Academic literature on the topic 'Iron-sulfur Protein Assembly'
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Journal articles on the topic "Iron-sulfur Protein Assembly"
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Liu, Jian She, Lin Qian, and Chun Li Zheng. "Biogenesis and Transfer of Iron-Sulfur Clusters from Acidithiobacillus ferrooxidans." Advanced Materials Research 825 (October 2013): 198–201. http://dx.doi.org/10.4028/www.scientific.net/amr.825.198.
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Iron-sulfur (Fe-S) proteins are ubiquitous and participate in multiple essential functions of life. However, little is currently known about the mechanisms of iron-sulfur biosynthesis and transfer in acidophilic microorganisms. In this study, the IscS, IscU and IscA proteins from Acidithiobacillus ferrooxidans were successfully expressed in Escherichia coli and purified by affinity chromatography. The IscS was a cysteine desulfurase which catalyzes desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly. Purified IscU did not have an iron-sulfur cluster but could act as a scaffold protein to assemble the [2Fe-2S] cluster in vitro. The IscA was a [4Fe-4S] cluster binding protein, but it also acted as an iron binding protein. Further studies indicated that the iron sulfur clusters could be transferred from pre-assembled scaffold proteins to apo-form iron sulfur proteins, the reconstituted iron sulfur proteins could restore their physiological activities.
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Song, Daisheng, and Frank S. Lee. "Mouse Knock-out of IOP1 Protein Reveals Its Essential Role in Mammalian Cytosolic Iron-Sulfur Protein Biogenesis." Journal of Biological Chemistry 286, no. 18 (March 2, 2011): 15797–805. http://dx.doi.org/10.1074/jbc.m110.201731.
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Iron-sulfur proteins play an essential role in a variety of biologic processes and exist in multiple cellular compartments. The biogenesis of these proteins has been the subject of extensive investigation, and particular focus has been placed on the pathways that assemble iron-sulfur clusters in the different cellular compartments. Iron-only hydrogenase-like protein 1 (IOP1; also known as nuclear prelamin A recognition factor like protein, or NARFL) is a human protein that is homologous to Nar1, a protein in Saccharomyces cerevisiae that, in turn, is an essential component of the cytosolic iron-sulfur protein assembly pathway in yeast. Previous siRNA-induced knockdown studies using mammalian cells point to a similar role for IOP1 in mammals. In the present studies, we pursued this further by knocking out Iop1 in Mus musculus. We find that Iop1 knock-out results in embryonic lethality before embryonic day 10.5. Acute, inducible global knock-out of Iop1 in adult mice results in lethality and significantly diminished activity of cytosolic aconitase, an iron-sulfur protein, in liver extracts. Inducible knock-out of Iop1 in mouse embryonic fibroblasts results in diminished activity of cytosolic but not mitochondrial aconitase and loss of cell viability. Therefore, just as with knock-out of Nar1 in yeast, we find that knock-out of Iop1/Narfl in mice results in lethality and defective cytosolic iron-sulfur cluster assembly. The findings demonstrate an essential role for IOP1 in this pathway.
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Dos Santos, Patricia C., Archer D. Smith, Jeverson Frazzon, Valerie L. Cash, Michael K. Johnson, and Dennis R. Dean. "Iron-Sulfur Cluster Assembly." Journal of Biological Chemistry 279, no. 19 (March 1, 2004): 19705–11. http://dx.doi.org/10.1074/jbc.m400278200.
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The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of [Fe-S] clusters uniquely destined for the maturation of the nitrogenase catalytic components. There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of [Fe-S] cluster biosynthetic scaffolds designated IscU. The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed [Fe-S] cluster biosynthetic scaffolds designated Nfu. Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation, although the N-terminal domain appears to have the dominant function. Thesein vivoexperiments were supported byin vitro[Fe-S] cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein.
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Srour, Batoul, Sylvain Gervason, Beata Monfort, and Benoit D’Autréaux. "Mechanism of Iron–Sulfur Cluster Assembly: In the Intimacy of Iron and Sulfur Encounter." Inorganics 8, no. 10 (October 3, 2020): 55. http://dx.doi.org/10.3390/inorganics8100055.
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Iron–sulfur (Fe–S) clusters are protein cofactors of a multitude of enzymes performing essential biological functions. Specialized multi-protein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein on which Fe–S clusters are assembled and a cysteine desulfurase that provides sulfur in the form of a persulfide. The sulfide ions are produced by reductive cleavage of the persulfide, which involves specific reductase systems. Several other components are required for Fe–S biosynthesis, including frataxin, a key protein of controversial function and accessory components for insertion of Fe–S clusters in client proteins. Fe–S cluster biosynthesis is thought to rely on concerted and carefully orchestrated processes. However, the elucidation of the mechanisms of their assembly has remained a challenging task due to the biochemical versatility of iron and sulfur and the relative instability of Fe–S clusters. Nonetheless, significant progresses have been achieved in the past years, using biochemical, spectroscopic and structural approaches with reconstituted system in vitro. In this paper, we review the most recent advances on the mechanism of assembly for the founding member of the Fe–S cluster family, the [2Fe2S] cluster that is the building block of all other Fe–S clusters. The aim is to provide a survey of the mechanisms of iron and sulfur insertion in the scaffold proteins by examining how these processes are coordinated, how sulfide is produced and how the dinuclear [2Fe2S] cluster is formed, keeping in mind the question of the physiological relevance of the reconstituted systems. We also cover the latest outcomes on the functional role of the controversial frataxin protein in Fe–S cluster biosynthesis.
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Balk, Janneke, Daili J. Aguilar Netz, Katharina Tepper, Antonio J. Pierik, and Roland Lill. "The Essential WD40 Protein Cia1 Is Involved in a Late Step of Cytosolic and Nuclear Iron-Sulfur Protein Assembly." Molecular and Cellular Biology 25, no. 24 (December 15, 2005): 10833–41. http://dx.doi.org/10.1128/mcb.25.24.10833-10841.2005.
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ABSTRACT The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1.
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Lu, Jianxin, Juanjuan Yang, Guoqiang Tan, and Huangen Ding. "Complementary roles of SufA and IscA in the biogenesis of iron–sulfur clusters in Escherichia coli." Biochemical Journal 409, no. 2 (December 21, 2007): 535–43. http://dx.doi.org/10.1042/bj20071166.
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Biogenesis of iron–sulfur clusters requires a concerted delivery of iron and sulfur to target proteins. It is now clear that sulfur in iron–sulfur clusters is derived from L-cysteine via cysteine desulfurases. However, the specific iron donor for the iron–sulfur cluster assembly still remains elusive. Previous studies showed that IscA, a member of the iron–sulfur cluster assembly machinery in Escherichia coli, is a novel iron-binding protein, and that the iron-bound IscA can provide iron for the iron–sulfur cluster assembly in a proposed scaffold IscU in vitro. However, genetic studies have indicated that IscA is not essential for the cell growth of E. coli. In the present paper, we report that SufA, an IscA paralogue in E. coli, may represent the redundant activity of IscA. Although deletion of IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA in E. coli results in a severe growth phenotype in minimal medium under aerobic growth conditions. Cell growth is restored when either IscA or SufA is re-introduced into the iscA−/sufA− double mutant, demonstrating further that either IscA or SufA is sufficient for their functions in vivo. Purified SufA, like IscA, is an iron-binding protein that can provide iron for the iron–sulfur cluster assembly in IscU in the presence of a thioredoxin reductase system which emulates the intracellular redox potential. Site-directed mutagenesis studies show that the SufA/IscA variants that lose the specific iron-binding activity fail to restore the cell growth of the iscA−/sufA− double mutant. The results suggest that SufA and IscA may constitute the redundant cellular activities to recruit intracellular iron and deliver iron for the iron–sulfur cluster assembly in E. coli.
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Stehling, Oliver, Daili J. A. Netz, Brigitte Niggemeyer, Ralf Rösser, Richard S. Eisenstein, Helene Puccio, Antonio J. Pierik, and Roland Lill. "Human Nbp35 Is Essential for both Cytosolic Iron-Sulfur Protein Assembly and Iron Homeostasis." Molecular and Cellular Biology 28, no. 17 (June 23, 2008): 5517–28. http://dx.doi.org/10.1128/mcb.00545-08.
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ABSTRACT The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.
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Lu, Jianxin, Jacob P. Bitoun, Guoqiang Tan, Wu Wang, Wenguang Min, and Huangen Ding. "Iron-binding activity of human iron–sulfur cluster assembly protein hIscA1." Biochemical Journal 428, no. 1 (April 28, 2010): 125–31. http://dx.doi.org/10.1042/bj20100122.
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A human homologue of the iron–sulfur cluster assembly protein IscA (hIscA1) has been cloned and expressed in Escherichia coli cells. The UV–visible absorption and EPR (electron paramagnetic resonance) measurements reveal that hIscA1 purified from E. coli cells contains a mononuclear iron centre and that the iron binding in hIscA1 expressed in E. coli cells can be further modulated by the iron content in the cell growth medium. Additional studies show that purified hIscA1 binds iron with an iron association constant of approx. 2×1019 M−1, and that the iron-bound hIscA1 is able to provide the iron for the iron–sulfur cluster assembly in a proposed scaffold protein, IscU of E. coli, in vitro. The complementation experiments indicate that hIscA1 can partially substitute for IscA in restoring the cell growth of E. coli in the M9 minimal medium under aerobic conditions. The results suggest that hIscA1, like E. coli IscA, is an iron-binding protein that may act as an iron chaperone for biogenesis of iron–sulfur clusters.
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Ishiyama, Akihiko, Chika Sakai, Yuichi Matsushima, Satoru Noguchi, Satomi Mitsuhashi, Yukari Endo, Yukiko K. Hayashi, et al. "IBA57 mutations abrogate iron-sulfur cluster assembly leading to cavitating leukoencephalopathy." Neurology Genetics 3, no. 5 (September 8, 2017): e184. http://dx.doi.org/10.1212/nxg.0000000000000184.
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Objective:To determine the molecular factors contributing to progressive cavitating leukoencephalopathy (PCL) to help resolve the underlying genotype-phenotype associations in the mitochondrial iron-sulfur cluster (ISC) assembly system.Methods:The subjects were 3 patients from 2 families who showed no inconsistencies in either clinical or brain MRI findings as PCL. We used exome sequencing, immunoblotting, and enzyme activity assays to establish a molecular diagnosis and determine the roles of ISC-associated factors in PCL.Results:We performed genetic analyses on these 3 patients and identified compound heterozygosity for the IBA57 gene, which encodes the mitochondrial iron-sulfur protein assembly factor. Protein expression analysis revealed substantial decreases in IBA57 protein expression in myoblasts and fibroblasts. Immunoblotting revealed substantially reduced expression of SDHB, a subunit of complex II, and lipoic acid synthetase (LIAS). Levels of pyruvate dehydrogenase complex-E2 and α-ketoglutarate dehydrogenase-E2, which use lipoic acid as a cofactor, were also reduced. In activity staining, SDH activity was clearly reduced, but it was ameliorated in mitochondrial fractions from rescued myoblasts. In addition, NFU1 protein expression was also decreased, which is required for the assembly of a subset of iron-sulfur proteins to SDH and LIAS in the mitochondrial ISC assembly system.Conclusions:Defects in IBA57 essentially regulate NFU1 expression, and aberrant NFU1 ultimately affects SDH activity and LIAS expression in the ISC biogenesis pathway. This study provides new insights into the role of the iron-sulfur protein assembly system in disorders related to mitochondrial energy metabolism associated with leukoencephalopathy with cavities.
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Bernard, Delphine G., Daili J. A. Netz, Thibaut J. Lagny, Antonio J. Pierik, and Janneke Balk. "Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis." Philosophical Transactions of the Royal Society B: Biological Sciences 368, no. 1622 (July 19, 2013): 20120259. http://dx.doi.org/10.1098/rstb.2012.0259.
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The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2 , with the Arabidopsis homologue AtDRE2 , but only when expressed together with the diflavin reductase AtTAH18 . Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.
Dissertations / Theses on the topic "Iron-sulfur Protein Assembly"
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Ding, Shu. "Thermodynamic studies on iron-sulfur cluster assembly proteins." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1316472363.
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Natarajan, Kshama. "Part I- cluster assembly in protein bound iron-sulfur clusters ; Part II- solution structural studies on the N-terminus perforin /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487951907957761.
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Dizin, Eric Michel. "Insights On Iron-Sulfur Cluster Assembly Donor Proteins." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1208532379.
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Foster, Matthew W. "Biosynthetic assembly and nitric oxide mediated degradation of iron-sulfur proteins /." The Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488199501404912.
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Vo, Amanda T. "Defining the role of cytosolic iron-sulfur cluster assembly targeting complex in identification of iron-sulfur cluster proteins." Thesis, 2018. https://hdl.handle.net/2144/33075.
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Iron sulfur (FeS) clusters are ubiquitous cofactors required for numerous fundamental biochemical processes, including DNA replication and repair, transcription, and translation. In the cell, these metallocofactors require a dedicated protein pathway for assembly. The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is conserved across higher-level eukaryotes and is responsible for building and inserting these cofactors into the FeS proteins that need them. A major unsolved problem in the FeS cluster biogenesis field is how so many diverse FeS proteins are identified for cluster insertion. Several studies have identified a multiprotein complex containing Cia1, Cia2, and Met18 as the CIA targeting complex responsible for FeS cluster recognition and target maturation. The CIA targeting complex has been shown to associate with an FeS cluster protein, Nar1. Nar1 is a CIA factor that plays an unknown role in cluster transfer. Little information is known about the structure of the CIA targeting complex its mechanism of FeS cluster protein recognition. In this thesis, I investigate the architecture of the CIA targeting complex as well as the role each subunit plays in identification of apo-proteins and iron-sulfur cluster insertion.
Previous proteomic and cell biological studies from the Lill lab propose that the CIA targeting complex exists as a mixture of discrete complexes in vivo. Each of these complexes is responsible for recognizing a distinct subset of targets. Herein, we utilize affinity co-purification and size exclusion chromatography investigate connectivity of the targeting complex, identify stable subcomplexes, and define their roles in recognizing our two model targets Rad3 and Leu1. We determine the CIA targeting complex contains one Met18, two Cia1, and four Cia2 polypepides. This complex is required to recognize Leu1. Our experiments reveal the formation of the stable subcomplexes Cia1-Cia2 and Met18-Cia2, which is sufficient to identify to Rad3. We also interrogate the role of Nar1 in binding to targets and cluster transfer, excluding the model that it acts as an adapter for cluster transfer.
Furthermore, using site directed mutagenesis, combined with our co-purification and in vivo assays, we map the key interfaces required to form the targeting complex and investigate how their mutations impacts CIA function in vivo. We identify the binding site of Cia1 on Cia2, as well as the general region in which Cia2 binds to Met18. Through these experiments, we shed light on the role these subunits of CIA targeting complex and Nar1 play in FeS target recognition and FeS cluster transfer.
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Saudino, Giovanni. "Protein expression and characterization for systems involved in the biogenesis of iron sulfur proteins." Doctoral thesis, 2021. http://hdl.handle.net/2158/1251249.
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Iron-sulfur clusters are essential cofactors found in all kingdoms of life; they had unique functional roles throughout evolution. These clusters, which are the second major form of complex iron cofactors in biology, are ubiquitous in all organisms, playing a key role in several biological pathways. Mutations on the protein involved in the iron-sulfur clusters biosynthesis pathway are associated with a group of multiple mitochondrial dysfunction syndromes (MMDS). These severe diseases could give infantile encephalopathy, lactic acidosis, leukodystrophy and death in early childhood. In human cells, cluster biosynthesis involves three different types of machinery: ISC (iron-sulfur cluster assembly machinery) located in the mitochondria, CIA (cytosolic iron-sulfur cluster assembly machinery) located in the cytosol and ISC export machinery located in the mitochondria inner membrane. The aim of the thesis was the deep investigation of the third step of the ISC assembly machinery. Indeed, using NMR, UV-vis and CD-vis spectroscopies in combination with size exclusion chromatography and multi-angle light scattering we assessed the key role of NFU1, ISCA1, ISCA2, FDX2 and LIAS in the above-mentioned machinery. Moreover, clinical BOLA3 Cys59Tyr mutation involved in the MMDS diseases has investigated at the atomistic and molecular levels. The gained data elucidated fundamental molecular details in the [4Fe-4S] cluster maturation and transfer to apo recipient proteins along the third step of ISC assembly machinery.
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Tam, William. "Characterization of an Iron-Sulfur Binding Protein in the Tail Tip Complex of Bacteriophage Lambda." Thesis, 2012. http://hdl.handle.net/1807/42907.
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The assembly of λ tail requires the action of 11 gene products which must interact in an organized fashion to assemble infectious tail particles. GpL is an essential protein for the formation of the tail tip complex and necessary for the assembly of λ tail. The work described here has shown that gpL and its homologues contain two domains where the C-terminal domain coordinates an oxygen-sensitive [4Fe-4S] 2+ cluster using 4 highly conserved cysteines. This is the first report of a bacteriophage morphogenetic protein to coordinate a [4Fe-4S]2+ cluster. Through two individual cysteine mutants, C184A and C228A, it was determined that these mutant proteins coordinate a [2Fe-2S]2+ cluster also using 4 cysteines; the fourth cysteine being non-conserved. λ tails assembled with cysteine mutant gpL resulted in a 1000-fold decrease in the titer of active tails and tail particles could not be detected by TEM indicating that λ tails cannot be assembled with cysteine mutant gpL. I propose that the coordination of a [4Fe-4S] cluster with the four conserved cysteines maintains a conformation in gpL that can optimally interact with other tail proteins for efficient tail assembly.
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CHANGMAI, Piya. "Functional analysis of Iron-Sulfur cluster assembly protein Isd11 in procyclic and bloodstream \kur{Trypanosoma brucei}." Master's thesis, 2009. http://www.nusl.cz/ntk/nusl-48197.
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BASU, Somsuvro. "Erv1 associated mitochondrial import-export pathway and the cytosolic iron-sulfur protein assembly machinery in Trypanosoma brucei." Doctoral thesis, 2014. http://www.nusl.cz/ntk/nusl-175336.
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This thesis highlights a divergent mitochondrial intermembrane assembly pathway in the parasitic protist Trypanosoma brucei. A comparative genomic study reveals the connection of Erv1 with the cytosolic iron-sulfur protein assembly (CIA) pathway in trypanosomatids. Further, the CIA machinery of T. brucei has been described using RNAi interference and other biochemical and complementation assays. Finally, part of the divergent CIA machinery has been identified in the human intestinal pathogen Giardia intestinalis by means of complementation assays in T. brucei.
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Bankapalli, Kondalarao. "Understanding the Role of ThiJ/DJ-1/PfpI Family Member Proteins in Regulating Redox Homeostasis, Mitochondrial Health and Lifespan in Saccharomyces cerevisiae." Thesis, 2017. http://etd.iisc.ac.in/handle/2005/4208.
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In a healthy cell, the ROS levels are stringently regulated by the action of various enzymatic or non-enzymatic antioxidant systems. Imbalance in the ROS homeostasis generates oxidative stress resulting in damage to cellular macromolecules. Besides, pro-oxidants, glyoxals which are normally generated as an intermediate compound in the glycolytic pathway and other metabolic activity are known to cause oxidative stress in the cell. Elevated oxidative stress is one of the prominent cellular aetiologies associated with premature aging, cardiovascular and retinal disorders, atherosclerosis, and several neurological disorders. Parkinson disease (PD) is one of well-known neurodegenerative diseases whose pathogenicity is correlated to mitochondrial dysfunction due to elevated oxidative stress in the neuronal cells. Several proteins which are associated with development of familial form of PD, DJ-1, a member of ThiJ/DJ-1/PfpI super family, is known to act as an oxidative sensor in humans. Interestingly, heat shock protein (Hsp)31 from S. cerevisiae which belongs to DJ-1 family was shown to provide a similar oxidative stress resistance in yeast. However, the mechanistic aspects how these family members functions as an oxidative stress sensor are not clearly defined. The main focus of my investigation is to understand the involvement of these DJ-1 proteins in regulation of redox homeostasis and mitochondrial health, which are major hallmarks in the pathogenesis of PD.
My major findings demonstrate the importance of Hsp31 family proteins in protecting cells against oxidative stress, which is induced by methylglyoxal (MG). The deletion of Hsp31 leads to a compromised growth phenotype in yeast upon MG induced stress. Moreover, Hsp31 exhibited robust GSH-independent glyoxalase activity both in vivo and in vitro. Besides, the glyoxalase activity is critical for glyoxal detoxification as well as suppression of ROS levels in cells. On the other hand, in agreement with the observed growth phenotypes, Hsp34 protein possesses a very mild glyoxalase activity as compared to Hsp31. Furthermore, active site mutational analysis reveals that methylglyoxalase activity of Hsp31 protein is critical for providing protection against oxidative stress in yeast. Importantly, endogenous expression of human DJ-1 could complement the growth of yeast under oxidative and glyoxal stress conditions signifying its functional conservation across species. Mechanistically, my findings highlight that Hsp31 regulates cellular GSH and NADPH homeostasis thereby protecting cells against oxidative stress. In addition, cellular localization experiment reveals that though Hsp31 is a cytosolic protein, it predominantly localizes into mitochondria under oxidative stress conditions and protects the organelle from severe oxidative damages.
Lastly, my findings uncover the role of Hsp31 paralogs in the maintenance of mitochondrial health integrity and other stress related pathways. To test their role in the mitochondrial health, I have analysed several parameters such as mass, dynamics and functionality. Interestingly, though the single deletions of these paralogs do not have significant effects over the mitochondrial phenotypes, the deletion of DJ-1 homologs in combination of hsp31 and hsp34 in yeast led to enhanced total as well as functional mitochondrial mass in cells. To address how mitochondrial mass enhancement occurs in the cells, the organelle turnover (mitophagy) was assessed. The microscopic and western analysis indicates, there was no alteration in mitophagy among the ∆hsp31∆hsp34 compared to WT. On the contrary, an enhancement in the basal levels of ROS stimulated increased biogenesis of mitochondria in ∆hsp31∆hsp34 cells was observed. Strikingly, ∆hsp31∆hsp34 cells also exhibit upregulation of mitochondrial fusion proteins resulting hyperfusion of mitochondria. Additionally, our results demonstrates that ∆hsp31∆hsp34 cells exhibited a long-term G2/M cell cycle arrest, which was rescued upon overexpression of mitochondrial fission protein, Dnm1. Lastly, absence of these paralogs in yeast, resulted in induction of apoptotic-like features in the cells and decreased lifespan in Saccharomyces cerevisiae. Altogether, my studies highlight the importance of DJ-1 class of proteins in maintaining the cellular redox status, mitochondrial integrity and cellular health in yeast.
In conclusion, overall my studies highlight that Hsp31 is a robust methylglyoxalase and regulates cellular NADPH and GSH pool thereby helps in the maintenance of redox homeostasis. Hsp31 predominantly translocate into mitochondria upon oxidative stress to protect the organelle from oxidative damages. Furthermore, my findings provide the first evidence over the involvement of DJ-1 family proteins in the regulation of mitochondrial health and dynamics, cell-cycle arres and reduced lifespan in yeast.
Book chapters on the topic "Iron-sulfur Protein Assembly"
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Saha, Prasenjit Prasad, Vinaya Vishwanathan, Kondalarao Bankapalli, and Patrick D’Silva. "Iron-Sulfur Protein Assembly in Human Cells." In Reviews of Physiology, Biochemistry and Pharmacology, 25–65. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/112_2017_5.
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Shen, Gaozhong, Mikhail L. Antonkine, Ilya R. Vassiliev, John H. Golbeck, and Donald A. Bryant. "A Rubredoxin-Like Protein Plays an Essential Role in Assembly of the FA, FB & FX Iron-Sulfur Clusters in Photosystem I." In Photosynthesis: Mechanisms and Effects, 3147–50. Dordrecht: Springer Netherlands, 1998. http://dx.doi.org/10.1007/978-94-011-3953-3_737.
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Tachezy, Jan, and Pavel Doležal. "Iron–Sulfur Proteins and Iron–Sulfur Cluster Assembly in Organisms with Hydrogenosomes and Mitosomes." In Origin of Mitochondria and Hydrogenosomes, 105–33. Berlin, Heidelberg: Springer Berlin Heidelberg, 2007. http://dx.doi.org/10.1007/978-3-540-38502-8_6.
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