identified based on their intrinsic Raman spectral signatures with an overall diagnostic accuracy of up to 83.7%. CONCLUSIONS: Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an ancillary test for analyzing single cells from thyroid FNA biopsies to better stratify "indeterminate" nodules and other cytologically challenging cases.


Assuntos
Neoplasias da Glândula Tireoide , Nódulo da Glândula Tireoide , Biópsia por Agulha Fina , Quimiometria , Humanos , Microscopia , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Nódulo da Glândula Tireoide/diagnóstico por imagem , Nódulo da Glândula Tireoide/patologia
5.
Opt Express ; 19(6): 5620-6, 2011 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-21445202

RESUMO

An upconversion random laser (RL) operating in the ultraviolet is reported for Nd3+ doped fluoroindate glass powder pumped at 575 nm. The RL is obtained by the resonant excitation of the Nd3+ state 2G7/2 followed by energy transfer among two excited ions such that one ion in the pair decays to a lower energy state and the other is promoted to state 4D7/2 from where it decays emitting light at 381 nm. The RL threshold of 30 kW/cm2 was determined by monitoring the photoluminescence intensity as a function of the pump laser intensity. The RL pulses have time duration of 29 ns that is 50 times smaller than the decay time of the upconversion signal when the sample is pumped with intensities below the RL laser threshold.

6.
Sci Rep ; 11(1): 1398, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446868

RESUMO

Hirschsprung disease (HD) is a congenital disorder in the distal colon that is characterized by the absence of nerve ganglion cells in the diseased tissue. The primary treatment for HD is surgical intervention with resection of the aganglionic bowel. The accurate identification of the aganglionic segment depends on the histologic evaluation of multiple biopsies to determine the absence of ganglion cells in the tissue, which can be a time-consuming procedure. We investigate the feasibility of using a combination of label-free optical modalities, second harmonic generation (SHG); two-photon excitation autofluorescence (2PAF); and Raman spectroscopy (RS), to accurately locate and identify ganglion cells in murine intestinal tissue without the use of exogenous labels or dyes. We show that the image contrast provided by SHG and 2PAF signals allows for the visualization of the overall tissue morphology and localization of regions that may contain ganglion cells, while RS provides detailed multiplexed molecular information that can be used to accurately identify specific ganglion cells. Support vector machine, principal component analysis and linear discriminant analysis classification models were applied to the hyperspectral Raman data and showed that ganglion cells can be identified with a classification accuracy higher than 95%. Our findings suggest that a near real-time intraoperative histology method can be developed using these three optical modalities together that can aid pathologists and surgeons in rapid, accurate identification of ganglion cells to guide surgical decisions with minimal human intervention.


Assuntos
Colo/diagnóstico por imagem , Colo/inervação , Doença de Hirschsprung/diagnóstico por imagem , Microscopia , Animais , Camundongos
7.
Sci Rep ; 11(1): 21544, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34732792

RESUMO

Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGFß signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.


Assuntos
Peixes/genética , Peixes/fisiologia , Duplicação Gênica , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/fisiologia , Cromossomos Sexuais , Animais , Mapeamento Cromossômico , Conservação dos Recursos Naturais , DNA/metabolismo , Evolução Molecular , Feminino , Pesqueiros , Marcadores Genéticos/genética , Genótipo , Masculino , Fenótipo , Filogenia , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Cromossomo Y
8.
Biomed Opt Express ; 11(12): 6962-6972, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33408973

RESUMO

Medullary thyroid carcinoma (MTC) is a rare form of thyroid malignancy that can be diagnostically challenging on fine needle aspiration (FNA) cytology. Ancillary tests such as elevated serum or immunohistochemical positive calcitonin have been helpful, yet they can occasionally provide false positive results. In search for an alternative method to improve diagnostic accuracy (DA), we applied hyperspectral Raman spectroscopy to characterize the biochemical composition of single cells from MTC and compared their spectral information to cells from other types of thyroid nodules. Hyperspectral Raman images of 117 MTC single cells from digested tissue were obtained with a line-scan hyperspectral Raman microscope and compared to 127 benign and 121 classic variant of papillary thyroid carcinoma (CVPTC) cells. When principal component analysis and linear discriminant analysis were used to classify the spectral data, MTC cells were differentiated from benign and CVPTC cells with 97% and 99% DA, respectively. In addition, MTC cells exhibited a prominent Raman peak at 1003 cm-1, whose intensity is 84% and 226% greater on average than that observed in benign and CVPTC cells, respectively. When specifically utilizing only this peak as a spectral marker, MTC cells were separated from benign and CVPTC cells with 87% and 95% DA, respectively. As this peak is linked to phenylalanine, which is known to be associated with calcitonin release in thyroid parafollicular cells, the increased intensity further suggests that this Raman peak could potentially be a new diagnostic marker for MTC. Furthermore, preliminary data from MTC cells (n=21) isolated from a simulated FNA procedure provided similar Raman signatures when compared to single cells from digestion. These results suggest that "Raman-based cytopathology" can be used as an adjunct technique to improve the diagnostic accuracy of FNA cytopathology at a single cell level.

9.
Biochem Pharmacol ; 182: 114230, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32979352

RESUMO

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.


Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Produtos Biológicos/farmacologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Lisossomos/imunologia , Peptídeo Hidrolases/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Asparaginase/química , Asparaginase/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Galinhas , Relação Dose-Resposta a Droga , Escherichia coli , Feminino , Cavalos , Humanos , Fenômenos Imunogenéticos/fisiologia , Células Jurkat , Lisossomos/química , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases/química , Peptídeo Hidrolases/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Estrutura Secundária de Proteína
10.
Biomed Opt Express ; 10(9): 4411-4421, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31565498

RESUMO

We report on the use of line-scan hyperspectral Raman microscopy in combination with multivariate statistical analyses for identifying and classifying single cells isolated from clinical samples of human thyroid nodules based on their intrinsic Raman spectral signatures. A total of 248 hyperspectral Raman images of single cells from benign thyroid (n = 127) and classic variant of papillary carcinoma (n = 121) nodules were collected. Spectral differences attributed to phenylalanine, tryptophan, proteins, lipids, and nucleic acids were identified for benign and papillary carcinoma cells. Using principal component analysis and linear discriminant analysis, cells were identified with 97% diagnostic accuracy. In addition, preliminary data of cells from follicular adenoma (n = 20), follicular carcinoma (n = 25), and follicular variant of papillary carcinoma (n = 18) nodules suggest the feasibility of further discrimination of subtypes. Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an objective approach for analyzing single cells from fine needle aspiration (FNA) biopsies to enable the minimally invasive diagnosis of "indeterminate" thyroid nodules and other challenging cases.

11.
PLoS One ; 13(5): e0196918, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29782551

RESUMO

Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static.


Assuntos
Proteínas de Bactérias/química , Peróxido de Hidrogênio/química , Peroxidases/química , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Oxirredução , Estrutura Secundária de Proteína , Xylella/enzimologia
12.
PLoS One ; 13(12): e0208316, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30521599

RESUMO

ß-thalassemia is a worldwide distributed monogenic red cell disorder, characterized by an absent or reduced beta globin chain synthesis. The unbalance of alpha-gamma chain and the presence of pathological free iron promote severe oxidative damage, playing crucial a role in erythrocyte hemolysis, exacerbating ineffective erythropoiesis and decreasing the lifespan of red blood cells (RBC). Catalase, glutathione peroxidase and peroxiredoxins act together to protect RBCs from hydrogen peroxide insult. Among them, peroxiredoxins stand out for their overall abundance and reactivity. In RBCs, Prdx2 is the third most abundant protein, although Prdxs 1 and 6 isoforms are also found in lower amounts. Despite the importance of these enzymes, Prdx1 and Prdx2 may have their peroxidase activity inactivated by hyperoxidation at high hydroperoxide concentrations, which also promotes the molecular chaperone activity of these proteins. Some studies have demonstrated the importance of Prdx1 and Prdx2 for the development and maintenance of erythrocytes in hemolytic anemia. Now, we performed a global analysis comparatively evaluating the expression profile of several antioxidant enzymes and their physiological reducing agents in patients with beta thalassemia intermedia (BTI) and healthy individuals. Furthermore, increased levels of ROS were observed not only in RBC, but also in neutrophils and mononuclear cells of BTI patients. The level of transcripts and the protein content of Prx1 were increased in reticulocyte and RBCs of BTI patients and the protein content was also found to be higher when compared to beta thalassemia major (BTM), suggesting that this peroxidase could cooperate with Prx2 in the removal of H2O2. Furthermore, Prdx2 production is highly increased in RBCs of BTM patients that present high amounts of hyperoxidized species. A significant increase in the content of Trx1, Srx1 and Sod1 in RBCs of BTI patients suggested protective roles for these enzymes in BTI patients. Finally, the upregulation of Nrf2 and Keap1 transcription factors found in BTI patients may be involved in the regulation of the antioxidant enzymes analyzed in this work.


Assuntos
Células Eritroides/metabolismo , Peroxirredoxinas/metabolismo , Talassemia beta/metabolismo , Talassemia beta/patologia , Adolescente , Adulto , Western Blotting , Criança , Pré-Escolar , Eritrócitos/citologia , Eritrócitos/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/metabolismo , Oxirredução , Peroxirredoxinas/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
13.
J Biomed Opt ; 21(7): 75012, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27435896

RESUMO

We evaluated the damage caused to optically trapped red blood cells (RBCs) after 1 or 2 min of exposure to near-infrared (NIR) laser beams at 785 or 1064 nm. Damage was quantified by measuring cell elasticity using an automatic, real-time, homemade, optical tweezer system. The measurements, performed on a significant number (hundreds) of cells, revealed an overall deformability decrease up to ∼104% after 2 min of light exposure, under 10 mW optical trapping for the 785-nm wavelength. Wavelength dependence of the optical damage is attributed to the light absorption by hemoglobin. The results provided evidence that RBCs have their biomechanical properties affected by NIR radiation. Our findings establish limits for laser applications with RBCs.


Assuntos
Eritrócitos/efeitos da radiação , Raios Infravermelhos , Lasers , Elasticidade , Hemoglobinas/metabolismo , Humanos , Pinças Ópticas
14.
Sci Rep ; 6: 33133, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27629822

RESUMO

Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.

15.
J Mol Biol ; 424(1-2): 28-41, 2012 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22985967

RESUMO

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the α-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8Å resolution of Tsa1(C47S) in the decameric form [(α(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that Glu50 and Arg146 participate in the stabilization of the Cys(P) α-helix. As a consequence, we raised the hypothesis that Glu50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6)M(-1)s(-1). Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that Glu50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx.


Assuntos
Arginina/metabolismo , Dissulfetos/metabolismo , Ácido Glutâmico/metabolismo , Peroxidases/metabolismo , Peroxirredoxinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Tiorredoxinas/metabolismo , Cromatografia em Gel , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Peroxidases/química , Proteínas de Saccharomyces cerevisiae/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA