An In Silico Analysis of Genetic Variants and Structural Modeling of the Human Frataxin Protein in Friedreich's Ataxia.

Friedreich's Ataxia (FRDA) stands out as the most prevalent form of hereditary ataxias, marked by progressive movement ataxia, loss of vibratory sensitivity, and skeletal deformities, severely affecting daily functioning. To date, the only medication available for treating FRDA is Omaveloxolone (Skyclarys®), recently approved by the FDA. Missense mutations within the human frataxin (FXN) gene, responsible for intracellular iron homeostasis regulation, are linked to FRDA development. These mutations induce FXN dysfunction, fostering mitochondrial iron accumulation and heightened oxidative stress, ultimately triggering neuronal cell death pathways. This study amalgamated 226 FXN genetic variants from the literature and database searches, with only 18 previously characterized. Predictive analyses revealed a notable prevalence of detrimental and destabilizing predictions for FXN mutations, predominantly impacting conserved residues crucial for protein function. Additionally, an accurate, comprehensive three-dimensional model of human FXN was constructed, serving as the basis for generating genetic variants I154F and W155R. These variants, selected for their severe clinical implications, underwent molecular dynamics (MD) simulations, unveiling flexibility and essential dynamic alterations in their N-terminal segments, encompassing FXN42, FXN56, and FXN78 domains pivotal for protein maturation. Thus, our findings indicate potential interaction profile disturbances in the FXN42, FXN56, and FXN78 domains induced by I154F and W155R mutations, aligning with the existing literature.

CRISPR/Cas9-based edition of frataxin gene in Dictyostelium discoideum.

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.

Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango.

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.

Ataxia de Friedreich, revisión y actualización de la literatura con búsqueda sistemática de casos en Latinoamérica / Friedreich's ataxia, review and literature update with a systematic search for cases in Latin America

La Ataxia de Friedreich (AF) es una enfermedad neurodegenerativa autosómica recesiva con compromiso multisistémico. En esta revisión, se actualizan aspectos epidemiológicos, fisiopatológicos y clínico-terapéuticos y se conduce una búsqueda sistemática de casos de AF reportados en Latinoamérica. La prevalencia de AF en poblaciones caucásicas es estimada entre 2 y 5 casos por 100 000 habitantes. En Latinoamérica se han publicado 35 estudios que reúnen 1481 casos en 6 países. Causada por la expansión anormal de repeticiones GAA en el gen FXN, la etiopatogenia está asociada a una reducción en los niveles de la proteína frataxina (que altera el metabolismo energético) y el acúmulo de hierro mitocondrial. El fenotipo clásico de AF suele comenzar antes de los 25 años, aunque hay otros de inicio tardío y retención de reflejos. La sintomatología se caracteriza por ataxia progresiva, alteración sensitiva, arreflexia, disartria, y alteraciones oculomotoras, además de compromiso cardiaco, endocrino y musculoesquelético. El diagnóstico requiere evaluación neurológica detallada, estudios neurofisiológicos, neuroimágenes y pruebas bioquímicas pero el enfoque determinante es el estudio genético que demuestre variantes genéticas bialélicas en el gen FXN. El manejo es multidisciplinario, orientado a aminorar los síntomas, prevenir complicaciones y brindar asesoramiento genético apropiado. Recientemente se ha aprobado el primer tratamiento farmacológico para AF con varios más en fases de experimentación.

SUMMARY Friedreich Ataxia (FA) is an autosomal recessive neurodegenerative disease with multisystemic involvement. This update of epidemiological, pathophysiological, and clinico-therapeutic aspects of FA, includes a systematic review of cases in Latin America. The estimated FA prevalence in Caucasian populations is between 2 to 5 cases per 100 000. In Latin America, 1481 cases have been published in 35 articles from six different countries. Caused by an abnormally repeated expansion of GAA trinucleotide inside the FXN gene, FA's etiopathogenesis is associated with reduced levels of the frataxin protein, which disturb the energy metabolism and result in mitochondrial iron accumulation. The classic phenotype usually shows symptoms before the age of 25, although there are others with a later onset. The main symptoms of AF are progressive ataxia, sensory disturbances, areflexia, dysarthria, and oculomotor alterations, in addition to cardiac, endocrine, and musculoskeletal compromise. Diagnostic workup requires a detailed neurological examination, neuroconduction studies, neuroimaging, and biochemical tests. The definitive diagnosis is provided by genetic testing showing biallelic variants within the FXN gene. The management is multidisciplinary, aimed at reducing symptoms, preventing complications, and providing an appropriate genetic counseling. Recently, the first pharmacological treatment for AF has been approved, with several others in clinical assessment trials.

Structural and Functional Characterization of CreFH1, the Frataxin Homolog from Chlamydomonas reinhardtii.

Frataxin plays a key role in cellular iron homeostasis of different organisms. It has been implicated in iron storage, detoxification, delivery for Fe-S cluster assembly and heme biosynthesis. However, its specific role in iron metabolism remains unclear, especially in photosynthetic organisms. To gain insight into the role and properties of frataxin in algae, we identified the gene CreFH1, which codes for the frataxin homolog from Chlamydomonas reinhardtii. We performed the cloning, expression and biochemical characterization of CreFH1. This protein has a predicted mitochondrial transit peptide and a significant structural similarity to other members of the frataxin family. In addition, CreFH1 was able to form a dimer in vitro, and this effect was increased by the addition of Cu2+ and also attenuated the Fenton reaction in the presence of a mixture of Fe2+ and H2O2. Bacterial cells with overexpression of CreFH1 showed increased growth in the presence of different metals, such as Fe, Cu, Zn and Ni and H2O2. Thus, results indicated that CreFH1 is a functional protein that shows some distinctive features compared to its more well-known counterparts, and would play an important role in response to oxidative stress in C. reinhardtii.

Conformational stability, dynamics and function of human frataxin: Tryptophan side chain interplay.

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.

Iron-Sulfur Cluster Complex Assembly in the Mitochondria of Arabidopsis thaliana.

In plants, the cysteine desulfurase (AtNFS1) and frataxin (AtFH) are involved in the formation of Fe-S groups in mitochondria, specifically, in Fe and sulfur loading onto scaffold proteins, and the subsequent formation of the mature Fe-S cluster. We found that the small mitochondrial chaperone, AtISD11, and AtFH are positive regulators for AtNFS1 activity in Arabidopsis. Moreover, when the three proteins were incubated together, a stronger attenuation of the Fenton reaction was observed compared to that observed with AtFH alone. Using pull-down assays, we found that these three proteins physically interact, and sequence alignment and docking studies showed that several amino acid residues reported as critical for the interaction of their human homologous are conserved. Our results suggest that AtFH, AtNFS1 and AtISD11 form a multiprotein complex that could be involved in different stages of the iron-sulfur cluster (ISC) pathway in plant mitochondria.

Relationship between activity and stability: Design and characterization of stable variants of human frataxin.

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.

Low frataxin mRNA expression is associated with inflammation and oxidative stress in patients with type 2 diabetes.

BACKGROUND: The mitochondrial protein frataxin is involved in iron metabolism, as well as regulation of oxidative stress. To elucidate the association of frataxin with the pathophysiology of diabetes, we evaluated the mRNA levels of frataxin in leukocytes of patients with type 2 diabetes (T2D). In addition, we investigated the relation between frataxin mRNA levels, inflammatory cytokines, and oxidative stress biomarkers. METHODS: A study including 150 subjects (115 patients with T2D and 35 healthy subjects) was performed to evaluate the frataxin mRNA levels in leukocytes. We assessed the relation between frataxin and interleukin (IL)-6, IL-1, tumour necrosis factor-alpha (TNF-α), total oxidation status (TOS), total antioxidant capacity (TAC), and serum iron. RESULTS: The frataxin mRNA levels in the T2D group were significantly lower than those in healthy subjects. It was also demonstrated that T2D patients with frataxin mRNA levels in the lowest quartile had significantly elevated levels of serum iron, TOS, and inflammatory cytokines, such as TNF-α, IL-1, and IL-6, while TAC levels were significantly lower in this quartile when compared with the upper quartile. CONCLUSIONS: Our findings showed that T2D patients with low frataxin mRNA levels showed a high degree of inflammation and oxidative stress. It is speculated that frataxin deficiency in T2D patients can contribute to the imbalance in mitochondrial iron homeostasis leading to the acceleration of oxidative stress and inflammation.

Frataxin Structure and Function.

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.

Ferrochelatase activity of plant frataxin.

Frataxin plays a key role in cellular iron homeostasis of different organisms. It is engaged in several activities at the FeS cluster assembly machinery and it is also involved in heme biosynthesis. In plants, two genes encoding ferrochelatases (FC1 and FC2) catalyze the incorporation of iron into protoporphyrin IX in the last stage of heme synthesis in chloroplasts. Despite ferrochelatases are absent from other cell compartments, a remaining ferrochelatase activity has been observed in plant mitochondria. Here we analyze the possibility that frataxin acts as the iron donor to protoporphyrin IX for the synthesis of heme groups in plant mitochondria. Our findings show that frataxin catalyzes the formation of heme in vitro when it is incubated with iron and protoporphyrin IX. When frataxin is combined with AtNFS1 and AtISD11 the ferrochelatse activity is increased. These results suggest that frataxin could be the iron donor in the final step of heme synthesis in plant mitochondria, and constitutes an important advance in the elucidation of the mechanisms of heme synthesis in plants.

Plant Frataxin in Metal Metabolism.

Frataxin is a highly conserved protein from prokaryotes to eukaryotes. Several functions related to iron metabolism have been postulated for this protein, including Fe-S cluster and heme synthesis, response to oxidative damage and oxidative phosphorylation. In plants, the presence of one or two isoforms of this protein with dual localization in mitochondria and chloroplasts has been reported. Frataxin deficiency affects iron metabolism in both organelles, leading to an impairment of mitochondrial respiration, and chlorophyll and photosynthetic electron transport deficiency in chloroplasts. In addition, plant frataxins can react with Cu2+ ions and dimerize, which causes the reduction of free Cu ions. This could provide an additional defense mechanism against the oxidation of Fe-S groups by Cu ions. While there is a consensus on the involvement of frataxin in iron homeostasis in most organisms, the interaction of plant frataxins with Cu ions, the presence of different isoforms, and/or the localization in two plant organelles suggest that this protein might have additional functions in vegetal tissues.

Identification of two frataxin isoforms in Zea mays: Structural and functional studies.

Frataxin is a ubiquitous protein that plays a role in Fe-S cluster biosynthesis and iron and heme metabolism, although its molecular functions are not entirely clear. In non-photosynthetic eukaryotes, frataxin is encoded by a single gene, and the protein localizes to mitochondria. Here we report the presence of two functional frataxin isoforms in Zea mays, ZmFH-1 and ZmFH-2. We confirmed our previous findings regarding plant frataxins: both proteins have dual localization in mitochondria and chloroplasts. Physiological, biochemical and biophysical studies show some differences in the expression pattern, protection against oxidants and in the aggregation state of both isoforms, suggesting that the two frataxin homologs would play similar but not identical roles in plant cell metabolism. In addition, two specific features of plant frataxins were evidenced: their ability to form dimers and their tendency to undergo conformational change under oxygen exposure.

The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin.

The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42-80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56-210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81-210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56-81, the moiety putatively responsible for promoting protein-protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or ß-strand structure. In this context, we explored the conformation and stability of hFXN56-210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90-210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81-210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function.