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• W2044992022 abstract "Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044G→C), compound heterozygous patients with severe myopathy have been identified to carry the c.149G→A missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms. Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044G→C), compound heterozygous patients with severe myopathy have been identified to carry the c.149G→A missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms. Iron-sulfur (Fe-S) clusters are indispensable and ubiquitous cofactors that are involved in a variety of regulatory processes, including catalysis and electron carrier activity (1.Craig E.A. Marszalek J. A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers.Cell. Mol. Life Sci. 2002; 59: 1658-1665Crossref PubMed Scopus (87) Google Scholar). Although the Fe-S clusters are relatively simple inorganic cofactors, their synthesis, assembly, and successive incorporation into apoproteins are highly intricate processes in living cells (2.Craig E.A. Voisine C. Schilke B. Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae.Biol. Chem. 1999; 380: 1167-1173Crossref PubMed Scopus (42) Google Scholar, 3.Lill R. Function and biogenesis of iron-sulphur proteins.Nature. 2009; 460: 831-838Crossref PubMed Scopus (835) Google Scholar). Fe-S clusters are usually integrated into proteins through coordination of the iron atoms by cysteine or histidine residues, although in more complex Fe-S clusters alternative ligands like Asp, Arg, and Ser and functional groups like CO and CN have been reported (4.Meyer J. Iron-sulfur protein folds, iron-sulfur chemistry, and evolution.J. Biol. Inorg. Chem. 2008; 13: 157-170Crossref PubMed Scopus (195) Google Scholar). Coordination of Fe-S clusters to electron transport chain complexes is indispensable for respiratory function within the cell. For example, complex I of bacteria contains nine Fe-S clusters, whereas eukaryotic complex I harbors eight Fe-S clusters, which are anchored to domains exposed to the cytosol or mitochondrial matrix, respectively (5.Hinchliffe P. Sazanov L.A. Organization of iron-sulfur clusters in respiratory complex I.Science. 2005; 309: 771-774Crossref PubMed Scopus (161) Google Scholar, 6.Sazanov L.A. Respiratory complex I: mechanistic and structural insights provided by the crystal structure of the hydrophilic domain.Biochemistry. 2007; 46: 2275-2288Crossref PubMed Scopus (178) Google Scholar). On the other hand, mammalian complex II possesses three distinct Fe-S clusters of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] type (7.Sun F. Huo X. Zhai Y. Wang A. Xu J. Su D. Bartlam M. Rao Z. Crystal structure of mitochondrial respiratory membrane protein complex II.Cell. 2005; 121: 1043-1057Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). Besides their importance in electron transfer, Fe-S proteins also play a pivotal role in enzyme-substrate reactions. Eukaryotic Fe-S cluster-containing enzymes, such as succinate dehydrogenase and aconitase, play a critical role in TCA 6The abbreviations used are: TCAtricarboxylic acidROSreactive oxygen speciesaaamino acidsNAO10-N-nonyl acridine orangeIPimmunoprecipitationAASatomic absorption spectroscopyMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideBN-PAGEblue native PAGEUTuntransfectedETCelectron transport chain. cycle metabolism (8.Beinert H. Kennedy M.C. Stout C.D. Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein.Chem. Rev. 1996; 96: 2335-2374Crossref PubMed Scopus (476) Google Scholar, 9.King A. Selak M.A. Gottlieb E. Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer.Oncogene. 2006; 25: 4675-4682Crossref PubMed Scopus (534) Google Scholar). Therefore, the biogenesis of these functionally important Fe-S clusters in mitochondria is an indispensable process for mitochondrial function as well as cell survival. tricarboxylic acid reactive oxygen species amino acids 10-N-nonyl acridine orange immunoprecipitation atomic absorption spectroscopy 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide blue native PAGE untransfected electron transport chain. Mitochondria are the major cellular compartment for Fe-S cluster biogenesis in eukaryotes, including the mammalian system. The central part of Fe-S protein biogenesis in human mitochondria is the de novo synthesis of the Fe-S cluster on a highly conserved scaffold protein, ISCU, before its transfer to apoproteins (10.Lill R. Dutkiewicz R. Elsässer H.P. Hausmann A. Netz D.J. Pierik A.J. Stehling O. Urzica E. Mühlenhoff U. Mechanisms of iron-sulfur protein maturation in mitochondria, cytosol and nucleus of eukaryotes.Biochim. Biophys. Acta. 2006; 1763: 652-667Crossref PubMed Scopus (134) Google Scholar). Mammalian ISCU is a nuclear encoded protein, predominantly localized in the mitochondrial matrix compartment, and comprises 167 amino acids with an N-terminal targeting signal. However, the presence of cytosolic ISCU has also been reported in humans (11.Tong W.H. Rouault T. Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells.EMBO J. 2000; 19: 5692-5700Crossref PubMed Scopus (168) Google Scholar). In Saccharomyces cerevisiae, there are two orthologs of human ISCU, namely Isu1 and Isu2, which are localized in the matrix compartment (12.Garland S.A. Hoff K. Vickery L.E. Culotta V.C. Saccharomyces cerevisiae ISU1 and ISU2: members of a well-conserved gene family for iron-sulfur cluster assembly.J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (167) Google Scholar). In yeast, the ISU1 and ISU2 double deletion mutant is inviable, thus signifying its central importance in the Fe-S cluster biogenesis (12.Garland S.A. Hoff K. Vickery L.E. Culotta V.C. Saccharomyces cerevisiae ISU1 and ISU2: members of a well-conserved gene family for iron-sulfur cluster assembly.J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (167) Google Scholar). The overall biogenesis process can be broadly categorized into two critical events: (a) the de novo assembly of an Fe-S cluster on a scaffold protein and (b) the transfer of the Fe-S cluster from the scaffold to target apoproteins (13.Fontecave M. Ollagnier-de-Choudens S. Iron-sulfur cluster biosynthesis in bacteria: mechanisms of cluster assembly and transfer.Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (146) Google Scholar). A cysteine desulfurase, Nfs1, assists in the sulfur transfer process to the scaffold protein (14.Biederbick A. Stehling O. Rösser R. Niggemeyer B. Nakai Y. Elsässer H.P. Lill R. Role of human mitochondrial Nfs1 in cytosolic iron-sulfur protein biogenesis and iron regulation.Mol. Cell. Biol. 2006; 26: 5675-5687Crossref PubMed Scopus (143) Google Scholar). This reaction is aided by direct interaction between Nfs1 and the scaffold protein (11.Tong W.H. Rouault T. Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells.EMBO J. 2000; 19: 5692-5700Crossref PubMed Scopus (168) Google Scholar, 15.Majewska J. Ciesielski S.J. Schilke B. Kominek J. Blenska A. Delewski W. Song J.Y. Marszalek J. Craig E.A. Dutkiewicz R. Binding of the chaperone Jac1 protein and cysteine desulfurase Nfs1 to the iron-sulfur cluster scaffold Isu protein is mutually exclusive.J. Biol. Chem. 2013; 288: 29134-29142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Upon transfer of iron and sulfur to the scaffold, the Fe-S cluster is formed by an unknown mechanism (13.Fontecave M. Ollagnier-de-Choudens S. Iron-sulfur cluster biosynthesis in bacteria: mechanisms of cluster assembly and transfer.Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (146) Google Scholar, 16.Johnson D.C. Dean D.R. Smith A.D. Johnson M.K. Structure, function, and formation of biological iron-sulfur clusters.Annu. Rev. Biochem. 2005; 74: 247-281Crossref PubMed Scopus (1088) Google Scholar). The iron-binding protein frataxin (Yfh1 in yeast and CyaY in bacteria) is believed to function as an iron donor (17.Bencze K.Z. Kondapalli K.C. Cook J.D. McMahon S. Millán-Pacheco C. Pastor N. Stemmler T.L. The structure and function of frataxin.Crit. Rev. Biochem. Mol. Biol. 2006; 41: 269-291Crossref PubMed Scopus (133) Google Scholar). The terminal transfer process is assisted by a dedicated chaperone system comprising the mtHsp70 (Ssq1 in yeast) and the DnaJ-like cochaperone, HSCB (Jac1 in yeast) (18.Craig E.A. Huang P. Aron R. Andrew A. The diverse roles of J-proteins, the obligate Hsp70 co-chaperone.Rev. Physiol. Biochem. Pharmacol. 2006; 156: 1-21PubMed Google Scholar, 19.Schilke B. Williams B. Knieszner H. Pukszta S. D'Silva P. Craig E.A. Marszalek J. Evolution of mitochondrial chaperones utilized in Fe-S cluster biogenesis.Curr. Biol. 2006; 16: 1660-1665Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The interaction of a J-protein cochaperone, such as Jac1 (HSCB in humans), with Fe-S scaffold Isu1 is conserved in evolution and indispensable in vivo (15.Majewska J. Ciesielski S.J. Schilke B. Kominek J. Blenska A. Delewski W. Song J.Y. Marszalek J. Craig E.A. Dutkiewicz R. Binding of the chaperone Jac1 protein and cysteine desulfurase Nfs1 to the iron-sulfur cluster scaffold Isu protein is mutually exclusive.J. Biol. Chem. 2013; 288: 29134-29142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 20.Ciesielski S.J. Schilke B.A. Osipiuk J. Bigelow L. Mulligan R. Majewska J. Joachimiak A. Marszalek J. Craig E.A. Dutkiewicz R. Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution.J. Mol. Biol. 2012; 417: 1-12Crossref PubMed Scopus (50) Google Scholar, 21.Andrew A.J. Dutkiewicz R. Knieszner H. Craig E.A. Marszalek J. Characterization of the interaction between the J-protein Jac1p and the scaffold for Fe-S cluster biogenesis, Isu1p.J. Biol. Chem. 2006; 281: 14580-14587Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Because Fe-S proteins play a critical role in a wide range of cellular activities, a mutation in different components of the synthesis machinery disrupts the process of Fe-S cluster biogenesis and is thus associated with multiple pathological conditions in humans. For instance, one mutation identified in the human mitochondrial iron-sulfur assembly enzyme, ISCU, is known to cause severe myopathy (ISCU myopathy; OMIM *611911). ISCU myopathy is a recessively inherited disorder characterized by lifelong exercise intolerance, where minor exertion causes pain of active muscles, shortness of breath, fatigue, and tachycardia (22.Mochel F. Knight M.A. Tong W.H. Hernandez D. Ayyad K. Taivassalo T. Andersen P.M. Singleton A. Rouault T.A. Fischbeck K.H. Haller R.G. Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance.Am. J. Hum. Genet. 2008; 82: 652-660Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 23.Olsson A. Lind L. Thornell L.E. Holmberg M. Myopathy with lactic acidosis is linked to chromosome 12q23.3–24.11 and caused by an intron mutation in the ISCU gene resulting in a splicing defect.Hum. Mol. Genet. 2008; 17: 1666-1672Crossref PubMed Scopus (102) Google Scholar). The disease is non-progressive, but in certain cases, metabolic acidosis, rhabdomyolysis, and myoglobinuria have also been reported (24.Kollberg G. Melberg A. Holme E. Oldfors A. Transient restoration of succinate dehydrogenase activity after rhabdomyolysis in iron-sulphur cluster deficiency myopathy.Neuromuscul. Disord. 2011; 21: 115-120Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 25.Larsson L.E. Linderholm H. Mueller R. Ringqvist T. Soernaes R. Hereditary metabolic myopathy with paroxysmal myoglobinuria due to abnormal glycolysis.J. Neurol. Neurosurg. Psychiatry. 1964; 27: 361-380Crossref PubMed Scopus (64) Google Scholar). Myopathy as a result of ISCU deficiency was found to have high incidence rates in individuals of Northern European ancestry with a carrier rate of 1:188 in the Northern Swedish population (23.Olsson A. Lind L. Thornell L.E. Holmberg M. Myopathy with lactic acidosis is linked to chromosome 12q23.3–24.11 and caused by an intron mutation in the ISCU gene resulting in a splicing defect.Hum. Mol. Genet. 2008; 17: 1666-1672Crossref PubMed Scopus (102) Google Scholar). Most affected individuals are homozygous for a mutation in intron 4 (g.7044G→C) of ISCU that results in synthesis of aberrantly spliced ISCU mRNA, successively causing accumulation of truncated non-functional ISCU protein (22.Mochel F. Knight M.A. Tong W.H. Hernandez D. Ayyad K. Taivassalo T. Andersen P.M. Singleton A. Rouault T.A. Fischbeck K.H. Haller R.G. Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance.Am. J. Hum. Genet. 2008; 82: 652-660Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 26.Kollberg G. Holme E. Antisense oligonucleotide therapeutics for iron-sulphur cluster deficiency myopathy.Neuromuscul. Disord. 2009; 19: 833-836Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 27.Sanaker P.S. Toompuu M. Hogan V.E. He L. Tzoulis C. Chrzanowska-Lightowlers Z.M. Taylor R.W. Bindoff L.A. Differences in RNA processing underlie the tissue specific phenotype of ISCU myopathy.Biochim. Biophys. Acta. 2010; 1802: 539-544Crossref PubMed Scopus (32) Google Scholar). Recently, a progressive myopathy associated with early onset of severe muscle weakness, extreme exercise intolerance, and cardiomyopathy has been reported in some patients. Interestingly, these patients were compound heterozygous for the common intronic splice mutation (g.7044G→C) on one allele, leading to truncated protein and a novel (c.149G→A) missense mutation in exon 3 on the other allele. The missense mutation in exon 3 changes a completely conserved glycine residue to a glutamate at the 50th position (G50E) in the amino acid sequence (28.Kollberg G. Tulinius M. Melberg A. Darin N. Andersen O. Holmgren D. Oldfors A. Holme E. Clinical manifestation and a new ISCU mutation in iron-sulphur cluster deficiency myopathy.Brain. 2009; 132: 2170-2179Crossref PubMed Scopus (84) Google Scholar). The transmission of the G50E mutation alone was found to be recessive because the carrier population did not show significant symptoms of the disease. However, the exact molecular mechanisms of disease development as a result of G50E mutation in ISCU in conjunction with the g.7044G→C allele in compound heterozygous patients have not been elucidated. Due to the critical function played by ISCU scaffold protein in the Fe-S cluster biogenesis process in humans, the G50E mutant is expected to contribute significantly toward ISCU myopathy. In this report, we delineate the impact of the G50E mutation on mitochondrial function by utilizing the HeLa cell line and yeast as a model system. Our findings highlight that the G50E mutation leads to severe growth defects, compromised Fe-S cluster-containing enzyme activity, sensitivity to oxidative stress, increased cellular reactive oxygen species (ROS), elevated iron level, and reduced interaction of scaffold protein with its interacting partners, thus contributing significantly toward mitochondrial myopathy. Moreover, at the protein level, the G50E mutation was found to form a higher order oligomeric structure that probably reduces the functionality of the protein. HeLa cells were transfected with pCI-neo-ISCU and pCI-neo-G50E ISCU using Lipofectamine 2000 for expression of wild type ISCU and G50E ISCU. Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Sigma). The cells were incubated at 37 °C in 5% CO2 for 48 h prior to experiments. The adherent cultures of HeLa cells were grown to ∼90% confluence. The cells were trypsinized, washed with ice-cold 1× phosphate-buffered saline (PBS), pH 7.4, and utilized for different experiments and mitochondria isolation. For genetic analysis in yeast, full-length human WT ISCU and yeast WT ISU1 were amplified from a HeLa cell cDNA library (Stratagene) and W303 yeast genomic DNA, respectively. ISCU and ISU1 with a C-terminal FLAG tag were cloned in pRS414 yeast expression vector under the TEF or GPD promoter containing a Trp marker for selection. The Δisu1/isu2 double deleted yeast haploid strain, W303 (trp1-1 ura3-1 leu2-3, 112 his3-11, 15 ade2-1 can1–100 GAL2+ met2-Δ1 lys2-Δ2 isu2::HIS3 isu1::LEU2), contained the plasmid pRS316-ISU1 for the maintenance of viability. For in vivo phenotype analysis, the haploid Δisu1/isu2 strain was transformed with pRS414-ISCU, pRS414-G50E ISCU, pRS414-ISU1, and pRS414-isu1G50E. The transformants were selected on tryptophan omission plates at 30 °C. The transformants (Trp+) were then subjected to spot test analysis using serial dilutions of the cells on different media like Trp−, Trp− containing H2O2, and medium containing 5-fluoroorotic acid (U.S. Biological) to eliminate the WT ISU1 containing the plasmid pRS316. For purification of ISCU and Isu1 using a bacterial expression system, the ORFs without N-terminal signal sequences of ISCU (aa 35–167) and ISU1 (aa 37–165) were inserted in between the BamHI-SalI restriction sites of the pRSFDuet-1 vector, carrying an N-terminal vector backbone His6 tag. The glutathione S-transferase (GST) fusion constructs of hNFS1 (aa 56–457 into BamHI-SalI), yNFS1 (aa 37–497 into BamHI-XhoI), HSCB (aa 22–235 into BamHI-SalI), and JAC1 (aa 1–184 into BamHI-XhoI) were generated by introducing the respective coding sequences downstream of the GST tag in the pGEX-KG vector, respectively. For mammalian transfection experiments, the full-length human ISCU and G50E ISCU were cloned downstream to a CMV-based promoter of pCI-neo vector (Promega) with a FLAG tag at the 3′-end of the open reading frame. The G50E point mutation was created through QuikChange site-directed mutagenesis, using high fidelity Pfu Turbo DNA polymerase (Stratagene). All of the clones were verified by DNA sequencing reactions carried out at Eurofins Inc. and Macrogen Inc. For purification of N-terminal His6-tagged human WT ISCU and G50E ISCU or yeast WT Isu1 and Isu1G50E, the proteins were expressed in Escherichia coli BL21(DE3) strain by allowing growth at 30 °C to an A600 of 0.6, followed by induction using 0.5 mm IPTG for 6 h. Cells were harvested by centrifugation and then lysed in buffer A (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 1 mm dithiothreitol, 20 mm imidazole, 1 mm PMSF, and 10% glycerol) along with 0.2 mg/ml lysozyme, followed by incubation at 4 °C for 1 h. The samples were gently treated with 0.2% deoxycholate, followed by DNase I (10 μg/ml) treatment for 15 min at 4 °C. The cells were further lysed by sonicating three times (for 15 s each) at 25% amplitude using an Ultrasonic processor with 2-min intervals in ice. The cell lysates were clarified by centrifuging at 22,000 × g for 30 min at 4 °C. The supernatant was incubated with nickel-nitrilotriacetic acid-Sepharose (GE Healthcare) for 2 h at 4 °C. Unbound proteins and nonspecific contaminants were removed by multiple washes of buffer A alone followed by sequential single washes of buffer B (buffer A along with 0.05% Triton X-100), buffer C (buffer A along with 1 mm ATP, 10 mm MgCl2), buffer D (buffer A along with 1 m NaCl), and buffer E (buffer C along with 40 mm imidazole). Finally, the bound proteins were eluted with buffer A containing 250 mm imidazole. Purification of other proteins was also achieved by the same strategy as mentioned above with minor modifications. For GST-tagged proteins, the purification was performed in a conventional manner using the GST-Sepharose beads (GE Healthcare). The bound proteins were stored in buffer containing 50 mm Tris-Cl, pH 7.5, and 100 mm NaCl at 4 °C. In order to analyze the extent of superoxide generation by the mitochondria, we utilized MitoSOX Red dye (Molecular Probes). This dye is specifically targeted to the mitochondria, where it undergoes oxidation by the mitochondrial superoxide radicals and fluoresces upon binding to mitochondrial DNA, with an emission maximum of 580 nm. Transfected HeLa cells cultured for 48 h and 0.1 OD (A600) of yeast cells from the early log phase were harvested and incubated with the dye (0.5 μm for HeLa and 2.5 μm for yeast) for 10 min, following which they were washed with 1× PBS and subjected to FACS analysis using a 488-nm argon laser for excitation (BD FACS Canto II). The respiratory inhibitor rotenone (1 mm) was used as a positive control for generating higher superoxide levels. The mean fluorescence intensity values of 10,000 events were recorded per sample and plotted to compare the relative ROS levels using the WinMDI version 2.9 software (29.Goswami A.V. Samaddar M. Sinha D. Purushotham J. D'Silva P. Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinson's disease.Hum. Mol. Genet. 2012; 21: 3317-3332Crossref PubMed Scopus (28) Google Scholar). HeLa cells harboring WT ISCU and G50E ISCU were seeded on a coverslip (5000 cells/well in 12-well plates) and cultured for 48 h. Medium was removed, cells were washed with 1× PBS, and cells were stained with 0.1 μm MitoSOX Red and 250 ng/ml Hoechst 33342 for 15 min in 300 μl of 1× PBS at 37 °C in a 5% CO2 incubator. Prior to analysis, cells were twice washed with 1× PBS. Images were acquired using a ×63 objective lens on a Zeiss apotome fluorescence microscope. For MitoSOX Red fluorescence imaging of yeast, 0.1 OD (A600) of cells from mid-log phase grown at 37 °C were incubated with 5 μm dye for 15 min. The cells were washed twice with 1× PBS before analysis. Images were acquired using a ×100 objective lens on a Leica fluorescence microscope. A similar strategy was used for H2DCF-DA and calcein blue staining. HeLa cells were treated with 1 μm H2DCF-DA for 15 min, and yeast cells were treated with 5 μm H2DCF-DA for 20 min, and for calcein blue staining, HeLa cells were treated with 3 μm calcein blue for 20 min. To test the specificity of calcein blue staining, the mutant cells were treated with a 1 mm concentration of the iron-specific chelator deferoxamine (DFO) (30.Uchiyama A. Kim J.S. Kon K. Jaeschke H. Ikejima K. Watanabe S. Lemasters J.J. Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury.Hepatology. 2008; 48: 1644-1654Crossref PubMed Scopus (116) Google Scholar). Images of HeLa cells were taken using a ×63 objective lens of the Zeiss apotome fluorescence microscope, and images of the yeast cell were taken with a ×100 objective lens on a Leica fluorescence microscope. Purified GST-hNFS1 (1.5 μm)/GST-yNfs1 (1.5 μm) and GST-HSCB (1 μm)/GST-Jac1 (2.5 μm) were incubated with a 10-μl bed volume of glutathione-agarose beads in 200 μl of GST buffer A (50 mm Tris-Cl, pH 7.5, 100 mm NaCl, 40 mm imidazole, 50 μm pyridoxal phosphate, 10% glycerol) and GST buffer B (50 mm Tris-Cl, pH 7.5, 100 mm NaCl, 40 mm imidazole, 0.2% Triton X-100, 5% glycerol) for an interaction study with either WT or mutant ISCU/Isu1, respectively. Unbound proteins were removed by washing the beads three times with the respective GST buffers. After removing unbound proteins, the samples were blocked with 0.1% BSA for 20 min at 20 °C, followed by washing two times with GST buffer to remove excess unbound BSA. The beads were resuspended in 200 μl of GST binding buffer and incubated with increasing concentrations of either WT or mutant ISCU/Isu1 for 30 min at 20 °C. The GST beads were washed three times with GST buffer and resolved on SDS-PAGE followed by Coomassie dye staining. Enzymatic assays of complex I, complex II, and complex IV were performed according to a protocol described previously (31.Spinazzi M. Casarin A. Pertegato V. Salviati L. Angelini C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells.Nat. Protoc. 2012; 7: 1235-1246Crossref PubMed Scopus (585) Google Scholar). Briefly, 15 μg of protein corresponding to mitochondria isolated from HeLa cells were subjected to three cycles of freeze-thawing before complex I activity was assayed. Mitochondria were resuspended in 50 mm of potassium phosphate buffer (pH 7.5) containing 100 μm NADH, 3 mg/ml fatty acid-free BSA, 300 μm sodium azide (NaN3). The reaction was started by adding 60 μm ubiquinone, and the decrease of absorbance at 340 nm was monitored for 2 min. To check for specificity of the reaction, the experiment was performed both in the presence and absence of the specific inhibitor rotenone (10 μm). For complex II activity analysis, 2 μg of protein corresponding to HeLa cell mitochondria and 10 μg of protein corresponding to yeast cell mitochondria were resuspended in 25 mm potassium phosphate buffer (pH 7.5) containing 20 mm succinate, 1 mg/ml fatty acid-free BSA, 300 μm NaN3, 10 μm rotenone, 10 μg/ml antimycin A, and 80 μm 2,6-dichlorophenolindophenol, followed by incubation at 37 °C for 10 min. The reaction was started by adding 60 μm ubiquinone, and the decrease in the absorbance at 600 nm was recorded for 3 min. For complex IV activity, 2 μg of protein corresponding to HeLa cell mitochondria and 10 μg of protein corresponding to yeast cell mitochondria were resuspended in 50 mm potassium phosphate buffer (pH 7). The reaction was started by adding 60 μm reduced cytochrome c, and the decrease in absorbance at 550 nm for 3 min was observed. To check the specificity of complex IV activity, this experiment was performed in the presence and in the absence of complex IV inhibitor NaN3 (300 μm). Aconitase activity was also measured following a similar protocol as described earlier (32.Pierik A.J. Netz D.J. Lill R. Analysis of iron-sulfur protein maturation in eukaryotes.Nat. Protoc. 2009; 4: 753-766Crossref PubMed Scopus (70) Google Scholar). Briefly, 25 μg of protein corresponding to HeLa cell mitochondria and 50 μg of protein corresponding to yeast cell mitochondria were dissolved in buffer A (50 mm Tris-Cl, pH 8.0, 50 mm NaCl) containing 1% deoxycholic acid and incubated for 10 min. The reaction was started by the addition of sodium citrate dihydrate, and the absorbance increase at 235 nm for 2 min was followed in a quartz cuvette. Mitochondrial ATP was quantified using the mitochondrial ToxGloTM assay kit (Promega). Briefly, 1–2 μg of mitochondria were resuspended in 80 μl of SE buffer (250 mm sucrose, 1 mm EDTA, 10 mm MOPS-KOH, pH 7.2) and mixed with 20 μl of 5× cytotoxicity reagent (bis-AAF-R110) for 1 min by orbital shaking (700 rpm). The reaction mixture was incubated at 37 °C for 30 min followed by equilibration of the assay plate at room temperature (25 °C) for 5 min. Subsequently, 100 μl of ATP detection reagent was added to each well" @default.
• W2044992022 created "2016-06-24" @default.
• W2044992022 creator A5016194841 @default.
• W2044992022 creator A5019135272 @default.
• W2044992022 creator A5021775086 @default.
• W2044992022 creator A5029772637 @default.
• W2044992022 creator A5044467719 @default.
• W2044992022 creator A5050430951 @default.
• W2044992022 date "2014-04-01" @default.
• W2044992022 modified "2023-10-10" @default.
• W2044992022 title "The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein (ISCU) Mutation Leads to Development of Mitochondrial Myopathy" @default.
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