The bacterial MrpORP is a novel Mrp/NBP35 protein involved in iron-sulfur biogenesis.
Ontology highlight
ABSTRACT:
SUBMITTER: Pardoux R
PROVIDER: S-EPMC6345978 | biostudies-literature | 2019 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Iron-sulfur (FeS) clusters are prosthetic groups critical for the function of many proteins in all domains of life. FeS proteins function in processes ranging from oxidative phosphorylation and cofactor biosyntheses to DNA/RNA metabolism and regulation of gene expression. In eukaryotic cells, mitochondria play a central role in the process of FeS biogenesis and support maturation of FeS proteins localized within mitochondria and in other cellular compartments. In humans, defects in mitochondrial FeS cluster biogenesis lead to numerous pathologies, which are often fatal. The generation of FeS clusters in mitochondria is a complex process. The [2Fe-2S] cluster is first assembled on a dedicated scaffold protein (Isu1) by the action of protein factors that interact with Isu1 to form the "assembly complex". Next, the FeS cluster is transferred onto a recipient apo-protein. Genetic and biochemical evidence implicates participation of a specialized J-protein co-chaperone Jac1 and its mitochondrial (mt)Hsp70 chaperone partner, and the glutaredoxin Grx5 in the FeS cluster transfer process. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Although a framework of protein components that are involved in the mitochondrial FeS cluster biogenesis has been established based on genetic and biochemical studies, detailed molecular mechanisms involved in this important and medically relevant process are not well understood. This review summarizes our molecular knowledge on chaperone proteins' functions during the FeS protein biogenesis.
| S-EPMC6006194 | biostudies-literature
Project description:BackgroundIron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.Methodology/principal findingsIn this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.ConclusionsOur data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.
| S-EPMC2764847 | biostudies-literature
Project description:Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.
| S-EPMC2648223 | biostudies-literature
Project description:Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 μM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.
| S-EPMC4583046 | biostudies-literature
Project description:Monothiol glutaredoxins play a crucial role in iron-sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in solution in the presence of physiological concentrations of glutathione and forming a transient, cluster-mediated protein-protein intermediate with two physiological protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.
| S-EPMC4035983 | biostudies-literature
Project description:Friedreich's ataxia is a neurodegenerative disorder caused by mutations in the frataxin gene that produces a predominantly mitochondrial protein whose primary function appears to be mitochondrial iron-sulfur cluster (ISC) biosynthesis. Previously we demonstrated that frataxin interacts with multiple components of the mammalian ISC assembly machinery. Here we demonstrate that frataxin interacts with the mammalian mitochondrial chaperone HSC20. We show that this interaction is iron-dependent. We also show that like frataxin, HSC20 interacts with multiple proteins involved in ISC biogenesis including the ISCU/Nfs1 ISC biogenesis complex and the GRP75 ISC chaperone. Furthermore, knockdown of HSC20 caused functional defects in activity of mitochondrial ISC-containing enzymes and also defects in ISC protein expression. Alterations up or down of frataxin expression caused compensatory changes in HSC20 expression inversely, as expected of two cooperating proteins operating in the same pathway and suggesting a potential therapeutic strategy for the disease. Knockdown of HSC20 altered cytosolic and mitochondrial iron pools and increased the expression of transferrin receptor 1 and iron regulatory protein 2 consistent with decreased iron bioavailability. These results indicate that HSC20 interacts with frataxin structurally and functionally and is important for ISC biogenesis and iron homeostasis in mammals. Furthermore, they suggest that HSC20 may act late in the ISC pathway as a chaperone in ISC delivery to apoproteins and that HSC20 should be included in multi-protein complex studies of mammalian ISC biogenesis.
| S-EPMC3298274 | biostudies-literature
Project description:The importance of mitochondrial iron-sulfur cluster (ISC) biogenesis for human health has been well established, but the roles of some components of this critical pathway still remain uncharacterized in mammals. Among them is human heat shock cognate protein 20 (hHSC20), the putative human homolog of the specialized DnaJ type co-chaperones, which are crucial for bacterial and fungal ISC assembly. Here, we show that the human HSC20 protein can complement for its counterpart in yeast, Jac1p, and interacts with its proposed human partners, hISCU and hHSPA9. hHSC20 is expressed in various human tissues and localizes mainly to the mitochondria in HeLa cells. However, small amounts were also detected extra-mitochondrially. RNA interference-mediated depletion of hHSC20 specifically reduced the activities of both mitochondrial and cytosolic ISC-containing enzymes. The recovery of inactivated ISC enzymes was markedly delayed after an oxidative insult of hHSC20-deficient cells. Conversely, overexpression of hHSC20 substantially protected cells from oxidative insults. These results imply that hHSC20 is an integral component of the human ISC biosynthetic machinery that is particularly important in the assembly of ISCs under conditions of oxidative stress. A cysteine-rich N-terminal domain, which clearly distinguishes hHSC20 from the specialized DnaJ type III proteins of fungi and most bacteria, was found to be important for the integrity and function of the human co-chaperone.
| S-EPMC2935859 | biostudies-literature
Project description:Soluble P loop NTPases represent a large protein family and are involved in diverse cellular functions. Here, we functionally characterized the first member of the Mrp/Nbp35 subbranch of this family, the essential Nbp35p of Saccharomyces cerevisiae. The protein resides in the cytosol and nucleus and carries an Fe/S cluster at its N terminus. Assembly of the Fe/S cluster requires the mitochondrial Fe/S cluster (ISC)-assembly and -export machineries. Depletion of Nbp35p strongly impairs the activity of the cytosolic Fe/S protein, isopropylmalate isomerase (Leu1p), whereas mitochondrial Fe/S enzymes are unaffected. Moreover, defects in the de novo maturation of various cytosolic and nuclear Fe/S proteins were observed in the absence of Nbp35p, demonstrating the functional involvement of Nbp35p in the biogenesis of extramitochondrial Fe/S proteins. Furthermore, Nbp35p genetically interacts with the closely similar P loop NTPase, Cfd1p, and the hydrogenase-like Nar1p, both of which were recently shown to perform a crucial function in cytosolic and nuclear Fe/S protein biogenesis. Hence, our study suggests that eukaryotic Nbp35 NTPases function in Fe/S protein maturation. The findings provide strong evidence for the existence of a highly conserved and essential machinery dedicated to assembling cytosolic and nuclear Fe/S proteins.
| S-EPMC552912 | biostudies-literature
Project description:Copper (Cu) ions serve as catalytic cofactors to drive key biochemical processes, and yet Cu levels that exceed cellular homeostatic control capacity are toxic. The underlying mechanisms for Cu toxicity are poorly understood. During pulmonary infection by the fungal pathogen Cryptococcus neoformans, host alveolar macrophages compartmentalize Cu to the phagosome, and the ability to detoxify Cu is critical for its survival and virulence. Here, we report that iron-sulfur (Fe-S) clusters are critical targets of Cu toxicity in both Saccharomyces cerevisiae and C. neoformans in a manner that depends on the accessibility of Cu to the Fe-S cofactor. To respond to this Cu-dependent Fe-S stress, C. neoformans induces the transcription of mitochondrial ABC transporter Atm1, which functions in cytosolic-nuclear Fe-S protein biogenesis in response to Cu and in a manner dependent on the Cu metalloregulatory transcription factor Cuf1. As Atm1 functions in exporting an Fe-S precursor from the mitochondrial matrix to the cytosol, C. neoformans cells depleted for Atm1 are sensitive to Cu even while the Cu-detoxifying metallothionein proteins are highly expressed. We provide evidence for a previously unrecognized microbial defense mechanism to deal with Cu toxicity, and we highlight the importance for C. neoformans of having several distinct mechanisms for coping with Cu toxicity which together could contribute to the success of this microbe as an opportunistic human fungal pathogen.IMPORTANCEC. neoformans is an opportunistic pathogen that causes lethal meningitis in over 650,000 people annually. The severity of C. neoformans infections is further compounded by the use of toxic or poorly effective systemic antifungal agents as well as by the difficulty of diagnosis. Cu is a natural potent antimicrobial agent that is compartmentalized within the macrophage phagosome and used by innate immune cells to neutralize microbial pathogens. While the Cu detoxification machinery of C. neoformans is essential for virulence, little is known about the mechanisms by which Cu kills fungi. Here we report that Fe-S cluster-containing proteins, including members of the Fe-S protein biogenesis machinery itself, are critical targets of Cu toxicity and therefore that this biosynthetic process provides an important layer of defense against high Cu levels. Given the role of Cu ionophores as antimicrobials, understanding how Cu is toxic to microorganisms could lead to the development of effective, broad-spectrum antimicrobials. Moreover, understanding Cu toxicity could provide additional insights into the pathophysiology of human diseases of Cu overload such as Wilson's disease.
| S-EPMC5666163 | biostudies-literature
Project description:The biogenesis of cytosolic iron-sulfur (Fe-S) proteins in mammalian cells is poorly understood. In Saccharomyces cerevisiae, there is a pathway dedicated to cytosolic Fe-S protein maturation that involves several essential proteins. One of these is Nar1, which intriguingly is homologous to iron-only hydrogenases, ancient enzymes that catalyze the formation of hydrogen gas in anaerobic bacteria. There are two orthologues of Nar1 in mammalian cells, iron-only hydrogenase-like protein 1 (IOP1) and IOP2 (also known as nuclear prelamin A recognition factor). We examined IOP1 for a potential role in mammalian cytosolic Fe-S protein biogenesis. We found that knockdown of IOP1 in both HeLa and Hep3B cells decreases the activity of cytosolic aconitase, an Fe-S protein, but not that of mitochondrial aconitase. Knockdown of IOP2, in contrast, had no effect on either. The decrease in aconitase activity upon IOP1 knockdown is rescued by expression of a small interference RNA-resistant version of IOP1. Upon loss of its Fe-S cluster, cytosolic aconitase is known to be converted to iron regulatory protein 1, and consistent with this, we found that IOP1 knockdown increases transferrin receptor 1 mRNA levels and decreases ferritin heavy chain protein levels. IOP1 knockdown also leads to a decrease in activity of xanthine oxidase, a distinct cytosolic Fe-S protein. Taken together, these results provide evidence that IOP1 is involved in mammalian cytosolic Fe-S protein maturation.
| S-EPMC2431034 | biostudies-literature