title: Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry and Physics, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Zuris
authors.forename: Yael
authors.initials: Y
authors.lastname: Harir
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Conlan
authors.forename: Maya
authors.initials: M
authors.lastname: Shvartsman
authors.forename: Dorit
authors.initials: D
authors.lastname: Michaeli
authors.forename: Sagi
authors.initials: S
authors.lastname: Tamir
authors.forename: Mark L
authors.initials: ML
authors.lastname: Paddock
authors.forename: José N
authors.initials: JN
authors.lastname: Onuchic
authors.forename: Ron
authors.initials: R
authors.lastname: Mittler
authors.forename: Zvi Ioav
authors.initials: ZI
authors.lastname: Cabantchik
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Jennings
authors.forename: Rachel
authors.initials: R
authors.lastname: Nechushtai
mesh_terms: D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D010539:Permeability / Q000187:drug effects; D000077205:Pioglitazone; D013329:Structure-Activity Relationship; D045162:Thiazolidinediones / Q000494:pharmacology
chemical_list: C525595:CISD1 protein, human; D005288:Ferredoxins; D007004:Hypoglycemic Agents; D007506:Iron-Sulfur Proteins; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; D045162:Thiazolidinediones; C026518:apoferredoxin; D006639:Histidine; D007501:Iron; D000077205:Pioglitazone