Mammalian iron-sulfur cluster biogenesis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a citric acid cycle enzyme, and it functions as a cytosolic aconitase in cells that are iron replete. In iron-depleted cells, IRP1 loses its iron sulfur cofactor, and the apoprotein switches to become an RNA binding protein. IRP1 involves a transition from a form of IRP1 in which a 4Fe-4S cluster is bound, to a form that loses both iron and aconitase activity. The 4Fe-4S containing protein does not bind RNA stem-loops known as IREs. Controlled degradation of the iron-sulfur cluster and mutagenesis reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster determines whether IRP1 will bind RNA. Over the past decade, we have identified mammalian enzymes of iron-sulfur cluster assembly that are homologous to the NifS, ISCU and Nif U, ferredoxins and ferredoxin reductase genes implicated in bacterial iron-sulfur cluster assembly, and we have shown that these gene products facilitate assembly of the iron- sulfur cluster of IRP1. We discovered that a mutation in the scaffold protein, ISCU, causes a rare myopathy. In both Friedreich ataxia and ISCU myopathy, our data indicate that mitochondrial iron overload occurs in conjunction with cytosolic iron depletion. In collaboration, we discovered that mutations in NFU1 and BOLA3 mutations cause a human disease characterized by lactic acidosis and lipoic acid deficiency. We predicted that other rare genetic diseases characterized by mitochondrial compromise were caused by mutations in the genes responsible for iron-sulfur cluster biogenesis, and we collaborated to discover that mutations of NFS1 cause neonatal mitochondrial disease. We are characterizing the steps that chaperone transfer of nascent iron-sulfur clusters from their association with the initial assembly apparatus to proteins that require iron-sulfur clusters for function. We discovered how SDHB acquires its three Fe-S clusters, and we have demonstrated that HSC20 cochaperone mediated iron sulfur cluster delivery is critical for iron sulfur acquisition of respiratory chains I-III. We are evaluating many more candidate recipients of iron sulfur clusters, and we expect our studies will greatly increase the number of known mammalian iron sulfur proteins. We established that Fe-S biogenesis occurs de novo in the cytosol, and that the chaperone HSC20 connects initial cytosolic biogenesis with the CIAO1-dependent Fe-S delivery platform in the cytosol by binding to a LYR motif in CIAO1. Thus Fe-S biogenesis occurs in parallel both in the mitochondrial matrix and in the cytosol of mammalian cells. Our work challenges the paradigm that initial Fe-S biogenesis occurs only in the mitochondrial matrix, and ABCB7 exports a component critical to Fe-S synthesis in mammalian cytosol. We are working to clarify the molecular interactions that promote transfer of Fe-S clusters to recipients using the HSC20 cochaperone system, followed in some cases by use of secondary scaffold proteins that confer specificity to subgroups of Fe-S recipients. Using informatics looking for iterations of the LYR motif, followed by overexpression and ICP-MS, we are in the process of identifying previously unrecognized Fe-S proteins, which we believe are common and represented in multiple key metabolic pathways of mammalian cells. We discovered that SARS-CoV-2 coopts the mammalian iron sulfur biogenesis machinery to supply iron sulfur cofactors for the viral nsp-12 replicase. The replicase incorporates two cubane iron sulfur cofactors, and they are needed for replicase function and for binding the associated helicase, nsp 13. The helicase, nsp13, ligates an iron sulfur cluster that is required for full function. Iron sulfur cofactors are vulnerable to oxidation. The stable nitroxide, Tempol, is an oxidant that degrades the iron sulfur cofactor of the replicase in vitro in primer extension assays. When tissue culture cells are infected with SARS-CoV-2, the addition of Tempol works as an antiviral by disabling the replicase. When Tempol was given to hamsters infected with SARS-CoV-2, Tempol attenuated the pathogenic effects of infection. We are working to develop Tempol as an oral antiviral for use against Covid infection. We are pursuing the hypothesis that many viruses utilize iron sulfur cofactors for function. Iron sulfur cofactors facilitate use of cellular reducing equivalents that the virus may utilize to support its energy requirements. Several other SARs-CoV-2 proteins are candidate iron sulfur proteins. We are studying HCoV-OC43, a viral cause of the common cold that is related to SARs-CoV-2. OC43 has a different mechanism for entering host cells, but the replicase, helicase, exoribonuclease and other non-structural proteins are highly conserved, making it a useful model for studies of coronaviral diseases. We aim to define the role of iron sulfur cofactors in coronaviral infections and to analyze structures and mechanisms of replication and translation through collaborations. Our ability to discover iron sulfur cofactors is due to our development of a system for predicting candidate proteins, over-expressing in human cell lines at 6% oxygen levels to mirror human tissues, and purification under anaerobic conditions that protect the iron sulfur cofactor from disassembly by oxidation. We discovered that CIAO1 deficient patients develop musculoskeletal disease, as do patients with BCS1L mutations. Numerous pathways in mammalian cells likely depend on function of proteins that are not yet recognized to depend on iron sulfur cofactors. Through publishing our research, we aim to galvanize discovery of iron sulfur proteins in multiple critical metabolic pathways in mammalian cells.