RESUMO

This paper addresses the dearth of studies examining the status of Operational Excellence (OE) implementation and offers a roadmap for enhancing OE maturity levels. The study encompassed three phases: (1) face-to-face interviews with industry OE experts in 57 companies across seven sectors, (2), classification of companies based on their maturity levels, and (3) development of a roadmap for companies to enhance their OE maturity. Through face-to-face interviews with OE experts in fifty-seven companies across various sectors, the study classified organizations into five maturity levels, with only 7 % reaching the champion level. The proposed roadmap, comprising twenty-three variables across six hierarchical levels, outlines a path for companies to progress towards champion-level OE maturity, with an estimated timeframe of 4.5-5 years from a zero level. This research contributes valuable insights into the OE implementation status in emerging countries and provides practical guidance for organizations aiming to elevate their OE maturity, emphasizing the importance of strategic planning, economic sustainability, environmental sustainability, and social sustainability variables tailored to the specific challenges and opportunities faced by businesses in emerging nations.

RESUMO

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Assuntos

COVID-19 , Saccharomycetales , Vacinas , Humanos , COVID-19/diagnóstico , COVID-19/prevenção & controle , Teste para COVID-19 , Pichia/genética , Pichia/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Recombinantes/química , Vacinas/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais

RESUMO

In this paper, we describe the development of a Dictyostelium discoideum strain deficient in frataxin protein (FXN). We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the D. discoideum fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition, the multicellular development is affected as well as growing on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued. The results of the study suggest that this new D. discoideum strain offers a wide range of possibilities to easily explore diverse FA FXN variants. This can facilitate the development of straightforward drug screenings to look for new therapeutic strategies.

RESUMO

BACKGROUND: An ostomy significantly influences a person's life, altering their biopsychosocial and sexual sphere and affecting their interpersonal relationships. MATERIALS AND METHODS: Through an observational, descriptive, and cross-sectional study, with a questionnaire aimed at professionals from a health area in Madrid, we analyzed: sociodemographic variables, knowledge of the professionals on the subject, referral of the patient according to the professional's assessment and feelings that the subject under study produces in the patient and in professionals. RESULTS: 49% claimed to have no knowledge about sexuality of the ostomyzed patients. 55.9% of those surveyed consider that the healthcare provider is the one who should introduce the topic of sexuality during the clinical interview. 48.5 and 85.2% are unaware of treatments for male and female sexual dysfunction, respectively. CONCLUSIONS: The data show that the training provided in the university centers is insufficient to deal effectively with this issue in the medical consultation. The participants manifest null or minimal knowledge about the sexual sphere in ostomized patients. Knowledge deficiencies are detected in relation to the sexuality of the ostomized patient, difficulty in talking about sex with these patients, and the importance that sanitary professionals give to the patient's sexual sphere, among others.

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ANTECEDENTES: Una ostomía influye significativamente en la vida de la persona, alterando su esfera biopsicosocial y sexual, y afectando a sus relaciones interpersonales. MATERIAL Y MÉTODO: Estudio observacional, descriptivo y transversal. Mediante un cuestionario dirigido a profesionales de un área sanitaria de Madrid, se analizan variables sociodemográficas, conocimientos de los profesionales, derivación del paciente a un especialista según la valoración del profesional encuestado y sentimientos que produce en ellos el tema de estudio. RESULTADOS: El 49% afirma tener conocimientos nulos sobre la sexualidad del paciente ostomizado. El 55.9% de los encuestados considera que el sanitario es quien debe introducir el tema de la sexualidad durante la entrevista clínica. El 48.5 y el 85.2% desconocen tratamientos para la disfunción sexual, masculina y femenina, respectivamente. CONCLUSIÓN: Los datos demuestran que la formación impartida en los centros universitarios es insuficiente para tratar de forma efectiva este tema en la consulta. Los participantes en el estudio muestran nulo o mínimo conocimiento sobre la esfera sexualidad en el paciente ostomizado Se detectan deficiencias de conocimiento en relación con la sexualidad del ostomizado, dificultad para hablar de sexo con el paciente y valor que da el profesional a la esfera sexual en su paciente, entre otras.

Assuntos

Comportamento Sexual , Sexualidade , Feminino , Humanos , Masculino , Estudos Transversais , Emoções , Pessoal de Saúde

RESUMO

Introducción: El estudio de predictores de desenlaces negativos en pacientes con insuficiencia cardiaca ha incluido la combinación de péptidos natriuréticos y el ancho de distribución eritrocitaria (RDW). Objetivo: Evaluar el uso combinado de la porción N-terminal del propéptido natriurético tipo B (NT-proBNP) y el RDW como pronóstico de fallecimiento por cualquier causa, hospitalización prolongada y reingreso al año del alta en pacientes con insuficiencia cardiaca aguda (ICA) descompensada. Métodos: Realizamos un estudio observacional retrospectivo. Construimos un índice combinado = NT-proBNP x RDW/100. Elaboramos curvas ROC, se estimó la sensibilidad y especificidad en base a los puntos de corte y se estimó el riesgo relativo para desarrollar los desenlaces. Comparamos las áreas bajo las curvas del índice combinado versus el NT-proBNP y RDW, por separado. Resultados: Analizamos los datos de 471 pacientes. El índice combinado tuvo su mejor corte en 927,79 para pronosticar fallecimiento durante el primer año de ingreso. Aquellos con valores ≥ 927,79 tuvieron un riesgo relativo de 32,7 (IC95%: 4,8 - 222,3). Para hospitalización ≥7 días el punto de corte fue 752,67, aquellos con este valor o superiores tuvieron un riesgo relativo de 22,4 (IC95%: 9,7 - 51,8). Para pronosticar reingreso al año del alta el corte fue 858,47 y el riesgo relativo fue 4,7 (IC95%: 3,3 - 6,8). Conclusiones: El índice combinado generó riesgos relativos que muestran una fuerte fuerza de asociación para fallecimiento por cualquier causa, hospitalización ≥ 7 días y reingresos al año del alta. Sin embargo, la superioridad para discriminar no fue concluyente respecto a los componentes individuales.

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Introduction: The study of predictors of negative outcomes in patients with heart failure has included the combination of natriuretic peptides and red cell distribution width (RDW). Objective: To evaluate the combined use of the amino-terminal pro-brain natriuretic peptide (NT-proBNP) and RDW as a prognostic factor for death from any cause, prolonged hospitalization, and readmission one year after discharge in patients with decompensated acute heart failure (AHF). Methods: We conducted a retrospective observational study. We constructed a combined index = NT-ProBNP x RDW/100. ROC curves were constructed, sensitivity and specificity were estimated based on the cut-off points, and the relative risk was estimated to develop the outcomes studied. We compared the area under curve of combined index versus NT-proBNP and RDW, separately. Results: We analyzed data from 471 patients. The combined index had its best cut of 927.79 to predict death during the first year of admission. Those with values ≥ 927,79 had a relative risk of 32.7 (95% CI: 4.8 - 222.3). To predict hospitalization ≥ 7 days, the cut-off point was 752.67; those with this value or higher had a relative risk of 22.4 (95% CI: 9.7 - 51.8). To predict readmission one year after discharge, the cutoff was 858.47 and the relative risk was 4.7 (95% CI: 3.3 - 6.8). Conclusions: The combined index used generate relative risks that show a strong strength of association for death from any cause, hospitalization ≥7 days, and readmissions one year after discharge. However, the superiority to discriminate was inconclusive with respect to the individual components.

RESUMO

Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe-4S]2+ prosthetic group which yields an inactive [3Fe-4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe-S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M-1 s-1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe-4S]2+ and [3Fe-4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe-S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe-4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe-4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.

Assuntos

Proteínas de Ligação ao Ferro , Proteínas Ferro-Enxofre , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Oxirredução , Superóxidos/metabolismo , Aconitato Hidratase/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Frataxina

RESUMO

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a KD value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.

Assuntos

Liases de Carbono-Enxofre , Proteínas de Ligação ao Ferro , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Frataxina

Assuntos

COVID-19 , Humanos , COVID-19/epidemiologia , Mão de Obra em Saúde , Pessoal de Saúde , Encaminhamento e Consulta , Academias e Institutos

RESUMO

In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(III) and Al(III). The interaction of the grafted TRX with iron(III) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(III) per TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(III), a Fe(III) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical.

Assuntos

Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/química , Ferro/química , Proteínas de Escherichia coli/química , Sítios de Ligação , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Compostos de Sulfidrila/química , Oxirredutases/metabolismo

RESUMO

OBJECTIVE: To identify the relationship between red blood cell distribution width (RDW, %), interleukin-6 (IL-6) (pg/ml), high sensitivity-c-reactive protein (hs-CRP) (mg/l), in-hospital mortality and disease severity among patients with heart failure (HF). METHODS: Prospective cohort. We included adults diagnosed with acute non-ischemic HF in 2015. The dependent variables were in-hospital mortality (yes or no) and disease severity. The latter was assessed with the Get With The Guidelines-HF score. We used hierarchical regression models to describe the pattern of association between biomarkers, mortality, and severity. We used the Youden index to identify the best cut-off for mortality prediction. RESULTS: We included 167 patients; the mean age was 72.61 (SD: 11.06). The majority of patients presented with New York Heart Association classification II (40.12%) or III (43.11%). After adjusting for age and gender, all biomarkers were associated with mortality. After adding comorbidities, only IL-6 was associated. The final model with all clinical variables showed no effect from any biomarker. The best cut-off for RDW, hs-CRP and IL-6 for mortality were 14.8, 68.7 and 52.9, respectively. IL-6 presented the highest sensitivity (100%), specificity (75.35%) and area under the curve (0.91). CONCLUSIONS: No biomarker is independent from the most important clinical variables; therefore it should not be used for management modifications.

Assuntos

Humanos , Carta

RESUMO

In humans, the loss of frataxin results in Friedreich's Ataxia, a neurodegenerative disease, in which a deficit in the iron-sulfur cluster assembly is observed. In this work, we analyzed three frataxin variants in which one tryptophan was replaced by a glycine: W155G, W168G and W173G. As expected, given its localization in the assembly site, W155G was not able to activate the desulfurase activity of the supercomplex for iron-sulfur cluster assembly. In turn, W168G, which was significantly more unstable than W155G, was fully active. W173G, which was highly unstable as W168G, showed a significantly decreased activity, only slightly higher than W155G. As W168G and W173G were highly sensitive to proteolysis, we investigated the protein motions by molecular dynamic simulations. We observed that W173G may display altered motions at the Trp155 site. Furthermore, we revealed a H-bond network in which Trp155 takes part, involving residues Gln148, Asn151, Gln153 and Arg165. We suggest that this motion modulation that specifically alters the population of different Trp155 rotamers can be directly transferred to the assembly site, altering the dynamics of the ISCU His137 key residue. This hypothesis was also contrasted by means of molecular dynamic simulations of frataxin in the context of the complete supercomplex. We propose that the supercomplex requires very definite motions of Trp155 to consolidate the assembly site.

Assuntos

Proteínas de Ligação ao Ferro/química , Triptofano/química , Humanos , Proteínas de Ligação ao Ferro/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Estabilidade Proteica , Frataxina

RESUMO

Several biological activities depend on iron-sulfur clusters ([Fe-S]). Even though they are well-known in several organisms their function and metabolic pathway were poorly understood in the majority of the organisms. We propose to use the amoeba Dictyostelium discoideum, as a biological model to study the biosynthesis of [Fe-S] at the molecular, cellular and organism levels. First, we have explored the D. discoideum genome looking for genes corresponding to the subunits that constitute the molecular machinery for Fe-S cluster assembly and, based on the structure of the mammalian supercomplex and amino acid conservation profiles, we inferred the full functionality of the amoeba machinery. After that, we expressed the recombinant mature form of D. discoideum frataxin protein (DdFXN), the kinetic activator of this pathway. We characterized the protein and its conformational stability. DdFXN is monomeric and compact. The analysis of the secondary structure content, calculated using the far-UV CD spectra, was compatible with the data expected for the FXN fold, and near-UV CD spectra were compatible with the data corresponding to a folded protein. In addition, Tryptophan fluorescence indicated that the emission occurs from an apolar environment. However, the conformation of DdFXN is significantly less stable than that of the human FXN, (4.0 vs. 9.0 kcal mol-1, respectively). Based on a sequence analysis and structural models of DdFXN, we investigated key residues involved in the interaction of DdFXN with the supercomplex and the effect of point mutations on the energetics of the DdFXN tertiary structure. More than 10 residues involved in Friedreich's Ataxia are conserved between the human and DdFXN forms, and a good correlation between mutational effect on the energetics of both proteins were found, suggesting the existence of similar sequence/function/stability relationships. Finally, we integrated this information in an evolutionary context which highlights particular variation patterns between amoeba and humans that may reflect a functional importance of specific protein positions. Moreover, the complete pathway obtained forms a piece of evidence in favor of the hypothesis of a shared and highly conserved [Fe-S] assembly machinery between Human and D. discoideum.

Assuntos

Dictyostelium/metabolismo , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sequência de Aminoácidos/genética , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Biologia Computacional , Cristalografia , Dictyostelium/genética , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Cinética , Simulação de Dinâmica Molecular , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes , Alinhamento de Sequência , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Frataxina

RESUMO

The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe-S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedreich's Ataxia, a cardio-neurodegenerative disease. Recently, the way in which FXN interacts with the rest of the subunits of the supercomplex was uncovered. This opens a window to explore relationships between structural dynamics and function. In this study, we prepared a set of FXN variants spanning a broad range of conformational stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and showed similar biological activity. Additionally, we prepared SILCAR, a variant that combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol-1 more stable and equally active. Some of the variants were significantly more resistant to proteolysis than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more rigid structure. It was corroborated by means of molecular dynamics simulations. Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants suggested similar internal motions in the microsecond to millisecond timescale. Instead, variant S157I showed higher denaturation resistance but a significant lower function, similarly to that observed for the FRDA variant N146K. We concluded that the contribution of particular side chains to the conformational stability of FXN might be highly subordinated to their impact on both the protein function and the stability of the functional supercomplex.

Assuntos

Proteínas de Ligação ao Ferro/química , Liases de Carbono-Enxofre/química , Biologia Computacional , Humanos , Proteínas de Ligação ao Ferro/genética , Simulação de Dinâmica Molecular , Mutação Puntual , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Proteólise , Frataxina

RESUMO

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF → LU and LU → FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues CP and CR. Our results suggest that the FF → LU transition has a higher associated energy barrier than the refolding LU → FF process in agreement with the relatively low experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type EcTPx.

Assuntos

Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Proteínas Periplásmicas/química , Peroxidases/química , Dobramento de Proteína , Simulação por Computador , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação/genética , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Conformação Proteica

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RESUMO

In recent years, the mammalian mitochondrial protein complex for iron-sulfur cluster assembly has been the focus of important studies. This is partly because of its high degree of relevance in cell metabolism and because mutations of the involved proteins are the cause of several human diseases. Cysteine desulfurase NFS1 is the key enzyme of the complex. At present, it is well-known that the active form of NFS1 is stabilized by the small protein ISD11. In this work, the structure of the human mitochondrial ACP-ISD11 heterodimer was determined at 2.0 Å resolution. ACP-ISD11 forms a cooperative unit stabilized by several ionic interactions, hydrogen bonds, and apolar interactions. The 4'-phosphopantetheine-acyl chain, which is covalently bound to ACP, interacts with several residues of ISD11, modulating together with ACP the foldability of ISD11. Recombinant human ACP-ISD11 was able to interact with the NFS1 desulfurase, thus yielding an active enzyme, and the NFS1/ACP-ISD11 core complex was activated by frataxin and ISCU proteins. Internal motions of ACP-ISD11 were studied by molecular dynamics simulations, showing the persistence of the interactions between both protein chains. The conformation of the dimer is similar to that found in the context of the (NFS1/ACP-ISD11)2 supercomplex core, which contains the Escherichia coli ACP instead of the human variant. This fact suggests a sequential mechanism for supercomplex consolidation, in which the ACP-ISD11 complex may fold independently and, after that, the NFS1 dimer would be stabilized.

Assuntos

Complexo I de Transporte de Elétrons/química , Proteínas Reguladoras de Ferro/química , Cristalografia por Raios X , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Ligação de Hidrogênio , Proteínas Reguladoras de Ferro/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica

RESUMO

Protein dynamics, folding, and thermodynamics represent a central aspect of biophysical chemistry. pH, temperature, and denaturant perturbations inform our understanding of diverse contributors to stability and rates. In this work, we performed a thermodynamic analysis using a combined experimental and computational approach to gain insights into the role of electrostatics in the folding reaction of a psychrophile frataxin variant from Psychromonas ingrahamii. This folding reaction is strongly modulated by pH with a single, narrow, and well-defined transition state with ∼80% compactness, ∼70% electrostatic interactions, and ∼60% hydration shell compared to the native state (αD = 0.82, αH = 0.67, and αΔCp = 0.59). Our results are best explained by a two-proton/two-state model with very different pKa values of the native and denatured states (∼5.5 and ∼8.0, respectively). As a consequence, the stability strongly increases from pH 8.0 to 6.0 (|ΔΔG°| = 5.2 kcal mol-1), mainly because of a decrease in the TΔS°. Variation of ΔH° and ΔS° at pH below 7.0 is dominated by a change in ΔHf⧧ and ΔSf⧧, while at pH above 7.0, it is governed by ΔHu⧧ and ΔSu⧧. Molecular dynamics simulations showed that these pH modulations could be explained by the fluctuations of two regions, rich in electrostatic contacts, whose dynamics are pH-dependent and motions are strongly correlated. Results presented herein contribute to the understanding of the stability and dynamics of this frataxin variant, pointing to an intrinsic feature of the family topology to support different folding mechanisms.

Assuntos

Proteínas de Ligação ao Ferro/química , Simulação de Dinâmica Molecular , Termodinâmica , Concentração de Íons de Hidrogênio , Dobramento de Proteína , Frataxina

RESUMO

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Assuntos

Oxirredução , Peroxirredoxinas/genética , Processamento de Proteína Pós-Traducional/genética , Tirosina/genética , Domínio Catalítico/genética , Cisteína/genética , Dissulfetos/química , Humanos , Mutação/genética , Nitratos/metabolismo , Peroxidase/genética , Peróxidos/metabolismo , Peroxirredoxinas/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/farmacologia , Transdução de Sinais/efeitos dos fármacos

RESUMO

Local events that affect specific regions of proteins are of utmost relevance for stability and function. The aim of this study is to quantitatively assess the importance of locally-focused dynamics by means of a simple chemical modification procedure. Taking human Frataxin as a working model, we investigated local fluctuations of the C-terminal region (the last 16 residues of the protein) by means of three L → C replacement mutants: L98C, L200C and L203C. The conformation and thermodynamic stability of each variant was assessed. All the variants exhibited native features and high stabilities: 9.1 (wild type), 8.1 (L198C), 7.0 (L200C) and 10.0 kcal mol-1 (L203C). In addition, kinetic rates of Cys chemical modification by DTNB and DTDPy were measured, conformational dynamics data were extracted and free energy for the local unfolding of the C-terminal region was estimated. The analysis of these results indicates that the conformation of the C-terminal region fluctuates with partial independence from global unfolding events. Additionally, numerical fittings of the kinetic model of the process suggest that the local transition occurs in the seconds to minutes timescale. In fact, standard free energy differences for local unfolding were found to be significantly lower than those of the global unfolding reaction, showing that chemical modification results may not be explained in terms of the global unfolding reaction alone. These results provide unequivocal experimental evidence of local phenomena with global effects and contribute to understanding how global and local stability are linked to protein dynamics.