In Vitro Studies of Cellular Iron–Sulfur Cluster Biosynthesis, Trafficking, and Transport

Wachnowsky, Christine; Cowan, J. A.

doi:10.1016/bs.mie.2017.06.045

Cited by 4 publications

(3 citation statements)

References 67 publications

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“…The aerobic lability of Fe-S clusters makes their identification difficult. Then, the anaerobic in vitro reconstitution of Fe-S clusters is a ‘gold standard’ method to assess the ability of a protein to bind to such clusters [ 38 , 39 ]. Thus, in order to determine if VDAC could accommodate an Fe-S cluster, we first overexpressed human VDAC1 and VDAC2 in E .…”

Section: Resultsmentioning

confidence: 99%

Dysfunction in the mitochondrial Fe-S assembly machinery leads to formation of the chemoresistant truncated VDAC1 isoform without HIF-1α activation

et al. 2018

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Biogenesis of iron-sulfur clusters (ISC) is essential to almost all forms of life and involves complex protein machineries. This process is initiated within the mitochondrial matrix by the ISC assembly machinery. Cohort and case report studies have linked mutations in ISC assembly machinery to severe mitochondrial diseases. The voltage-dependent anion channel (VDAC) located within the mitochondrial outer membrane regulates both cell metabolism and apoptosis. Recently, the C-terminal truncation of the VDAC1 isoform, termed VDAC1-ΔC, has been observed in chemoresistant late-stage tumor cells grown under hypoxic conditions with activation of the hypoxia-response nuclear factor HIF-1α. These cells harbored atypical enlarged mitochondria. Here, we show for the first time that depletion of several proteins of the mitochondrial ISC machinery in normoxia leads to a similar enlarged mitochondria phenotype associated with accumulation of VDAC1-ΔC. This truncated form of VDAC1 accumulates in the absence of HIF-1α and HIF-2α activations and confers cell resistance to drug-induced apoptosis. Furthermore, we show that when hypoxia and siRNA knock-down of the ISC machinery core components are coupled, the cell phenotype is further accentuated, with greater accumulation of VDAC1-ΔC. Interestingly, we show that hypoxia promotes the downregulation of several proteins (ISCU, NFS1, FXN) involved in the early steps of mitochondrial Fe-S cluster biogenesis. Finally, we have identified the mitochondria-associated membrane (MAM) localized Fe-S protein CISD2 as a link between ISC machinery downregulation and accumulation of anti-apoptotic VDAC1-ΔC. Our results are the first to associate dysfunction in Fe-S cluster biogenesis with cleavage of VDAC1, a form which has previously been shown to promote tumor resistance to chemotherapy, and raise new perspectives for targets in cancer therapy.

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Section: Resultsmentioning

confidence: 99%

Dysfunction in the mitochondrial Fe-S assembly machinery leads to formation of the chemoresistant truncated VDAC1 isoform without HIF-1α activation

et al. 2018

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“…The former is likely a metal binding site, and indeed a recent cryogenic electron microscopy (cryo-EM) structure of the trypanosomal METTL17 homologue revealed coordination of Zn 2+ through these residues (Saurer et al, 2019). It is known that Zn 2+ can replace a labile Fe-S cluster in aerobically purified proteins (Imlay, 2006; Nicolet and Fontecilla-Camps, 2014; Wachnowsky and Cowan, 2017). The LYR motif (not to be confused for LYRM protein) is often found in Fe-S cluster apoproteins (Maio et al, 2014) which is proposed to engage the Fe-S cluster handoff machinery.…”

Section: Resultsmentioning

confidence: 99%

“…Affinity chromatography of METTL17 gave a clean preparation of the protein that resulted in both a single band on an SDS-PAGE gel and a monodisperse analytical gel filtration peak corresponding to a molecular weight near 50 kDa (Fig 5A). Purified METTL17 precipitates upon exposure to air as is common for air-sensitive Fe-S proteins (Wachnowsky and Cowan, 2017). Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed the presence of 3.7 Fe ions per polypeptide (Fig 5B).…”

Section: Resultsmentioning

confidence: 99%

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation

Ast

Itoh

Sadre

et al. 2022

Preprint

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Friedreich's ataxia (FA) is the most common monogenic mitochondrial disease. FA is caused by a depletion of the mitochondrial protein frataxin (FXN), an iron-sulfur (Fe-S) cluster biogenesis factor. To better understand the cellular consequences of FA, we performed quantitative proteome profiling of human cells depleted for FXN. Nearly every known Fe-S cluster-containing protein was depleted in the absence of FXN, indicating that as a rule, cluster binding confers stability to Fe-S proteins. Proteomic and genetic interaction mapping identified impaired mitochondrial translation downstream of FXN loss, and specifically highlighted the methyltransferase-like protein METTL17 as a candidate effector. Using comparative sequence analysis, mutagenesis, biochemistry and cryogenic electron microscopy we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+cluster required for its stability on the mitoribosome. Notably, METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN null cells. Our data suggest that METTL17 serves as an Fe-S cluster checkpoint: promoting the translation and assembly of Fe-S cluster rich OXPHOS proteins only when Fe-S cluster levels are replete.

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Glutaredoxins with iron-sulphur clusters in eukaryotes - Structure, function and impact on disease

Berndt

Christ

Rouhier

et al. 2021

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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In Vitro Studies of Cellular Iron–Sulfur Cluster Biosynthesis, Trafficking, and Transport

Cited by 4 publications

References 67 publications

Dysfunction in the mitochondrial Fe-S assembly machinery leads to formation of the chemoresistant truncated VDAC1 isoform without HIF-1α activation

Dysfunction in the mitochondrial Fe-S assembly machinery leads to formation of the chemoresistant truncated VDAC1 isoform without HIF-1α activation

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation

Glutaredoxins with iron-sulphur clusters in eukaryotes - Structure, function and impact on disease

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