Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum

Bax?2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax?2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax?2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax?2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax?2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax?2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of Bax?2 in Purkinje cells.     

Three homologous genes encoding functional ∆8-sphingolipid desaturase in Populus tomentosa

?⁸-sphingolipid desaturase is characterized by its ability to catalyze desaturation at the C8 position of the long-chain base of sphingolipids in plants. No previous studies have been conducted on genes encoding Δ⁸-sphingolipid desaturases in the woody plant Populus tomentosa. In this study, three genes that encode Δ⁸-sphingolipid desaturase were isolated from P. tomentosa. Among these genes, PtD8A and PtD8B showed high sequence similarity; whereas PtD8C exhibited large sequence divergence. RT-PCR results showed that PtD8A and PtD8B were expressed in all tissues detected, whereas PtD8C was not expressed in roots. Heterologous expression in yeast revealed that PtD8A/B/C were functional Δ⁸-sphingolipid desaturases, and can catalyze the C18-phytosphingenine desaturation to produce 8(Z)- and 8(E)-C18-phytosphingenine. However, the conversion rate and ratios of the two products differed. Compared with control cells, transgenic yeasts expressing PtD8A/B/C exhibited enhanced aluminum tolerance. Our findings further elucidated the biochemical functions and evolutionary history of Δ⁸-sphingolipid desaturases in plants. Candidate genes for breeding new poplar germplasm resources with enhanced tolerance ability to aluminium were also provided.     

Proteome analysis of Azotobacter vinelandii ∆arrF mutant that overproduces poly-β-hydroxybutyrate polymer

Azotobacter vinelandii ArrF is an iron-responsive small RNA that is under negative control of Ferric uptake regulator protein. A. vinelandii ∆arrF mutant that had a deletion of the entire arrF gene was known to overproduce poly-β-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E₁ component of pyruvate dehydrogenase complex, which leads to the production of NADPH and acetyl-CoA, were upregulated, while proteins in the tricarboxylic acid cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were highly overexpressed in the mutant. Antioxidant proteins such as Fe-containing superoxide dismutase (FeSOD), a putative oxidoreductase, alkyl hydroperoxide reductase, flavorprotein WrbA, and cysteine synthase were also overexpressed in the ∆arrF mutant, indicating that the PHB accumulation is stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase, though the reasons for their overexpression are unclear. Among genes upregulated in the mutant, sodB coding for FeSOD and phbF encoding PHB synthesis regulator PhbF were negatively regulated by small RNA ArrF probably in an antisense mechanism. The deletion of arrF gene, therefore, would increase PhbF and FeSOD levels, which favors PHB synthesis in the mutant. On the other hand, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that multiple factors including the direct effect of small RNA ArrF might be responsible for the PHB overproduction in the mutant.     

p∆TubHA4C, a new versatile vector for constitutive expression in Drosophila

Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. In this report we describe a new Drosophila expression vector (p?TubHA4C) for ubiquitous expression of coding sequences under the control of a minimal 0.9 kb promoter of α1 tubulin (α1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the ?Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9 kb ΔTub promoter induced similar expression levels to the intact 2.6 kb α1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible ?TubHA4C vector.     

Amexanthomycins A–J,pentangular polyphenols produced by Amycolatopsis mediterranei S699∆rifA

Ten new pentangular polyphenols, namely amexanthomycins A–J (1–10) were isolated from the strain Amycolatopsis mediterranei S699∆rifA constructed by deleting the polyketide synthase genes responsible for the biosynthesis of rifamycins. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic data and high-resolution ESIMS. Amexanthomycins A–C (1–3) showed inhibitory activity against human DNA topoisomerases.

     

Trans-thylakoid ∆pH dependent oscillation of FPSI/FPSII under continuous irradiance in isolated thylakoids

Energy distribution between photosystems (PSI & PSII) under prolonged and continuous white light irradiance was assessed by monitoring the progress of their fluorescence emission (F_PSI/F_PSII) at 77 K. Our observations indicate F_PSI/F_PSII to oscillate with the progress of irradiance treatments at all intensities tested (100, 200, 500, and 800 μE m^?2 S^?1). The amplitude of the oscillation increased with the progress, whereas the periodicity of the oscillation increased with the intensity of the incident irradiance. Spectral analysis indicated fluctuation of F_PSI to be the major determinant of the observed oscillation. The first rise and fall of F_PSI/F_PSII overlapped with phosphorylation and dephosphorylation of LHCII, but oscillation of F_PSI/F_PSII continued for several cycles without any further phosphorylation of LHCII. Moreover, in presence of DCMU where linear electron flow (LEF) is suppressed and LHCII phosphorylation is completely abolished, the oscillation of F_PSI/F_PSII was not abolished. These data indicated that LHCII phosphorylation was not essential for the observed oscillation of energy distribution between the photosystems. In contrast, in the presence of inhibitors of cyclic electron flow (CEF) like Antimycin A (AA) and rotenone, the oscillation of F_PSI/F_PSII was either abolished or severely dampened. Additionally, the oscillation was also abolished in presence of uncouplers like NH₄Cl and nigericin that cancels the trans-thylakoid ?pH. Thus, trans-thylakoid ?pH, generated through CEF, appear to be an important determinant of oscillation of F_PSI/F_PSII in isolated thylakoids. The phenomenon of oscillation could be associated with a CEF mediated chromatic adaptation of PSI in presence of excess irradiance.     

Revealing the complementation of ferredoxin by cytochrome b 5 in the Spirulina-∆6-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b 5 domain and Spirulina-∆6-acyl-lipid desaturase

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b ₅. The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-∆⁶ desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-∆⁶ desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b ₅ domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-∆⁶ desaturase fused or co-expressed with cytochrome b ₅ in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b ₅. Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b ₅ domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b ₅ domain in the desaturation process of Spirulina-∆⁶ desaturase. Furthermore, the free form of cytochrome b ₅ domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.     

Ifenprodil Attenuates Methamphetamine-Induced Behavioral Sensitization and Activation of Ras-ERK-∆FosB Pathway in the Caudate Putamen

Addiction is a debilitating, chronic psychiatric disorder that is difficult to cure completely owing to the high rate of relapse. Behavioral sensitization is considered to may underlie behavioral changes, such as relapse, caused by chronic abuse of psychomotor stimulants. Thus, its animal models have been widely used to explore the etiology of addiction. Recently, increasing evidence has demonstrated that N-methyl-d-aspartate receptors (NMDARs) play an important role in addiction to psychomotor stimulants. However, the role of GluN2B-containing receptors and their downstream signaling pathway(s) in behavioral sensitization induced by methamphetamine (METH) have not been investigated yet. In this study, we used different doses of ifenprodil (2.5, 5, 10 mg/kg), a selective antagonist of the GluN2B subunit, to investigate the role of GluN2B-containing NMDARs in METH-induced behavioral sensitization. We then examined changes in the levels of Ras, phosphorylated extracellular signal-regulated kinase (pERK)/ERK, and ?FosB in the caudate putamen (CPu) by western blot. We found that 2.5 or 10 mg/kg ifenprodil significantly attenuated METH-induced behavioral sensitization, whereas the mice treated with a moderate dose of ifenprodil (5 mg/kg) displayed no significant changes. Further results of western blot experiments showed that repeated administration of METH caused the increases in the levels of Ras, pERK/ERK and ?FosB in the CPu, and these changes were inhibited by only the 2.5 mg/kg dose of ifenprodil. In conclusion, these results demonstrated that 2.5 mg/kg ifenprodil could attenuate METH-induced behavioral sensitization. Moreover, GluN2B-containing NMDARs and their downstream Ras-ERK-?FosB signaling pathway in the CPu might be involved in METH-induced behavioral sensitization.     

Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum ‘Xanthi’) plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.     

Expression of yeast acyl-CoA-∆9 desaturase leads to accumulation of unusual monounsaturated fatty acids in soybean seeds

An acyl-CoA-Δ9 desaturase from Saccharomyces cerevisiae was expressed by subcellular-targeting in soybean (Glycine max) seeds with the goal of increasing palmitoleic acid (16:1Δ9), a high-valued fatty acid (FA), and simultaneously decreasing saturated FA in oil. The expression resulted in the conversion of palmitic acid (16:0) to 16:1Δ9 in soybean seeds. 16:1Δ9 and its elongation product cis-vaccenic acid (18:1Δ11) were increased to 17 % of the total fatty acids by plastid-targeted expression of the enzyme. Other lipid changes include the decrease of polyunsaturated FA and saturated FA, suggesting that a mechanism exists downstream in oil biosynthesis to compensate the FA alternation. This is the first time a cytosolic acyl-CoA-?9 desaturase is functionally expressed in plastid and stronger activity was achieved than its cytosolic expression. The present study provides a new strategy for converting 16:0 to 16:1Δ9 by engineering acyl-CoA-Δ9 desaturase in commercialized oilseeds.     

Modification of secondary head-forming activity of microinjected ∆β-catenin mRNA by co-injected spermine and spermidine in Xenopus early embryos

Polyamines are essential for cell growth and differentiation. In Xenopus early embryos, per embryo level of spermine is extremely low as compared with that of spermidine. To disclose the possible function of polyamines in Xenopus early embryos, we tested the effect of co-injection of spermine and spermidine on the functioning of exogenously microinjected in vitro-synthesized, ?β-catenin mRNA which is known to induce a secondary head after being microinjected into a ventral vegetal blastomere in 8-celled Xenopus embryos. Microinjection of ?β-catenin mRNA in fact induced a secondary axis with a secondary head, and co-injection of spermine or spermidine suppresses induction of the secondary head and secondary axis without drastic effects like induction of immediate cell death or execution of apoptosis at blastula stage. The inhibitory effects were dosage dependent, and at lower doses the inhibition was mainly on secondary head formation rather than on secondary axis formation. We performed similar experiments using GFP mRNA and confirmed that expression of GFP mRNA was also suppressed by co-injection of spermine. We mixed ?β-catenin mRNA with different amounts of spermine and performed electrophoresis on agarose gels, with a finding that the prior mixing greatly suppressed mRNA migration. These results suggest that an excess amount of spermine as well as spermidine exerts inhibitory effects on mRNA translation, and that the inhibition may be due to direct binding of polyamines to mRNA and a reduction of negative charges on mRNA molecules that might also induce the formation of cross link-like networks among mRNAs.     

Methyl-end desaturases with ∆12 and ω3 regioselectivities enable the de novo PUFA biosynthesis in the cephalopod Octopus vulgaris

The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C₂₀) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C₁₈ PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase (“ωx2”) mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase (“ωx1”) catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C₂₀ ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-¹⁴C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.     

A test for enhancement of cytotype regulation in Drosophila melanogaster by the transposase-encoding P Element ∆2-3

Transposable P elements are regulated in the germ line by piRNAs, which are small RNAs that associate with the Piwi class of proteins. This regulation, called the P cytotype, is enhanced by genetic interactions between P elements that are primary sources of these RNAs and other P elements. The enhanced regulation is thought to reflect amplification of the primary piRNAs by cleavage of mRNAs derived from the other P elements through a mechanism called the ping-pong cycle. We tested the transposase-encoding P element known as ?2-3 for its ability to enhance cytotype regulation anchored in P elements inserted at the telomere of the left arm of the X chromosome (TP elements). The ?2-3 P element lacks the intron between exons 2 and 3 in the structurally complete P element (CP). Unlike the CP element, it does not markedly enhance cytotype regulation anchored in TP elements, nor does it transmit transposase activity through the egg cytoplasm. However, mRNAs from both the CP and ?2-3 elements are maternally deposited in embryos. These observations suggest that maternally transmitted CP mRNA enhances cytotype regulation by participating in the ping-pong cycle and that it encodes the P transposase in the embryonic germ line, whereas maternally transmitted ?2-3 mRNA does not, possibly because it is not efficiently directed into the primordial embryonic germ line. Strong transposon regulation may, therefore, require ping-pong cycling with maternally inherited mRNAs in the embryo.     

Ocular Disposition of ∆<Superscript>8</Superscript>-Tetrahydrocannabinol from Various Topical Ophthalmic Formulations

The purposes of this project are to enhance the trans-membrane penetration of Δ⁸-Tetrahydrocannabinol (Δ⁸-THC) and to study the effect of various lipid based systems in delivering the compound, non-invasively, to anterior and posterior ocular chambers. Solid lipid nanoparticles (SLNs), fast gelling films were manufactured using high pressure homogenization and melt cast techniques, respectively. The formulations were characterized for drug content, entrapment efficiency, particle size and subsequently evaluated in vitro for trans-corneal permeation. In vivo, the drug disposition was tested via topical administration in albino rabbits. The eye globes were enucleated at the end of experiment and tissues were analyzed for drug content. All formulations showed favorable physicochemical characteristics in terms of particle size, entrapment efficiency, and drug content. In vitro, the formulations exhibited a transcorneal flux that depended on the formulation’s drug load. An increase in drug load from 0.1 to 0.75% resulted in 12- to16-folds increase in permeation. In vivo, the film was able to deliver THC to all the tissues with high accumulations in cornea and sclera. The SLNs showed a greater ability in delivering THC to all the tissues, at a significantly lower drug load, due to their colloidal size range, which in turn enhanced corneal epithelial membrane penetration. The topical formulations evaluated in the present study were able to successfully deliver Δ⁸-THC in therapeutically meaningful concentrations (EC₅₀ values for CB₁: 6 nM and CB₂: 0.4 nM) to all ocular tissues except the vitreous humor, with pronounced tissue penetration achieved using SLNs as a Δ⁸-THC delivery vehicle.     

Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabissativa

?1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ?1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75? resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.     

Analysis of the intracellular localization of p73 N-terminal protein isoforms TAp73 and ∆Np73 in medulloblastoma cell lines

The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein–protein interactions in medulloblastoma cells.     

Understanding causes of tree growth response to gap formation: ∆13C-values in tree rings reveal a predominant effect of light

Key message

Carbon isotope ratios in growth rings of a tropical tree species show that treefall gaps stimulate diameter growth mainly through changes in the availability of light and not water. The formation of treefall gaps in closed canopy forests usually entails considerable increases in light and nutrient availability for remaining trees, as well as altered plant water availability, and is considered to play a key role in tree demography. The effects of gaps on tree growth are highly variable and while usually stimulatory they may also include growth reductions. In most studies, the causes of changes in tree growth rates after gap formation remain unknown. We used changes in carbon isotope ¹³C discrimination (Δ¹³C) in annual growth rings to understand growth responses after gap formation of Peltogyne cf. heterophylla, in a moist forest of Northern Bolivia. We compared growth and Δ¹³C of the 7 years before and after gap formation. Forty-two trees of different sizes were studied, half of which grew close (<10 m) to single treefall gaps (gap trees), the other half more than 40 m away from gaps (controls). We found variable responses among gap trees in growth and Δ¹³C. Increased growth was mainly associated with decreased Δ¹³C, suggesting that the growth response was driven by increased light availability, possibly in combination with improved nutrient availability. Most trees showing zero or negative growth change after gap formation had increased Δ¹³C, suggesting that increased water stress did not play a role, but rather that light conditions had not changed much or nutrient availability was insufficient to support increased growth. Combining growth rates with Δ¹³C proved to be a valuable tool to identify the causes of temporal variation in tree growth.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ∆yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.