UC San Diego

There exist three different NEET family proteins in human cells. Named for their shared characteristic amino acid sequence (Asp-Glu-Glu-Thr), they were first identified as pioglitazone drug targets, one of the thiazolidinedione class of insulin-sensitizing therapeutics for Type 2 diabetes. Although each share similar structural organization and coordinate two [2Fe-2S] clusters with a unique 3Cys:1His coordination chemistry, their cellular localization varies and their functional roles are dissimilar. MitoNEET (mNT, CISD1), was the first characterized and is an outer mitochondrial membrane-anchored dimer with the C-terminal iron-sulfur cluster domain in the cytosol. Nutrient Deprivation Autophagy Factor 1 (NAF-1, Miner1, CISD2) is anchored to the mitochondrial-associated membranes of the endoplasmic reticulum (ER) with the iron-sulfur cluster domain in the cytosol. The third human NEET family protein is Mitochondrial Inner NEET (MiNT, Miner2, CISD3), is distinguished among the three, as it is the only soluble NEET, exists as a monomer, and is found inside the mitochondrial matrix.

The NEET proteins are critical for cellular iron regulation and reactive oxygen species (ROS) protection, redox sensing, metabolic regulation, and are critical for maintenance of respiratory electron transfer machinery. A missense mutation in NAF-1 causes Wolfram syndrome 2 and other NEET pathologies include neurodegeneration, diabetes, premature aging, as well as play a role in the proliferation of several cancers. Collectively these indicated a crucial underlying cellular role for iron management, redox regulation, and metabolism.

Until recently the role that mNT and NAF-1 play in the cell was unresolved. Though plenty of in situ and in vivo findings were published, elucidation of the precise cellular role of these proteins proved fruitless to scientific investigation. The major clues to their putative role were that 3Cys:1His iron-sulfur coordination is utilized by other iron-sulfur cluster transfer proteins, and a NEET family protein found in Arabidopsis thaliana (At-NEET) transfers iron into the mitochondria. Furthermore, RNAi knockdown of At-NEET caused free iron overload in the cell and led to ROS accumulation. Both mNT and NAF-1 have recently been identified as key links between the iron-sulfur cluster (ISC) biogenesis pathways in the mitochondria and the cytosol by transferring [2Fe-2S] clusters to human anamorsin, an early key piece of machinery that is responsible for providing [2Fe-2S] to the cytosolic iron-sulfur assembly (CIA) pathway. It has also been shown that mNT can transfer clusters to NAF-1, establishing that these proteins connect the flow of iron-sulfur away from the mitochondria to cytosolic targets.

The method by which iron-sulfur clusters are exported out of the mitochondrion to the cytosol is currently now well characterized. To determine whether human mNT could translocate iron-sulfur clusters into the mitochondrion through the outer membrane voltage dependent anion channel (VDAC), we examined the ability of mNT to control membrane potential across a bilayer embedded with VDAC. We characterized the mNT-VDAC binding with microscale thermophoresis (MST) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) in addition to computational fragment-docking direct-coupling analysis (Fd-DCA) to propose a model for mNT-VDAC binding. Through these data, our model suggests that mNT interacts measurably with the inner VDAC channel in addition to controlling VDAC gating in a redox-dependent manner, while NAF-1 does neither.

To further identify and characterize cytosolic iron-sulfur protein transfer partners, we selected human glutaredoxin-3 (GRX3), a protein recently determined to carry [2Fe-2S] clusters and contribute to the CIA pathway. We identified that GRX3 selectively transfers clusters to mNT, but not NAF-1, further establishing diverged cellular roles for these two proteins. This is also the only protein reported to date that can transfer [2Fe-2S] clusters to mNT. These findings further solidify the role of the NEET proteins link the ISC and CIA, and demonstrate that there is directionality of iron-sulfur transfer at the interface of the ISC-CIA pathways. This directionality suggests a path for feedback and recycling of cytosolic iron-sulfur.