[New Drug Discovery Targeting Iron in Bacterial Infectious Diseases].

Iron is necessary for all living organisms, and bacteria that cause infections in human hosts also need ferrous ions for their growth and proliferation. In the human body, most ferric ions (Fe3+) are tightly bound to iron-binding proteins such as hemoglobin, transferrin, lactoferrin, and ferritin. Pathogenic bacteria express highly specific iron uptake systems, including siderophores and specific receptors. Most bacteria secrete siderophores, which are low-molecular weight metal-chelating agents, to capture Fe3+ outside cell. Siderophores are mainly classified as either catecholate or hydroxamate. Vibrio vulnificus, a Gram-negative pathogenic bacterium, is responsible for serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In our study, we generated deletion mutants of the genes encoding proteins involved in the vulnibactin mediated iron-utilization system, such as ferric-vulnibactin receptor protein (VuuA), periplasmic ferric-vulnibactin binding protein (FatB), ferric-vulnibactin reductase (VuuB), and isochorismate synthase (ICS). ICS and VuuA are required under low-iron conditions for ferric-utilization in M2799, but the alternative proteins FatB and VuuB can function as a periplasmic binding protein and a ferric-chelate reductase, respectively. VatD, which functions as ferric-hydroxamate siderophores periplasmic binding protein, was shown to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the ferric-hydroxamate siderophore reductase IutB was observed to participate in ferric-vulnibactin reduction in the absence of VuuB. We propose that ferric-siderophore periplasmic binding proteins and ferric-chelate reductases represent potential targets for drug discovery in the context of infectious diseases.

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.

The Regulation of the Disease-Causing Gene FXN.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease caused in almost all patients by expanded guanine-adenine-adenine (GAA) trinucleotide repeats within intron 1 of the FXN gene. This results in a relative deficiency of frataxin, a small nucleus-encoded mitochondrial protein crucial for iron-sulfur cluster biogenesis. Currently, there is only one medication, omaveloxolone, available for FRDA patients, and it is limited to patients 16 years of age and older. This necessitates the development of new medications. Frataxin restoration is one of the main strategies in potential treatment options as it addresses the root cause of the disease. Comprehending the control of frataxin at the transcriptional, post-transcriptional, and post-translational stages could offer potential therapeutic approaches for addressing the illness. This review aims to provide a general overview of the regulation of frataxin and its implications for a possible therapeutic treatment of FRDA.

Ferrostatin-1 specifically targets mitochondrial iron-sulfur clusters and aconitase to improve cardiac function in Sirtuin 3 cardiomyocyte knockout mice.

AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial iron­sulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and iron­sulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.

Determination of the levels and possible associations of alpha2-macroglobulin with autoantibodies in the serum of patients with various forms of autoimmune thyroiditis.

Antibodies to thyroid peroxidase (AB-TPO), antibodies to thyroglobulin (AB-TG), and the content of α2-macroglobulin (α2-MG) have been studied in serum samples of patients with autoimmune thyroiditis (AIT). All the patients were divided into 3 groups depending on age: 25-35, 36-50, 51-65 years. We found a significant change in the thyroid panel parameters in AIT, but without significant changes in the average concentration of α2-MG in the age groups of patients. This may be due to the accumulation and retention of complexes of defective forms of α2-MG in the circulation associated with their decreased ability to bind to receptors.

Pharmacotherapeutic strategies for Friedreich Ataxia: a review of the available data.

INTRODUCTION: Friedreich ataxia (FRDA) is a rare autosomal recessive disease, marked by loss of coordination as well as impaired neurological, endocrine, orthopedic, and cardiac function. There are many symptomatic medications for FRDA, and many clinical trials have been performed, but only one FDA-approved medication exists. AREAS COVERED: The relative absence of the frataxin protein (FXN) in FRDA causes mitochondrial dysfunction, resulting in clinical manifestations. Currently, the only approved treatment for FRDA is an Nrf2 activator called omaveloxolone (Skyclarys). Patients with FRDA also rely on various symptomatic medications for treatment. Because there is only one approved medication for FRDA, clinical trials continue to advance in FRDA. Although some trials have not met their endpoints, many current and upcoming clinical trials provide exciting possibilities for the treatment of FRDA. EXPERT OPINION: The approval of omaveloxolone provides a major advance in FRDA therapeutics. Although well tolerated, it is not curative. Reversal of deficient frataxin levels with gene therapy, protein replacement, or epigenetic approaches provides the most likely prospect for enduring, disease-modifying therapy in the future.

Expression and processing of mature human frataxin after gene therapy in mice.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results suggest that AAVrh.10hFXN can likely induce expression of therapeutic levels of mature hFXN in mice.

Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2.

Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381NFS1 to Cys138ISCU2 and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138ISCU2 rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin's molecular role in this fundamental process.

[Molecular mimicry between human thyroid peroxidase, thyroglobulin, cosinophil peroxidase, IL-24 and microorganisms antigens]. / Mimetismo molecular entre peroxidasa tiroidea humana, tiroglobulina, peroxidasa de eosinófilos, IL-24 y antígenos de microorganismos.

OBJECTIVE: Identify molecular mimicry between TPO, eosinophil peroxidase (EPX), thyroglobulin and IL24 and microorganism antigens. METHODS: Through in silico analysis, we performed local alignments between human and microorganism antigens with PSI-BLAST. Proteins that did not present a 3D structure were modeled by homology through the Swiss Modeller server and epitope prediction was performed through Ellipro. Epitopes were located in the 3D models using PYMOL software. RESULTS: A total of 38 microorganism antigens (parasites, bacteria) had identities between 30% and 45%, being the highest with Anisakis simplex. The alignment between 2 candidate proteins from A. simplex and EPX presented significant values, with identities of 43 and 44%. In bacteria, Campylobacter jejuni presented the highest identity with thyroglobulin (35%). 220 linear and conformational epitopes of microorganism antigens were predicted. Peroxidasin-like proteins from Toxocara canis and Trichinella pseudospiralis presented 10 epitopes similar to TPO and EPX, as possible molecules triggering cross-reactivity. No virus presented identity with the human proteins studied. CONCLUSION: TPO and EPX antigens shared potential cross-reactive epitopes with bacterial and nematode proteins, suggesting that molecular mimicry could be a mechanism that explains the relationship between infections and urticaria/hypothyroidism. In vitro work is needed to demonstrate the results obtained in the in silico analysis.

OBJETIVO: Identificar mimetismo molecular entre TPO, eosinofil peroxidasa (EPX), tiroglobulina e IL24 y antígenos de microorganismos. MÉTODOS: A través de análisis in silico, realizamos los alineamientos locales entre los antígenos humanos y de microorganismos con PSI-BLAST. Las proteínas que no presentaban estructura 3D, fueron modeladas por homología a través del servidor Swiss Modeller y se realizó una predicción de epítopes a través de Ellipro. Los epítopes se localizaron en los modelos 3D utilizando el software PYMOL. RESULTADOS: Un total de 38 antígenos de microorganismos (parásitos y bacterias), tuvieron identidades entre 30 y 45%, siendo los más altos con Anisakis simplex. El alineamiento entre dos proteínas candidatas de A. simplex y EPX presentaron valores importantes, con identidades de 43 y 44%. En las bacterias, Campylobacter jejuni presentó la mayor identidad con tiroglobulina (35%). Se predijeron 220 epítopes lineales y conformacionales de antígenos de microorganismos. Las proteínas similares a la peroxidasina de Toxocara canis y Trichinella pseudospiralis presentaron diez epítopes similares a TPO y EPX, como posibles moléculas desencadenantes de una reactividad cruzada. Ningún virus presentó identidad con las proteínas humanas estudiadas. CONCLUSIÓN: Los antígenos TPO y EPX compartieron potenciales epítopes de reacción cruzada con proteínas bacterianas y nematodos, lo que sugiere que el mimetismo molecular podría ser un mecanismo que explique la relación entre infecciones y la urticaria/hipotiroidismo. Se necesitan trabajos in vitro que demuestren los resultados obtenidos en el análisis in silico.

Generation and characterization of iPSC lines from Friedreich's ataxia patient (FRDA) with GAA.TTC repeat expansion in the Frataxin (FXN) gene's first intron (IGIBi016-A) and a non-FRDA healthy control individual (IGIBi017-A).

Friedreich's ataxia is a spinocerebellar degenerative disease caused by microsatellite (GAA.TTC)n repeat expansion in the first intron of FXN gene. Here, we developed iPSC lines from an FRDA patient (IGIBi016-A) and non-FRDA healthy control (IGIBi017-A). Both iPSC lines displayed typical iPSC morphology, expression of pluripotency markers, regular karyotypes (46, XY; 46, XX), capacity to grow into three germ layers, and FRDA hallmark -GAA repeat expansion and decreased FXN mRNA. Through these iPSC lines, FRDA phenotypes may be replicated in the in vitro assays, by creating neuron subtypes, cardiomyocytes and 3D organoids, for molecular and cellular biomarkers and therapeutic applications.

Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity.

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Inhibition of CISD1 attenuates cisplatin-induced hearing loss in mice via the PI3K and MAPK pathways.

Cisplatin is an effective chemotherapeutic drug for different cancers, but it also causes severe and permanent hearing loss. Oxidative stress and mitochondrial dysfunction in cochlear hair cells (HCs) have been shown to be important in the pathogenesis of cisplatin-induced hearing loss (CIHL). CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) plays a critical role in mitochondrial oxidative capacity and cellular bioenergetics. Targeting CISD1 may improve mitochondrial function in various diseases. However, the role of CISD1 in cisplatin-induced ototoxicity is unclear. Therefore, this study was performed to assess the role of CISD1 in cisplatin-induced ototoxicity. We found that CISD1 expression was significantly increased after cisplatin treatment in both HEI-OC1 cells and cochlear HCs. Moreover, pharmacological inhibition of CISD1 with NL-1 inhibited cell apoptosis and reduced mitochondrial reactive oxygen species accumulation in HEI-OC1 cells and cochlear explants. Inhibition of CISD1 with small interfering RNA in HEI-OC1 cells had similar protective effects. Furthermore, NL-1 protected against CIHL in adult C57 mice, as evaluated by the auditory brainstem response and immunofluorescent staining. Mechanistically, RNA sequencing revealed that NL-1 attenuated CIHL via the PI3K and MAPK pathways. Most importantly, NL-1 did not interfere with the antitumor efficacy of cisplatin. In conclusion, our study revealed that targeting CISD1 with NL-1 reduced reactive oxygen species accumulation, mitochondrial dysfunction, and apoptosis via the PI3K and MAPK pathways in HEI-OC1 cell lines and mouse cochlear explants in vitro, and it protected against CIHL in adult C57 mice. Our study suggests that CISD1 may serve as a novel target for the prevention of CIHL.

Inhibition of CISD1 alleviates mitochondrial dysfunction and ferroptosis in mice with acute lung injury.

The NET family member, CDGSH iron-sulfur domain-containing protein 1 (CISD1), is located in theoutermembrane of mitochondria, where it regulates energy and iron metabolism. CISD1 has vital functions in certain human diseases; however, its function in acute lung injury (ALI) is unknown. ALI pathogenesis critically involves mitochondrial dysfunction and ferroptosis, which might be regulated by CISD1. Therefore, we investigated CISD1's function in mitochondrial dysfunction and ferroptosis regulation in lipopolysaccharide (LPS)-induced ALI. We found that CISD1 was upregulated in LPS-induced ALI,and silencing Cisd1 prevented cell apoptosis and increased cell viability. When CISD1was inhibited by mitoNEET ligand-1 (NL-1) there was a significant mitigation of pathological injury and lung edema, and reduced numbers of total cells, polymorphonuclear leukocytes, and a decreased protein content in the bronchoalveolar lavage fluid (BALF). Moreover, inhibition of CISD1 markedly decreased the interleukin (IL)6, IL-1ß, and tumor necrosis factor alpha (TNF-α) levels in the lungs and BALF of ALI-model mice. Silencing of Cisd1 prevented LPS-induced mitochondrial membrane potential depolarization, cellular ATP reduction, and reactive oxygen species (ROS) accumulation, suggesting mitochondrial protection. ALI activated ferroptosis, as evidenced by the increased lipid-ROS, intracellular Fe2+ level, reduced Gpx4 (glutathione peroxidase 4) expression, and the glutathione/glutathione disulfide ratio. Interestingly, inhibition of CISD1 reduced LPS-induced ferroptosis in vivo and in vitro. In conclusion, inhibition of CISD1 alleviated mitochondrial dysfunction and ferroptosis in LPS-induced ALI, identifying CISD1 as possible target for therapy of LPS-induced ALI.

Searching for Frataxin Function: Exploring the Analogy with Nqo15, the Frataxin-like Protein of Respiratory Complex I from Thermus thermophilus.

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.

Nutraceutical and Health-Promoting Potential of Lactoferrin, an Iron-Binding Protein in Human and Animal: Current Knowledge.

Lactoferrin is a natural cationic iron-binding glycoprotein of the transferrin family found in bovine milk and other exocrine secretions, including lacrimal fluid, saliva, and bile. Lactoferrin has been investigated for its numerous powerful influences, including anticancer, anti-inflammatory, anti-oxidant, anti-osteoporotic, antifungal, antibacterial, antiviral, immunomodulatory, hepatoprotective, and other beneficial health effects. Lactoferrin demonstrated several nutraceutical and pharmaceutical potentials and have a significant impact on improving the health of humans and animals. Lactoferrin plays a critical role in keeping the normal physiological homeostasis associated with the development of pathological disorders. The current review highlights the medicinal value, nutraceutical role, therapeutic application, and outstanding favorable health sides of lactoferrin, which would benefit from more exploration of this glycoprotein for the design of effective medicines, drugs, and pharmaceuticals for safeguarding different health issues in animals and humans.

Frataxin analysis using triple quadrupole mass spectrometry: application to a large heterogeneous clinical cohort.

BACKGROUND: Friedreich ataxia is a progressive multisystem disorder caused by deficiency of the protein frataxin; a small mitochondrial protein involved in iron sulfur cluster synthesis. Two types of frataxin exist: FXN-M, found in most cells, and FXN-E, found almost exclusively in red blood cells. Treatments in clinical trials include frataxin restoration by gene therapy, protein replacement, and epigenetic therapies, all of which necessitate sensitive assays for assessing frataxin levels. METHODS: In the present study, we have used a triple quadrupole mass spectrometry-based assay to examine the features of both types of frataxin levels in blood in a large heterogenous cohort of 106 patients with FRDA. RESULTS: Frataxin levels (FXN-E and FXN M) were predicted by GAA repeat length in regression models (R2 values = 0.51 and 0.27, respectively), and conversely frataxin levels predicted clinical status as determined by modified Friedreich Ataxia Rating scale scores and by disability status (R2 values = 0.13-0.16). There was no significant change in frataxin levels in individual subjects over time, and apart from start codon mutations, FXN-E and FXN-M levels were roughly equal. Accounting for hemoglobin levels in a smaller sub-cohort improved prediction of both FXN-E and FXN-M levels from R2 values of (0.3-0.38 to 0.20-0.51). CONCLUSION: The present data show that assay of FXN-M and FXN-E levels in blood provides an appropriate biofluid for assessing their repletion in particular clinical contexts.

A modified mouse model of Friedreich's ataxia with conditional Fxn allele homozygosity delays onset of cardiomyopathy.

Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional Fxn allele and one floxed-out/null allele. These mice quickly develop cardiomyopathy that becomes fatal by 9-11 wk of age. Here, we generated a cardiac-specific model with floxed Fxn allele homozygosity (MCK-Fxnflox/flox). MCK-Fxnflox/flox mice were phenotypically normal at 9 wk of age, despite no detectable FXN protein expression. Between 13 and 15 wk of age, these mice began to display progressive cardiomyopathy, including decreased ejection fraction and fractional shortening and increased left ventricular mass. MCK-Fxnflox/flox mice began to lose weight around 16 wk of age, characteristically associated with heart failure in other cardiac-specific FA models. By 18 wk of age, MCK-Fxnflox/flox mice displayed elevated markers of Fe-S deficiency, cardiac stress and injury, and cardiac fibrosis. This modified model reproduced important pathophysiological and biochemical features of FA over a longer timescale than previous cardiac-specific mouse models, offering a larger window for studying potential therapeutics.NEW & NOTEWORTHY Previous cardiac-specific frataxin knockout models exhibit rapid and fatal cardiomyopathy by 9 wk of age. This severe phenotype poses challenges for the design and execution of intervention studies. We introduce an alternative cardiac-specific model, MCK-Fxnflox/flox, with increased longevity and delayed onset of all major phenotypes. These phenotypes develop to the same severity as previous models. Thus, this new model provides the same cardiomyopathy-associated mortality with a larger window for potential studies.

Human frataxin, the Friedreich ataxia deficient protein, interacts with mitochondrial respiratory chain.

Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease caused by an expanded GAA repeat in the first intron of the FXN gene, leading to transcriptional silencing and reduced expression of frataxin. Frataxin participates in the mitochondrial assembly of FeS clusters, redox cofactors of the respiratory complexes I, II and III. To date it is still unclear how frataxin deficiency culminates in the decrease of bioenergetics efficiency in FRDA patients' cells. We previously demonstrated that in healthy cells frataxin is closely attached to the mitochondrial cristae, which contain both the FeS cluster assembly machinery and the respiratory chain complexes, whereas in FRDA patients' cells with impaired respiration the residual frataxin is largely displaced in the matrix. To gain novel insights into the function of frataxin in the mitochondrial pathophysiology, and in the upstream metabolic defects leading to FRDA disease onset and progression, here we explored the potential interaction of frataxin with the FeS cluster-containing respiratory complexes I, II and III. Using healthy cells and different FRDA cellular models we found that frataxin interacts with these three respiratory complexes. Furthermore, by EPR spectroscopy, we observed that in mitochondria from FRDA patients' cells the decreased level of frataxin specifically affects the FeS cluster content of complex I. Remarkably, we also found that the frataxin-like protein Nqo15 from T. thermophilus complex I ameliorates the mitochondrial respiratory phenotype when expressed in FRDA patient's cells. Our data point to a structural and functional interaction of frataxin with complex I and open a perspective to explore therapeutic rationales for FRDA targeted to this respiratory complex.