Review
Outlining the Complex Pathway of Mammalian Fe-S Cluster Biogenesis

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Highlights

  • Iron-sulfur (Fe-S) proteins have critical roles in essential metabolic pathways ranging from DNA and RNA metabolism to mitochondrial function, and regulation of cell growth and division.

  • Mammalian cells likely contain many more Fe-S proteins than have been identified to date, due to loss of fragile iron–sulfur clusters (ISCs) during purification.

  • Structural determinations of the architecture of the initial ISC biogenesis complex, comprising NFS1, ISD11, ACP, ISCU, and FXN, have shed light on the interactions that govern de novo ISC assembly.

  • In mammalian cells, a full complement of initial ISC biogenesis factors localizes to the cytosol, where it initiates de novo ISC assembly.

  • Further biochemical and clinical investigations of ISC-associated disorders and their phenotypes will elucidate the distinct pathways that assemble and deliver ISCs to the numerous enzymes that require them for function.

Iron–sulfur (Fe-S) clusters (ISCs) are ubiquitous cofactors essential to numerous fundamental cellular processes. Assembly of ISCs and their insertion into apoproteins involves the function of complex cellular machineries that operate in parallel in the mitochondrial and cytosolic/nuclear compartments of mammalian cells. The spectrum of diseases caused by inherited defects in genes that encode the Fe-S assembly proteins has recently expanded to include multiple rare human diseases, which manifest distinctive combinations and severities of global and tissue-specific impairments. In this review, we provide an overview of our understanding of ISC biogenesis in mammalian cells, discuss recent work that has shed light on the molecular interactions that govern ISC assembly, and focus on human diseases caused by failures of the biogenesis pathway.

Section snippets

Biological Roles of Iron–Sulfur Proteins and Relevance to Human Diseases

ISCs are among the oldest cofactors known in biology and have been proposed to have had a critical role in the emergence of life on Earth more than 3 billion years ago, when they were incorporated into early metabolic pathways by primitive organisms [1]. Their versatile chemical properties have fostered their pervasive use in almost all organisms to execute an impressive number of reactions involved in fundamental cellular processes, such as respiration, photosynthesis, metabolism, and nitrogen

Specific Examples of Dysfunctional Fe-S Proteins in Human Diseases

ISCs commonly mediate redox reactions in mitochondrial respiration, photosynthesis, and nitrogen fixation, where they act as electron relay chains that are ensheathed within an otherwise impervious protein matrix. A redox switch in the oxidation state of [4Fe-4S] clusters was also recently found to modulate the DNA-binding affinity of DNA-processing enzymes, including glycosylases, helicases, and primases [5]. Additionally, Fe-S enzymes have crucial roles in translation, as in the case of the

ISC Biogenesis on the Main Scaffold Protein ISCU in Mammalian Cells

ISC biogenesis is an evolutionarily highly conserved process. The first insights into the proteins that contributed to the pathway were obtained through analysis of a bacterial operon needed for Fe-S biogenesis in nitrogen-fixing bacteria [25]. Most protein functions defined in bacterial ISC biogenesis have been conserved in mammalian cells.

De novo ISC assembly is a complex, multiprotein-mediated process that involves an initial critical step catalyzed by a pyridoxal-phosphate (PLP)-dependent

Architecture of the Human Mitochondrial ISC Assembly Machinery

The NFS1/ISD11/ACP/ISCU complex is a symmetric hetero-octamer, comprising two copies of each of the four constituent proteins [32,34] (Figure 2B). NFS1, ISD11, and ACP form a homohexameric core, with ISCU bound to each end of the complex. FXN, which acts as an allosteric regulator of ISC biogenesis by driving efficient sulfur transfer from Cys381 of the mobile S-loop of NFS1 to Cys138 of ISCU (Figure 1), was recently co-crystallized with the NFS1/ISD11/ACP/ISCU complex [34] (Figure 2C). FXN

Insights into the FXN-Mediated Activation of the Core ISC Complex

In the closed conformation of the NFS1/ISD11/ACP/ISCU/FXN complex captured in a cryo-electron microscopy (cryo-EM) structure [34] (Figure 2C), FXN occupied the interface between NFSI and ISCU and contacted one of the two NFS1 protomers at the catalytic S-loop, supporting the idea that FXN functions as an allosteric modulator of ISC biogenesis [27] (Figure 1, Figure 2C). FXN also bound to two key regions of ISCU, one of which is the conserved ISCU-Alanine-loop (Ala66-Asp71), containing the Fe-S

A Specialized Chaperone/Cochaperone System Assists Delivery of ISCs to Recipient Proteins

Transfer of newly assembled ISCs downstream of the main scaffold protein ISCU in mammalian cells relies on the activity of a highly conserved chaperone/cochaperone system analogous to the yeast Ssq1/Jac1 and the bacterial HscA/HscB complexes [49., 50., 51.]. In mammalian cells, the multifunctional member of the HSP70 family, HSPA9 (also known as mortalin or GRP75), works with the specialized DnaJ-type III protein, HSC20 (also referred to as DNAJC20 or HSCB), to either directly facilitate ISC

Distinct Pathways Assemble [4Fe-4S] Clusters in Mitochondria for Specific Fe-S Recipient Proteins

The initial model for the biogenesis of [4Fe-4S] clusters in mitochondria, largely based on studies in S. cerevisiae, proposed that three human proteins, ISCA1, ISCA2, and IBA57, function as secondary carriers and form a heterocomplex that enable the assembly of [4Fe-4S] clusters, using as building blocks [2Fe-2S] cofactors assembled upon ISCU and transferred by GLRX5 [57,58]. However, the functional association between ISCA1, ISCA2, and IBA57 has been recently questioned [59., 60., 61., 62.].

Longstanding Controversy over the Existence of a Parallel System for ISC Biogenesis in the Cytosolic/Nuclear Compartment of Mammalian Cells

Two contrasting models have emerged to describe cytoplasmic Fe-S biogenesis [67,76] (Figure 3). One model, largely based on studies in yeast, proposed that the assembly of extramitochondrial Fe-S proteins relied on the mitochondrial ISC machinery for the synthesis of a sulfur-containing compound (named X-S) that was exported to the cytoplasm by the ABC transporter Atm1 (ABCB7 in human) and utilized for the assembly of a [4Fe-4S] cluster transiently bound to the heterotetrameric Nbp35/Cfd1

Concluding Remarks

The fact that an increasing number of newly recognized rare diseases results from compromise of the Fe-S biogenesis pathway offers the prospect that better understanding of the complexity of ISC biogenesis will lead to more effective diagnostics and treatments for a related group of human diseases. However, prior to that, the complexity of these pathways, including identifying the source of iron to build the clusters, characterizing motifs that guide Fe-S cofactors to recipients, and teasing

Acknowledgments

The authors would like to acknowledge support from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Glossary

Cysteine desulfurase
an enzyme that catalyzes one of the early steps in Fe-S cluster biogenesis, namely the abstraction of the inorganic sulfur, required to build the cluster, from cysteine. The gene is named NFS1 in humans, Nfs1 in yeast, and IscS in prokaryotes. In humans, the same NFS1 nuclear gene encodes two isoforms that localize either to the cytosol/nucleus or to mitochondria, as a result of initiation of translation from two alternative initiation codons on the mRNA.
De novo ISC assembly

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