ABSTRACT OF THE DISSERTATION

Biochemical and Structural Characterization of the NEET Protein Family and their Role in Iron Homeostasis and Disease

by

Colin Harrison Lipper

Doctor of Philosophy in Chemistry

University of California, San Diego, 2017

Professor Patricia A. Jennings, Chair

The NEET proteins are a unique family of iron-sulfur proteins that bind a [2Fe-2S] cluster with an unusual 3Cys-1His coordination. The family consists of three human members, mitoNEET (mNT), nutrient-deprivation autophagy factor-1 (NAF-1) and Mitochondrial inner NEET (MiNT). These proteins have important roles in the regulation of iron and reactive oxygen species homeostasis and are involved in the pathology of several human diseases including cancer, diabetes, Wolfram syndrome 2 and neurodegeneration. These proteins have the ability to act as redox-sensors and transfer their Fe/[2Fe-2S] clusters to apo-acceptor proteins. We discovered that both mNT and NAF-1 can donate their clusters to human Anamorsin, a protein that is essential for the cytosolic assembly of Fe-S clusters. This connects the mitochondrial iron-sulfur cluster assembly system (ISC) to the dependent cytosolic iron-sulfur cluster assembly system (CIA). Additionally we identify an oxidation-dependent interaction with the outer-mitochondrial membrane pore protein VDAC1. The VDAC1 channel permeability is gated by the interaction with mNT. This NEET protein can transfer its [2Fe-2S] cluster into the mitochondria, causing iron to accumulate. Addition of a VDAC inhibitor prevents this mitochondrial transfer in situ and inhibits the interaction between mNT and VDAC1 in vitro. This indicates that VDAC channels are a direct route for mitochondrial iron transfer from mNT. Moreover, we find using hydrogen deuterium exchange mass spectrometry that mNT binds across the VDAC1 channel on the cytosolic side. Until now, very little was known about the third NEET family member, MiNT. We determined the crystal structure of this protein, which is significantly different than the other two human family members. It is monomeric with two cluster-binding domains in a single polypeptide, while mNT and NAF-1 are each homodimers with one cluster-binding domain per protomer. We find that MiNT can transfer its [2Fe-2S] cluster to human mitochondrial matrix ferredoxins, which are important for mitochondrial assembly of both iron-sulfur clusters and heme. MiNT is upregulated in breast cancer cells; we find that suppression of MiNT expression in cancer cells results in accumulation of iron and reactive oxygen species, as well as decreased mitochondrial membrane potential. Taken together, our findings further our understanding of how the NEET proteins function at a molecular level in maintaining iron homeostasis.