The maintenance of a properly folded proteome is critical for cellular function and organismal health, and its age-dependent collapse is associated with a wide range of diseases. Here, we find that despite the central role of Coenzyme A as a molecular cofactor in hundreds of cellular reactions, limiting Coenzyme A levels in C. elegans and in human cells, by inhibiting the conserved pantothenate kinase, promotes proteostasis. Impairment of the cytosolic iron-sulfur clusters formation pathway, which depends on Coenzyme A, similarly promotes proteostasis and acts in the same pathway. Proteostasis improvement by Coenzyme A/iron-sulfur cluster deficiencies are dependent on the conserved HLH-30/TFEB transcription factor. Strikingly, under these conditions, HLH-30 promotes proteostasis by potentiating the expression of select chaperone genes providing a chaperone-mediated proteostasis shield, rather than by its established role as an autophagy and lysosome biogenesis promoting factor. This reflects the versatile nature of this conserved transcription factor, that can transcriptionally activate a wide range of protein quality control mechanisms, including chaperones and stress response genes alongside autophagy and lysosome biogenesis genes. These results highlight TFEB as a key proteostasis-promoting transcription factor and underscore it and its upstream regulators as potential therapeutic targets in proteostasis-related diseases.

Iron-sulfur (Fe-S) clusters are ubiquitous and are necessary to sustain basic life processes. The intracellular Fe-S clusters do not form spontaneously and many proteins are required for their biosynthesis and delivery. The bacterial P-loop NTPase family protein ApbC participates in Fe-S cluster assembly and transfers the cluster into apoproteins, with the Walker A motif and CxxC motif being essential for functionality of ApbC in Fe-S protein biogenesis. However, the structural basis underlying the ApbC activity and the motifs\' role remains unclear. Here, we report the crystal structure of Escherichia coli ApbC at 2.8 Å resolution. The dimeric structure is in a W shape and the active site is located in the 2-fold center. The function of the motifs can be annotated by structural analyses. ApbC has an additional N-terminal domain that differs from other P-loop NTPases, possibly conferring its inherent specificity in vivo.

The functionality of many cellular proteins depends on cofactors; yet, they have only been implicated in a minority of Mendelian diseases. Here, we describe the first 2 inherited disorders of the cytosolic iron-sulfur protein assembly system.
Genetic testing via genome sequencing was applied to identify the underlying disease cause in 3 patients with microcephaly, congenital brain malformations, progressive developmental and neurologic impairments, recurrent infections, and a fatal outcome. Studies in patient-derived skin fibroblasts and zebrafish models were performed to investigate the biochemical and cellular consequences.
Metabolic analysis showed elevated uracil and thymine levels in body fluids but no pathogenic variants in DPYD, encoding dihydropyrimidine dehydrogenase. Genome sequencing identified compound heterozygosity in 2 patients for missense variants in CIAO1, encoding cytosolic iron-sulfur assembly component 1, and homozygosity for an in-frame 3-nucleotide deletion in MMS19, encoding the MMS19 homolog, cytosolic iron-sulfur assembly component, in the third patient. Profound alterations in the proteome, metabolome, and lipidome were observed in patient-derived fibroblasts. We confirmed the detrimental effect of deficiencies in CIAO1 and MMS19 in zebrafish models.
A general failure of cytosolic and nuclear iron-sulfur protein maturation caused pleiotropic effects. The critical function of the cytosolic iron-sulfur protein assembly machinery for antiviral host defense may well explain the recurrent severe infections occurring in our patients.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.

[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe4 S4 ] center with a binuclear iron center ([2Fe]H ). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H -subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3 Å resolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.

Xanthine oxidoreductase (XOR) catalyzes the oxidation of purines (hypoxanthine and xanthine) to uric acid. XOR is widely used in various therapeutic and biotechnological applications. In this study, we characterized the biophysical and mechanistic properties of a novel bacterial XOR from Sulfobacillus acidophilus TPY (SaXOR). Our results showed that SaXOR is a heterotrimer consisting of three subunits, namely XoA, XoB, and XoC, which denote the molybdenum cofactor (Moco), 2Fe-2S, and FAD-binding domains, respectively. XoC was found to be stable when co-expressed with XoB, forming an XoBC complex. Furthermore, we prepared a fusion of XoB and XoC via a flexible linker (fusXoBC) and evaluated its function in comparison to that of XoBC. Spectroscopic analysis revealed that XoB harbors two 2Fe-2S clusters, whereas XoC bears a single-bound FAD cofactor. Electron transfer from reduced forms of XoC, XoBC, and fusXoBC to molecular oxygen (O2 ) during oxidative half-reaction yielded no flavin semiquinones, implying ultrafast single-electron transfer from 2Fe-2Sred to FAD. In the presence of XoA, XoBC and fusXoBC exhibited comparable XoA affinity and exploited a shared overall mechanism. Nonetheless, the linkage may accelerate the two-step, single-electron transfer cascade from 2Fe-2Sred to FAD while augmenting protein stability. Collectively, our findings provide novel insights into SaXOR properties and oxidation mechanisms divergent from prior mammalian and bacterial XOR paradigms.

Atomically defined large metal clusters have applications in new reaction development and preparation of materials with tailored properties. Expanding the synthetic toolbox for reactive high nuclearity metal complexes, we report a new class of Fe clusters, Tp*4 W4 Fe13 S12 , displaying a Fe13 core with M-M bonds that has precedent only in main group and late metal chemistry. M13 clusters with closed shell electron configurations can show significant stability and have been classified as superatoms. In contrast, Tp*4 W4 Fe13 S12 displays a large spin ground state of S=13. This compound performs small molecule activations involving the transfer of up to 12 electrons resulting in significant cluster rearrangements.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an Fe4S4 cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.

We have examined how the refined B-factor changes as a function of Z (the atomic number of a scatterer) at the sulfur site of the [4Fe:4S] cluster of the nitrogenase iron protein by refinement. A simple model is developed that quantitatively captures the observed relationship between Z and B, based on a Gaussian electron density distribution with a constant electron density at the position of the scatterer. From this analysis, the fractional changes in B and Z are found to be similar. The utility of B-factor refinement to potentially distinguish atom types reflects the Z dependence of X-ray atomic scattering factors; the weaker dependence of electron atomic scattering factors on Z implies that distinctions between refined values of B in an electron scattering structure will be less sensitive to the atomic identity of a scatterer than for the case with X-ray-diffraction. This behavior provides an example of the complementary information that can be extracted from different types of scattering studies.