RESUMEN
The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron-sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: "GGG" and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron-sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron-sulfur cluster assembly site but also modulates the internal motions of ISCU2.
Asunto(s)
Proteínas Hierro-Azufre , Humanos , Proteínas Hierro-Azufre/química , Liasas de Carbono-Azufre/metabolismo , Azufre/química , Hierro/química , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/genéticaRESUMEN
TDP-43 is an RNA-binding protein that presents four domains comprising an N-terminal region, two RNA recognition motifs and a C-terminal region. The N-terminal domain (NTD) has a relevant role in the oligomerization and splicing activity of TDP-43. In this work, we have expressed, purified and biophysically characterized the region that includes residues 1 to 102 that contains the nuclear localization signal (residues 80-102, NLS). Furthermore, we have evaluated the oligomerization equilibrium for this protein fragment. Also, we have determined changes in the tertiary structure and its stability in a broad range of pH values by means of different spectroscopic methods. Additionally, we compared this fragment with the one that lacks the NLS employing experimental and computational methods. Finally, we evaluated the motion of dimeric forms to get insights into the conformational flexibility of this TDP-43 module in an oligomeric state. Our results suggest that this domain has a conformational plasticity in the vicinity of the single tryptophan of this domain (Trp68), which is enhanced by the presence of the nuclear localization signal. All these results help to understand the molecular features of the NTD of TDP-43.
Asunto(s)
Señales de Localización Nuclear , Triptófano , Conformación Proteica , Proteínas de Unión al ADN/metabolismoRESUMEN
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.
Asunto(s)
Liasas de Carbono-Azufre , Proteínas de Unión a Hierro , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/metabolismo , FrataxinaRESUMEN
Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.
Asunto(s)
FN-kappa B , Ubiquitina , FN-kappa B/genética , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Transducción de Señal/fisiología , Mitocondrias/metabolismo , UbiquitinaciónRESUMEN
Gluten related-disorders have a prevalence of 1-5 % worldwide triggered by the ingestion of gluten proteins in wheat, rye, barley, and some oats. In wheat gluten, the most studied protein is gliadin, whose immunodominant 33-mer amino acid fragment remains after digestive proteolysis and accumulates in the gut mucosa. Here, we report the formation of 33-mer thin-plate superstructures using intrinsic tyrosine (Tyr) steady-state fluorescence anisotropy and cryo-TEM in combination with water tension measurements. Furthermore, we showed that fluorescence decay measurements of 33-mer intrinsic fluorophore Tyr provided information on the early stages of the formation of the thin-plate structures. Finally, conformational analysis of Tyr residues using minimalist models by molecular dynamic simulations (MD) demonstrated that changes in Tyr rotamer states depend on the oligomerization stage. Our findings further advance the understanding of the formation of the 33-mer gliadin peptide superstructures and their relation to health and disease.
Asunto(s)
Gliadina , Glútenes , Gliadina/química , Gliadina/metabolismo , Glútenes/química , Triticum , Proteínas , Péptidos/química , Fragmentos de Péptidos/químicaRESUMEN
SCOPE: Proteolysis-resistant gliadin peptides are intensely investigated in biomedical research relates to celiac disease and gluten-related disorders. Herein, the first integrated supramolecular investigation of pepsin-digested gliadin peptides (p-gliadin) is presented in combination with its functional behavior in the Caco-2 cell line. METHODS AND RESULTS: First, gliadins are degraded by pepsin at pH 3, and the physicochemical properties of p-gliadin are compared with gliadin. An integrated approach using interfacial, spectroscopic, and microscopic techniques reveals that the p-gliadin forms spontaneously soluble large supramolecular structures, mainly oligomers and fibrils, capable of binding amyloid-sensitive dyes. The self-assembly of p-gliadin starts at a concentration of 0.40 µg mL-1 . Second, the stimulation of Caco-2 cells with the p-gliadin supramolecular system is performed, and the mRNA expression levels of a panel of genes are tested. The experiments show that p-gliadin composed of supramolecular structures triggers significant mRNA up-regulation (p < 0.05) of pro-apoptotic biomarkers (ratio Bcl2/Bak-1), chemokines (CCL2, CCL3, CCL4, CCL5, CXCL8), and the chemokine receptor CXCR3. CONCLUSIONS: This work demonstrates that p-gliadin is interfacial active, forming spontaneously amyloid-type structures that trigger genes in the Caco-2 cell line involved in recruiting specialized immune cells.
Asunto(s)
Gliadina/química , Nanoestructuras , Pepsina A/metabolismo , Apoptosis , Células CACO-2 , Enfermedad Celíaca/inmunología , Factores Quimiotácticos , Regulación de la Expresión Génica , Humanos , Inflamación , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , ProteolisisRESUMEN
In recent years, the evaluation of the structural properties of food has become of crucial importance in the understanding of food-related disorders. One of the most exciting systems is gliadin, a protein in wheat gluten, that plays a protagonist role in gluten-related disorders with a worldwide prevalence of 5%, including autoimmune celiac disease (CeD) (1%) and non-celiac wheat sensitivity (0.5-13%). It is accepted that gliadin is not fully digested by humans, producing large peptides that reach the gut mucosa. The gliadin peptides cross the lamina propria eliciting different immune responses in susceptible patients. Many clinical and biomedical efforts aim to diagnose and understand gluten-related disorders; meanwhile, the early stages of the inflammatory events remain elusive. Interestingly, although the primary sequence of many gliadin peptides is well known, it was only recently revealed the self-assembly capability of two pathogenic gliadin fragments and their connection to the early stage of diseases. This review is dedicated to the most relevant biophysical characterization of the complex gliadin digest and the two most studied gliadin fragments, the immunodominant 33-mer peptide and the toxic p31-43 in connection with inflammation and innate immune response. Here, we want to emphasize that combining different biophysical methods with cellular and in vivo models is of key importance to get an integrative understanding of a complex biological problem, as discussed here.
RESUMEN
The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson's disease (PD), multiple system atrophy, Alzheimer's disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.
Asunto(s)
Agregado de Proteínas , Agregación Patológica de Proteínas/etiología , Agregación Patológica de Proteínas/metabolismo , Multimerización de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , Amiloidosis , Animales , Susceptibilidad a Enfermedades , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Extracción Líquido-Líquido , Modelos Moleculares , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Conformación Proteica , Relación Estructura-Actividad , alfa-Sinucleína/aislamiento & purificaciónRESUMEN
The self-assembly of proteins is an essential process for a variety of cellular functions including cell respiration, mobility and division. On the other hand, protein or peptide misfolding and aggregation is related to the development of Parkinson's disease and Alzheimer's disease, among other aggregopathies. As a consequence, significant research efforts are directed towards the understanding of this process. In this review, we are focused on the use of UV-Visible Absorption Spectroscopy, Fluorescence Spectroscopy and Circular Dichroism to evaluate the self-organization of proteins and peptides in solution. These spectroscopic techniques are commonly available in most chemistry and biochemistry research laboratories, and together they are a powerful approach for initial as well as routine evaluation of protein and peptide self-assembly and aggregation under different environmental stimulus. Furthermore, these spectroscopic techniques are even suitable for studying complex systems like those in the food industry or pharmaceutical formulations, providing an overall idea of the folding, self-assembly, and aggregation processes, which is challenging to obtain with high-resolution methods. Here, we compiled and discussed selected examples, together with our results and those that helped us better to understand the process of protein and peptide aggregation. We put particular emphasis on the basic description of the methods as well as on the experimental considerations needed to obtain meaningful information, to help those who are just getting into this exciting area of research. Moreover, this review is particularly useful to those out of the field who would like to improve reproducibility in their cellular and biomedical experiments, especially while working with peptide and protein systems as an external stimulus. Our final aim is to show the power of these low-resolution techniques to improve our understanding of the self-assembly of peptides and proteins and translate this fundamental knowledge in biomedical research or food applications.
Asunto(s)
Péptidos/química , Proteínas/química , Dicroismo Circular , Péptidos/síntesis química , Agregado de Proteínas , Proteínas/síntesis química , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Celiac disease (CeD) is a highly prevalent chronic immune-mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α-gliadin peptide, p31-43, induces proinflammatory responses, observed by in vitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31-43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31-43 secondary structure by different biophysical and in silico techniques. By dynamic light scattering and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy analysis showed p31-43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self-organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with ß-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased ß-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.
Asunto(s)
Enfermedad Celíaca/tratamiento farmacológico , Gliadina/farmacología , Inmunidad Innata/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Células CACO-2 , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Gliadina/genética , Gliadina/inmunología , Gliadina/ultraestructura , Humanos , Inmunidad Innata/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Microscopía de Fuerza Atómica , Conformación Molecular/efectos de los fármacos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/ultraestructura , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Conformación Proteica en Lámina beta , Soluciones/química , Agua/químicaRESUMEN
The proteolytic resistant 33-mer gliadin peptide is an immunodominant fragment in gluten and responsible for the celiac disease and other gluten-related disorders. Meanwhile, the primary structure of the 33-mer is associated with the adaptive immune response in celiac patients, and the structural transformation of the 33-mer into protofilaments activates a primordial innate immune response in human macrophages. This means that accumulation, oligomerisation and structural transformation of the 33-mer could be the unknown first event that triggers the disease. Herein, we reveal the early stepwise mechanism of 33-mer oligomerisation by combining multiple computational simulations, tyrosine cross-linking, fluorescence spectroscopy and circular dichroism experiments. Our theoretical findings demonstrated that the partial charge distribution along the 33-mer molecule and the presence of glutamine that favours H-bonds between the oligomers are the driving forces that trigger oligomerisation. The high content of proline is critical for the formation of the flexible PPII secondary structure that led to a ß structure transition upon oligomerisation. Experimentally, we stabilised the 33-mer small oligomers by dityrosine cross-linking, detecting from dimers to higher molecular weight oligomers, which confirmed our simulations. The relevance of 33-mer oligomers as a trigger of the disease as well as its inhibition may be a novel therapeutic strategy for the treatment of gluten-related disorders.
RESUMEN
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.
Asunto(s)
Complejo I de Transporte de Electrón/química , Proteínas Reguladoras del Hierro/química , Cristalografía por Rayos X , Complejo I de Transporte de Electrón/metabolismo , Humanos , Enlace de Hidrógeno , Proteínas Reguladoras del Hierro/metabolismo , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Multimerización de ProteínaRESUMEN
The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in iron homeostasis and biosynthesis of FeS clusters, however its role in both pathways is not unequivocally defined. In this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations.
Asunto(s)
Ataxia de Friedreich , Proteínas de Unión a Hierro/química , Hierro/química , Mutación , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Hierro/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios Proteicos , FrataxinaRESUMEN
Peptide structural transformation and aggregation is associated with a large number of outsider aetiology diseases, and it is intrinsically linked to amyloid peptide aggregation. Diphenylalanine self-assembled structures are used as robust minimalist beta amyloids not only to elucidate protein aggregation but also to generate hydrogels. Herein, we employed a neutral model peptide Ac-Phe-Phe-Cys-NH2 (Ac-FFC-NH2) to elucidate the role of intermolecular disulfide bonds in protein fibrillation. The Ac-FFC-NH2 peptide initially self-assembles into nanospheres that evolve to amyloid type fibrils under mild oxidative conditions. Incubation of the peptide in the presence of the chemical reduction agent TCEP inhibits the formation of the fibrils, detecting only spherical nanostructures with no secondary structure. Importantly, we triggered the transformation of the preformed linear straight amyloid fibrils to non-fibrillar structures by TCEP treatment. Under this condition, the amyloid bundles are transformed into rings, which evolve to a new spherical microstructure. We showed that the chemical reduction of intermolecular S-S in internal amyloid sequences might favour the off-path intermediates of amyloid fibril growth, even when the fibrils are formed. Our findings demonstrated that in internal amyloid sequences, the formation of intermolecular S-S promotes the formation of amyloid type fibrils; meanwhile, its reduction stabilises non-fibrillar structures. Altogether, this work provides fundamental understanding at the molecular and supramolecular level, thus facilitating the rational design of therapeutic tools for protein aggregation diseases.
Asunto(s)
Amiloide/química , Disulfuros/química , Oligopéptidos/química , Oligopéptidos/síntesis química , Oxidación-Reducción , Multimerización de ProteínaRESUMEN
Celiac disease (CD) is a chronic enteropathy elicited by a Th1 response to gluten peptides in the small intestine of genetically susceptible individuals. However, it remains unclear what drives the induction of inflammatory responses of this kind against harmless antigens in food. In a recent work, we have shown that the p31-43 peptide (p31-43) from α-gliadin can induce an innate immune response in the intestine and that this may initiate pathological adaptive immunity. The receptors and mechanisms responsible for the induction of innate immunity by p31-43 are unknown and here we present evidence that this may reflect conformational changes in the peptide that allow it to activate the NLRP3 inflammasome. Administration of p31-43, but not scrambled or inverted peptides, to normal mice induced enteropathy in the proximal small intestine, associated with increased production of type I interferon and mature IL-1ß. P31-43 showed a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro and induced activation of the ASC speck complex. In parallel, the enteropathy induced by p31-43 in vivo did not occur in the absence of NLRP3 or caspase 1 and was inhibited by administration of the caspase 1 inhibitor Ac-YVAD-cmk. Collectively, these findings show that p31-43 gliadin has an intrinsic propensity to form oligomers which trigger the NLRP3 inflammasome and that this pathway is required for intestinal inflammation and pathology when p31-43 is administered orally to mice. This innate activation of the inflammasome may have important implications in the initial stages of CD pathogenesis.
Asunto(s)
Caspasa 1/metabolismo , Gliadina/metabolismo , Inflamasomas/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fragmentos de Péptidos/metabolismo , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Apoptosis , Enfermedad Celíaca/etiología , Enfermedad Celíaca/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Gliadina/química , Gliadina/ultraestructura , Mucosa Intestinal/ultraestructura , Intestino Delgado , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/ultraestructura , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Mammalian frataxin is a small mitochondrial protein involved in iron sulfur cluster assembly. Frataxin deficiency causes the neurodegenerative disease Friedreich's Ataxia. Valuable knowledge has been gained on the structural dynamics of frataxin, metal-ion-protein interactions, as well as on the effect of mutations on protein conformation, stability and internal motions. Additionally, laborious studies concerning the enzymatic reactions involved have allowed for understanding the capability of frataxin to modulate Fe-S cluster assembly function. Remarkably, frataxin biological function depends on its interaction with some proteins to form a supercomplex, among them NFS1 desulfurase and ISCU, the scaffolding protein. By combining multiple experimental tools including high resolution techniques like NMR and X-ray, but also SAXS, crosslinking and mass-spectrometry, it was possible to build a reliable model of the structure of the desulfurase supercomplex NFS1/ACP-ISD11/ISCU/frataxin. In this chapter, we explore these issues showing how the scientific view concerning frataxin structure-function relationships has evolved over the last years.
Asunto(s)
Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Ataxia de Friedreich/genética , Humanos , Proteínas de Unión a Hierro/genética , Dispersión del Ángulo Pequeño , Relación Estructura-Actividad , Difracción de Rayos X , FrataxinaRESUMEN
Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier protein (ACP). We carried out the coexpression of human mitochondrial ACP and ISD11 in E. coli. This work shows that ACP and ISD11 form a soluble, structured, and stable complex able to bind to the human NFS1 subunit modulating its activity. Results suggest that ACP plays a key-role in ISD11 folding and stability in vitro. These findings offer the opportunity to study the mechanism of interaction between ISD11 and NFS1.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Proteínas Reguladoras del Hierro/metabolismo , Liasas de Carbono-Azufre/metabolismo , Humanos , Mitocondrias/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Multimerización de ProteínaRESUMEN
Gliadin, an immunogenic protein present in wheat, is not fully degraded by humans and after the normal gastric and pancreatic digestion, the immunodominant 33-mer gliadin peptide remains unprocessed. The 33-mer gliadin peptide is found in human faeces and urine, proving not only its proteolytic resistance in vivo but more importantly its transport through the entire human body. Here, we demonstrate that 33-mer supramolecular structures larger than 220 nm induce the overexpression of nuclear factor kappa B (NF-κB) via a specific Toll-like Receptor (TLR) 2 and (TLR) 4 dependent pathway and the secretion of pro-inflammatory cytokines such as IP-10/CXCL10 and TNF-α. Using helium ion microscopy, we elucidated the initial stages of oligomerisation of 33-mer gliadin peptide, showing that rod-like oligomers are nucleation sites for protofilament formation. The relevance of the 33-mer supramolecular structures in the early stages of the disease is paving new perspectives in the understanding of gluten-related disorders.
Asunto(s)
Gliadina/metabolismo , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Humanos , Inmunidad Innata/fisiología , FN-kappa B/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Friedreich's ataxia is a disease caused by a decrease in the levels of expression or loss of functionality of the mitochondrial protein frataxin (FXN). The development of an active and stable recombinant variant of FXN is important for protein replacement therapy. Although valuable data about the mature form FXN81-210 has been collected, not enough information is available about the conformation of the frataxin precursor (FXN1-210). We investigated the conformation, stability and function of a recombinant precursor variant (His6-TAT-FXN1-210), which includes a TAT peptide in the N-terminal region to assist with transport across cell membranes. His6-TAT-FXN1-210 was expressed in Escherichia coli and conditions were found for purifying folded protein free of aggregation, oxidation or degradation, even after freezing and thawing. The protein was found to be stable and monomeric, with the N-terminal stretch (residues 1-89) mostly unstructured and the C-terminal domain properly folded. The experimental data suggest a complex picture for the folding process of full-length frataxin in vitro: the presence of the N-terminal region increased the tendency of FXN to aggregate at high temperatures but this could be avoided by the addition of low concentrations of GdmCl. The purified precursor was translocated through cell membranes. In addition, immune response against His6-TAT-FXN1-210 was measured, suggesting that the C-terminal fragment was not immunogenic at the assayed protein concentrations. Finally, the recognition of recombinant FXN by cellular proteins was studied to evaluate its functionality. In this regard, cysteine desulfurase NFS1/ISD11/ISCU was activated in vitro by His6-TAT-FXN1-210. Moreover, the results showed that His6-TAT-FXN1-210 can be ubiquitinated in vitro by the recently identified frataxin E3 ligase RNF126, in a similar way as the FXN1-210, suggesting that the His6-TAT extension does not interfere with the ubiquitination machinery.