Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and a labile [2Fe-2S] cluster. The rare 1-His and 3-Cys coordination of mNT's [2Fe-2S] leads to cluster lability that is strongly dependent on the presence of the single histidine...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2011-08, Vol.108 (32), p.13047-13052
Main Authors: Zuris, John A, Harir, Yael, Conlan, Andrea R, Shvartsman, Maya, Michaeli, Dorit, Tamir, Sagi, Paddock, Mark L, Onuchic, José N, Mittler, Ron, Cabantchik, Zvi Ioav, Jennings, Patricia A, Nechushtai, Rachel
Format: Article
Language:English
Subjects:
Biological Sciences
Cell lines
Diabetes
drugs
Ferredoxins - chemistry
Ferredoxins - metabolism
Fluorescence
HEK293 Cells
histidine
Histidine - metabolism
Humans
Hypoglycemic Agents - pharmacology
iron
Iron - metabolism
iron overload
Iron-Sulfur Proteins - chemistry
Iron-Sulfur Proteins - metabolism
Kidney cells
Kinetics
Ligands
mechanism of action
Mitochondria
Mitochondria - drug effects
Mitochondria - metabolism
mitochondrial membrane
Mitochondrial Proteins - metabolism
Models, Biological
Mutagenesis
Mutant Proteins - chemistry
Mutant Proteins - metabolism
mutants
Oxidation-Reduction - drug effects
Oxidative stress
Permeability - drug effects
polyacrylamide gel electrophoresis
Prescription drugs
Proteins
spectroscopy
Structure-Activity Relationship
Thiazolidinediones - pharmacology
Transfer rates
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Summary:MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and a labile [2Fe-2S] cluster. The rare 1-His and 3-Cys coordination of mNT's [2Fe-2S] leads to cluster lability that is strongly dependent on the presence of the single histidine ligand (His87). These properties of mNT are similar to known [2Fe-2S] shuttle proteins. Here we investigated whether mNT is capable of cluster transfer to acceptor protein(s). Facile [2Fe-2S] cluster transfer is observed between oxidized mNT and apo-ferredoxin (a-Fd) using UV-VIS spectroscopy and native-PAGE, as well as with a mitochondrial iron detection assay in cells. The transfer is unidirectional, proceeds to completion, and occurs with a second-order-reaction rate that is comparable to known iron-sulfur transfer proteins. Mutagenesis of His87 with Cys (H87C) inhibits transfer of the [2Fe-2S] clusters to a-Fd. This inhibition is beyond that expected from increased cluster kinetic stability, as the equivalently stable Lys55 to Glu (K55E) mutation did not inhibit transfer. The H87C mutant also failed to transfer its iron to mitochondria in HEK293 cells. The diabetes drug pioglitazone inhibits iron transfer from WT mNT to mitochondria, indicating that pioglitazone affects a specific property, [2Fe-2S] cluster transfer, in the cellular environment. This finding is interesting in light of the role of iron overload in diabetes. Our findings suggest a likely role for mNT in [2Fe-2S] and/or iron transfer to acceptor proteins and support the idea that pioglitazone's antidiabetic mode of action may, in part, be to inhibit transfer of mNT's [2Fe-2S] cluster.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1109986108