RESUMEN
The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Anticuerpos de Dominio Único , Proteínas tau , Humanos , Epítopos/química , Epítopos/inmunología , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/inmunología , Péptidos/química , Péptidos/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Proteínas tau/química , Proteínas tau/inmunologíaRESUMEN
Proteinaceous aggregates accumulate in neurodegenerative diseases such as Alzheimer's Disease (AD), inducing cellular defense mechanisms and altering the redox status. S100 pro-inflammatory cytokines, particularly S100B, are activated during AD, but recent findings reveal an unconventional molecular chaperone role for S100B in hindering Aß aggregation and toxicity. This suggests a potential protective role for S100B at the onset of Aß proteotoxicity, occurring in a complex biochemical environment prone to oxidative damage. Herein, we report an investigation in which extracellular oxidative conditions are mimicked to test if the susceptibility of S100B to oxidation influences its protective activities. Resorting to mild oxidation of S100B, we observed methionine oxidation as inferred from mass spectrometry, but no cysteine-mediated crosslinking. Structural analysis showed that the folding, structure, and stability of oxidized S100B were not affected, and nor was its quaternary structure. However, studies on Aß aggregation kinetics indicated that oxidized S100B was more effective in preventing aggregation, potentially linked to the oxidation of Met residues within the S100:Aß binding cleft that favors interactions. Using a cell culture model to analyze the S100B functions in a highly oxidative milieu, as in AD, we observed that Aß toxicity is rescued by the co-administration of oxidized S100B to a greater extent than by S100B. Additionally, results suggest a disrupted positive feedback loop involving S100B which is caused by its oxidation, leading to the downstream regulation of IL-17 and IFN-α2 expression as mediated by S100B.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Estrés Oxidativo , Agregado de Proteínas , Oxidación-Reducción , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismoRESUMEN
A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity.
Asunto(s)
Glutaril-CoA Deshidrogenasa , Lisina , Sirtuinas , Animales , Glutaril-CoA Deshidrogenasa/metabolismo , Lisina/metabolismo , Ratones , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Sirtuinas/metabolismo , Triptófano/metabolismoRESUMEN
Aggregation of the microtubule-associated protein tau is implicated in several neurodegenerative tauopathies including Alzheimer's disease (AD). Recent studies evidenced tau liquid-liquid phase separation (LLPS) into droplets as an early event in tau pathogenesis with the potential to enhance aggregation. Tauopathies like AD are accompanied by sustained neuroinflammation and the release of alarmins at early stages of inflammatory responses encompass protective functions. The Ca2+ -binding S100B protein is an alarmin augmented in AD that was recently implicated as a proteostasis regulator acting as a chaperone-type protein, inhibiting aggregation and toxicity through interactions of amyloidogenic clients with a regulatory surface exposed upon Ca2+ -binding. Here we expand the regulatory functions of S100B over protein condensation phenomena by reporting its Ca2+ -dependent activity as a modulator of tau LLPS induced by crowding agents (PEG) and metal ions (Zn2+ ). We observe that apo S100B has a negligible effect on PEG-induced tau demixing but that Ca2+ -bound S100B prevents demixing, resulting in a shift of the phase diagram boundary to higher crowding concentrations. Also, while incubation with apo S100B does not compromise tau LLPS, addition of Ca2+ results in a sharp decrease in turbidity, indicating that interactions with S100B-Ca2+ promote transition of tau to the mixed phase. Further, electrophoretic analysis and FLIM-FRET studies revealed that S100B incorporates into tau liquid droplets, suggesting an important stabilizing and chaperoning role contributing to minimize toxic tau aggregates. Resorting to Alexa488-labeled tau we observed that S100B-Ca2+ reduces the formation of tau fluorescent droplets, without compromising liquid-like behavior and droplet fusion events. The Zn2+ -binding properties of S100B also contribute to regulate Zn2+ -promoted tau LLPS as droplets are decreased by Zn2+ buffering by S100B, in addition to the Ca2+ -triggered interactions with tau. Altogether this work uncovers the versatility of S100B as a proteostasis regulator acting on protein condensation phenomena of relevance across the neurodegeneration continuum.
Asunto(s)
Enfermedad de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100RESUMEN
The interpretation of a salt's effect on protein stability traditionally discriminates low concentration regimes (<0.3 M), dominated by electrostatic forces, and high concentration regimes, generally described by ion-specific Hofmeister effects. However, increased theoretical and experimental studies have highlighted observations of the Hofmeister phenomena at concentration ranges as low as 0.001 M. Reasonable quantitative predictions of such observations have been successfully achieved throughout the inclusion of ion dispersion forces in classical electrostatic theories. This molecular description is also on the basis of quantitative estimates obtained resorting to surface/bulk solvent partition models developed for ion-specific Hofmeister effects. However, the latter are limited by the availability of reliable structures representative of the unfolded state. Here, we use myoglobin as a model to explore how ion-dependency on the nature of the unfolded state affects protein stability, combining spectroscopic techniques with molecular dynamic simulations. To this end, the thermal and chemical stability of myoglobin was assessed in the presence of three different salts (NaCl, (NH4)2SO4 and Na2SO4), at physiologically relevant concentrations (0-0.3 M). We observed mild destabilization of the native state induced by each ion, attributed to unfavorable neutralization and hydrogen-bonding with the protein side-chains. Both effects, combined with binding of Na+, Cl- and SO42- to the thermally unfolded state, resulted in an overall destabilization of the protein. Contrastingly, ion binding was hindered in the chemically unfolded conformation, due to occupation of the binding sites by urea molecules. Such mechanistic action led to a lower degree of destabilization, promoting surface tension effects that stabilized myoglobin according to the Hofmeister series. Therefore, we demonstrate that Hofmeister effects on protein stability are modulated by the heterogeneous physico-chemical nature of the unfolded state. Altogether, our findings evidence the need to characterize the structure of the unfolded state when attempting to dissect the molecular mechanisms underlying the effects of salts on protein stability.
Asunto(s)
Mioglobina/química , Estabilidad Proteica , Desplegamiento Proteico , Sales (Química)/química , Electricidad EstáticaRESUMEN
S100B is an astrocytic extracellular Ca2+-binding protein implicated in Alzheimer's disease, whose role as a holdase-type chaperone delaying Aß42 aggregation and toxicity was recently uncovered. Here, we employ computational biology approaches to dissect the structural details and dynamics of the interaction between S100B and Aß42. Driven by previous structural data, we used the Aß25-35 segment, which recapitulates key aspects of S100B activity, as a starting guide for the analysis. We used Haddock to establish a preferred binding mode, which was studied with the full length Aß using long (1 µs) molecular dynamics (MD) simulations to investigate the structural dynamics and obtain representative interaction complexes. From the analysis, Aß-Lys28 emerged as a key candidate for stabilizing interactions with the S100B binding cleft, in particular involving a triad composed of Met79, Thr82 and Glu86. Binding constant calculations concluded that coulombic interactions, presumably implicating the Lys28(Aß)/Glu86(S100B) pair, are very relevant for the holdase-type chaperone activity. To confirm this experimentally, we examined the inhibitory effect of S100B over Aß aggregation at high ionic strength. In agreement with the computational predictions, we observed that electrostatic perturbation of the Aß-S100B interaction decreases anti-aggregation activity. Altogether, these findings unveil features relevant in the definition of selectivity of the S100B chaperone, with implications in Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Biología Computacional/métodos , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/metabolismo , Simulación de Dinámica Molecular , Fragmentos de Péptidos/metabolismo , Agregación Patológica de ProteínasRESUMEN
S100 proteins assume a diversity of oligomeric states including large order self-assemblies, with an impact on protein structure and function. Previous work has uncovered that S100 proteins, including S100B, are prone to undergo ß-aggregation under destabilizing conditions. This propensity is encoded in aggregation-prone regions (APR) mainly located in segments at the homodimer interface, and which are therefore mostly shielded from the solvent and from deleterious interactions, under native conditions. As in other systems, this characteristic may be used to develop peptides with pharmacological potential that selectively induce the aggregation of S100B through homotypic interactions with its APRs, resulting in functional inhibition through a loss of function. Here we report initial studies towards this goal. We applied the TANGO algorithm to identify specific APR segments in S100B helix IV and used this information to design and synthesize S100B-derived APR peptides. We then combined fluorescence spectroscopy, transmission electron microscopy, biolayer interferometry, and aggregation kinetics and determined that the synthetic peptides have strong aggregation propensity, interact with S100B, and may promote co-aggregation reactions. In this framework, we discuss the considerable potential of such APR-derived peptides to act pharmacologically over S100B in numerous physiological and pathological conditions, for instance as modifiers of the S100B interactome or as promoters of S100B inactivation by selective aggregation.
Asunto(s)
Enfermedades Neurodegenerativas/tratamiento farmacológico , Péptidos/farmacología , Subunidad beta de la Proteína de Unión al Calcio S100/antagonistas & inhibidores , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Péptidos/química , Péptidos/genética , Agregado de Proteínas/efectos de los fármacos , Conformación Proteica , Pliegue de Proteína , Subunidad beta de la Proteína de Unión al Calcio S100/genética , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismoRESUMEN
Riboflavin is the biological precursor of two important flavin cofactors-flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)-that are critical prosthetic groups in several redox enzymes. While dietary supplementation with riboflavin is a recognized support therapy in several inborn errors of metabolism, it has yet unproven benefits in several other pathologies affecting flavoproteins. This is the case for glutaric aciduria type I (GA-I), a rare neurometabolic disorder associated with mutations in the GCDH gene, which encodes for glutaryl-coenzyme A (CoA) dehydrogenase (GCDH). Although there are a few reported clinical cases that have responded to riboflavin intake, there is still not enough molecular evidence supporting therapeutic recommendation. Hence, it is necessary to elucidate the molecular basis in favor of riboflavin supplementation in GA-I patients. Here, using a combination of biochemical and biophysical methodologies, we investigate the clinical variant GCDH-p.Val400Met as a model for a phenotype associated with severe deflavinylation. Through a systematic analysis, we establish that recombinant human GCDH-p.Val400Met is expressed in a nonfunctional apo form, which is mainly monomeric rather than tetrameric. However, we show that exogenous FAD is a driver for structural reorganization of the mutant enzyme with concomitant functional recovery, improved thermolability, and resistance to trypsin digestion. Overall, these results establish proof of principle for the beneficial effects of riboflavin supplementation in GA-I patients.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Encefalopatías Metabólicas , Glutaril-CoA Deshidrogenasa/deficiencia , Glutaril-CoA Deshidrogenasa/genética , Riboflavina , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Encefalopatías Metabólicas/metabolismo , Glutaril-CoA Deshidrogenasa/química , Glutaril-CoA Deshidrogenasa/efectos de los fármacos , Glutaril-CoA Deshidrogenasa/metabolismo , Humanos , Mutación , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Recombinantes , Riboflavina/farmacologíaRESUMEN
Metal ions are well known modulators of protein aggregation and are key players in Alzheimer's Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.
Asunto(s)
Agregado de Proteínas/fisiología , Zinc/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Benzotiazoles/metabolismo , Calcio/metabolismo , Dicroismo Circular , Humanos , Cinética , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Transthyretin (TTR) has a neuroprotective role in the central nervous system (CNS) in Alzheimer's disease (AD) and cerebral ischemia. Increased levels of TTR and activated insulin-like growth factor I receptor (IGF-IR) are associated with reduced neurodegeneration in an AD mouse model. In the present study, we found that TTR and IGF-I have a synergistic effect on activation of one of the IGF-IR signaling pathways. Hippocampus of TTR null mice present decreased levels of phosphorylated IGF-IR and Akt when compared with TTR wild type littermate animals. Cell studies reveal the synergistic effect of TTR and IGF-I in promoting IGF-IR signaling even under glutamate induced toxicity. TTR:IGF-IR complexes are identified and a bio-layer interferometry assay demonstrated an interaction between TTR and IGF-IR with a KD ranging from 99 to 744nM. In summary, our results point to a new TTR role through the IGF-I axis, mediated through TTR-IGF-IR interactions.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Hipocampo/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Prealbúmina/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Modelos Animales de Enfermedad , Hipocampo/patología , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Ratones Noqueados , Prealbúmina/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/genéticaRESUMEN
Calcium deregulation is a central feature among neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Calcium accumulates in the spinal and brain stem motor neurons of ALS patients triggering multiple pathophysiological processes which have been recently shown to include direct effects on the aggregation cascade of superoxide dismutase 1 (SOD1). SOD1 is a Cu/Zn enzyme whose demetallated form is implicated in ALS protein deposits, contributing to toxic gain of function phenotypes. Here we undertake a combined experimental and computational study aimed at establishing the molecular details underlying the regulatory effects of Ca(2+) over SOD1 aggregation potential. Isothermal titration calorimetry indicates entropy driven low affinity association of Ca(2+) ions to apo SOD1, at pH7.5 and 37°C. Molecular dynamics simulations denote a noticeable loss of native structure upon Ca(2+) association that is especially prominent at the zinc-binding and electrostatic loops, whose decoupling is known to expose the central SOD1 ß-barrel triggering aggregation. Structural mapping of the preferential apo SOD1 Ca(2+) binding locations reveals that among the most frequent ligands for Ca(2+) are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. Calcium interactions thus diminish gatekeeping roles of these residues, by shielding repulsive interactions via stacking between aggregating ß-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in ALS inclusions. Interestingly, many fALS mutations occur at these positions, disclosing how Ca(2+) interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic ALS pathomechanisms.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Calcio/metabolismo , Agregación Patológica de Proteínas/metabolismo , Superóxido Dismutasa/química , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/patología , Entropía , Humanos , Simulación de Dinámica Molecular , Neuronas Motoras/química , Neuronas Motoras/patología , Mutación , Agregación Patológica de Proteínas/genética , Unión Proteica , Estructura Secundaria de Proteína , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid-ß (Aß) is found in AD brains, and Cu-Aß could contribute to this oxidative stress, as it is able to produce in vitro H2O2 and HOË in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu-Aß-catalyzed H2O2 production is however not known, although it was proposed that H2O2 is directly formed from O2 via a 2-electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase-1 (SOD1) to show, for the first time, that H2O2 production by Cu-Aß in the presence of ascorbate occurs mainly via a free O2Ë(-) intermediate. This finding radically changes the view on the catalytic mechanism of H2O2 production by Cu-Aß, and opens the possibility that Cu-Aß-catalyzed O2Ë(-) contributes to oxidative stress in AD, and hence may be of interest.
Asunto(s)
Péptidos beta-Amiloides/química , Cobre/química , Peróxido de Hidrógeno/química , Oxígeno/química , Péptidos/química , Superóxidos/química , Superóxido Dismutasa/químicaRESUMEN
Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca(2+) dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca(2+). We show that at physiological pH, Ca(2+) induces conformational changes that increase SOD1 ß-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca(2+) boosts the onset of SOD1 aggregation. In agreement, Ca(2+) decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca(2+) induced aggregates consisting preferentially of antiparallel ß-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca(2+)-induced aggregates, thus indicating that Ca(2+) diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca(2+) levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca(2+) may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.
Asunto(s)
Esclerosis Amiotrófica Lateral , Complejos Multiproteicos/química , Superóxido Dismutasa/química , Calcio , Dicroismo Circular , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Oligomeric clusters of amyloid-ß (Aß) are one of the major biomarkers for Alzheimer's disease (AD). However, proficient methods to detect Aß-oligomers in brain tissue are lacking. Here we show that synthetic M13 bacteriophages displaying Aß-derived peptides on their surface preferentially interact with Aß-oligomers. When exposed to brain tissue isolated from APP/PS1-transgenic mice, these bacteriophages detect small-sized Aß-aggregates in hippocampus at an early age, prior to the occurrence of Aß-plaques. Similarly, the bacteriophages reveal the presence of such small Aß-aggregates in post-mortem hippocampus tissue of AD-patients. These results advocate bacteriophages displaying Aß-peptides as a convenient and low-cost tool to identify Aß-oligomers in post-mortem brain tissue of AD-model mice and AD-patients.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Humanos , Ratones , Animales , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Bacteriófago M13/metabolismo , Ratones Transgénicos , Encéfalo/metabolismoRESUMEN
S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H(I) and H(IV). Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca(2+) exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca(2+) promotes anti-parallel ß-sheet conformations that repress fibrillation. At pH 7, Ca(2+) rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca(2+). Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.
Asunto(s)
Amiloide/química , Calcio/química , Proteínas de Ciclo Celular/química , Proteínas S100/química , Superóxido Dismutasa/química , Tiazoles/química , Amiloide/metabolismo , Amiloide/ultraestructura , Esclerosis Amiotrófica Lateral/metabolismo , Benzotiazoles , Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Humanos , Cinética , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteína A6 de Unión a Calcio de la Familia S100 , Proteínas S100/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of ß-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a ß-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD.
Asunto(s)
Drosophila/genética , Flavoproteínas Transportadoras de Electrones/genética , Flavinas/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Mutación , Alelos , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Carnitina/análogos & derivados , Carnitina/metabolismo , Drosophila/metabolismo , Flavoproteínas Transportadoras de Electrones/metabolismo , Flavina-Adenina Dinucleótido/genética , Flavina-Adenina Dinucleótido/metabolismo , Flavinas/metabolismo , Genotipo , Modelos Moleculares , Datos de Secuencia Molecular , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/metabolismo , FenotipoRESUMEN
Superoxide dismutase 1 (SOD1) aggregation is one of the pathological markers of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. The underlying molecular grounds of SOD1 pathologic aggregation remains obscure as mutations alone are not exclusively the cause for the formation of protein inclusions. Thus, other components in the cell environment likely play a key role in triggering SOD1 toxic aggregation in ALS. Recently, it was found that ALS patients present a specific altered metabolomic profile in the cerebrospinal fluid (CSF) where SOD1 is also present and potentially interacts with metabolites. Here we have investigated how some of these small molecules affect apoSOD1 structure and aggregation propensity. Our results show that as co-solvents, the tested small molecules do not affect apoSOD1 thermal stability but do influence its tertiary interactions and dynamics, as evidenced by combined biophysical analysis and proteolytic susceptibility. Moreover, these compounds influence apoSOD1 aggregation, decreasing nucleation time and promoting the formation of larger and less soluble aggregates, and in some cases polymeric assemblies apparently composed by spherical species resembling the soluble native protein. We conclude that some components of the ALS metabolome that shape the chemical environment in the CSF may influence apoSOD1 conformers and aggregation.
Asunto(s)
Aminoácidos/líquido cefalorraquídeo , Metaboloma , Monosacáridos/líquido cefalorraquídeo , Azúcares Ácidos/líquido cefalorraquídeo , Superóxido Dismutasa/líquido cefalorraquídeo , Aminoácidos/metabolismo , Esclerosis Amiotrófica Lateral/líquido cefalorraquídeo , Esclerosis Amiotrófica Lateral/patología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Monosacáridos/metabolismo , Mutación , Unión Proteica , Azúcares Ácidos/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Extracellular aggregation of the amyloid-ß 1-42 (Aß42) peptide is a major hallmark of Alzheimer's disease (AD), with recent data suggesting that Aß intermediate oligomers (AßO) are more cytotoxic than mature amyloid fibrils. Understanding how chaperones harness such amyloid oligomers is critical toward establishing the mechanisms underlying regulation of proteostasis in the diseased brain. This includes S100B, an extracellular signaling Ca2+-binding protein which is increased in AD as a response to neuronal damage and whose holdase-type chaperone activity was recently unveiled. Driven by this evidence, we here investigate how different S100B chaperone multimers influence the formation of oligomers during Aß42 fibrillation. Resorting to kinetic analysis coupled with simulation of AßO influx distributions, we establish that supra-stoichiometric ratios of dimeric S100B-Ca2+ drastically decrease Aß42 oligomerization rate by 95% and AßO levels by 70% due to preferential inhibition of surface-catalyzed secondary nucleation, with a concomitant redirection of aggregation toward elongation. We also determined that sub-molar ratios of tetrameric apo-S100B decrease Aß42 oligomerization influx down to 10%, while precluding both secondary nucleation and, more discreetly, fibril elongation. Coincidently, the mechanistic predictions comply with the independent screening of AßO using a combination of the thioflavin-T and X-34 fluorophores. Altogether, our findings illustrate that different S100B multimers act as complementary suppressors of Aß42 oligomerization and aggregation, further underpinning their potential neuroprotective role in AD.
RESUMEN
Medullary Thyroid Carcinoma (MTC) is a tumor of the neuroendocrine system. In recent years, the need to assess the MTC diagnostic-related parameters has emerged with the aim to elucidate the mechanisms involved in this pathology. The objective of this study was to evaluate the role of Matrix Metalloproteinases (MMPs) 2 and 9, their tissue inhibitors of matrix metalloproteinases (TIMPs), S100 protein, and amyloid in the diagnostic of MTC. Thirty-two samples with MTC (72% women) were included in this cross-sectional study and divided by groups: T category 1 (T1)≤20 mm and T category 2 (T2) 20 to 40 mm of tumor size. MMPs 2 and 9, TIMPs 2 and 1, S100 protein, and calcitonin in tissues were obtained by immunohistochemical techniques. The presence of amyloid in tissue sections was detected on Thioflavin T-stained slides under fluorescent microscope. Percentage of positive cells (P) observed for MMP-2 was higher in those samples presenting T2 MTC with respect to those with T1 MTC ( P <0.05). Moreover, P-MMP-2 showed a direct correlation with higher T category of MTC (Rho=0.439, P < 0.001), whereas P-MPP-9 was directly correlated with S100 protein and the intensity of calcitonin in tissues (Rho=0.419, P =0.017; Rho=0.422, P =0.016, respectively. Therefore, MMPs were directly correlated with some traditional biomarkers of MTC. In this regard, P-MMP-2 was more expressed in type 2 MTC. Combining the analysis of traditional and other useful biomarkers of MTC as MMPs 2 and 9 could be a useful strategy in the diagnostic of MTC.
Asunto(s)
Carcinoma Neuroendocrino , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Neoplasias de la Tiroides , Inhibidores Tisulares de Metaloproteinasas , Femenino , Humanos , Masculino , Amiloide/metabolismo , Biomarcadores de Tumor/análisis , Calcitonina , Carcinoma Neuroendocrino/diagnóstico , Carcinoma Neuroendocrino/metabolismo , Carcinoma Neuroendocrino/patología , Estudios Transversales , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas S100 , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
Medullary Thyroid Carcinoma (MTC) constitutes around 5% of all thyroid cancers. Trace elements assessment has emerged as a useful strategy in the diagnostics of MTC combined with Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) analysis. The aim of this study was to compare the presence and content of trace elements (i.e., Copper (Cu), Zinc (Zn), Iron (Fe), and Manganese (Mn)) in MTC with respect to control samples and their potential relationship with markers of MTC in tissues. The study included 26 patients who had undergone thyroidectomy, due to the diagnosis of MTC and 17 patients as control. We combined tumour pathology and staging, immunohistochemical analysis of calcitonin, MMPs, and TIMPs, with analytical biochemistry using Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) to determine the levels of trace elements. No differences by MTC type for MMPs and their TIPMs, although strong TIMP-1 and TIMP-2 immunohistochemical expression of MTC were unveiled. Additionally, Zn, Fe, and Mn tended to be decreased, and Cu to be increased in samples presenting MTC with respect to controls. Moreover, Zn was the unique trace element which seemed to be correlated with MMPs and TIMPs. Trace elements such as Zn, Fe, and Mn are decreased in tissues affected by MTC. In addition, Zn may be the trace element which saves more relationship with the proportion and intensity of MMPs, being considered altogether useful biomarkers of MTC. We therefore suggest the analysis of novel and traditional markers of MTC as a novel approach in this pathology.