Defining a pathway for mitochondrial heme trafficking

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ABSTRACT Hemeproteins play vital transport, enzymatic, and signaling roles that fundamentally impact our cardiovascular, pulmonary, digestive, neurological, and immune systems. Cells must transport mitochondrial heme to proteins that mature and function outside the mitochondria. Our goal is to understand how intracellular heme delivery takes place and is regulated in mammals. Cytosolic heme delivery proteins have been postulated but their identities are unclear. We found that GAPDH binds mitochondrially-generated heme and that its heme binding is essential for delivery to eight different hemeprotein targets. We hypothesize that GAPDH-dependent heme delivery is fundamental for hemeprotein function, and we propose to discern mechanisms, scope, and regulation of GAPDH-dependent heme delivery. Our experiments utilize purified proteins and cell culture, which provides a facile path to molecular-level discoveries and a robust means to validate their biological relevance. We have ways to control cell heme production, monitor GAPDH-heme binding and transfer in live cells and between proteins, assess heme delivery to targets, characterize GAPDH-target complexes, and determine their relevance for heme delivery. AIM 1. How does GAPDH participate in intracellular heme delivery? We hypothesize GAPDH may bind directly to the target proteins to deliver heme and found it does so with at least four proteins (apo-sGCβ, IDO1, TDO, and Mb) in cells and purified form. We will: (i) identify interface regions in each GAPDH-hemeprotein complex using HDx-MS, MS-cleavable crosslinking, and NMR approaches; (ii) use mutagenesis to test the role of GAPDH-target protein contacts in heme deliveries; (iii) structurally characterize the GAPDH-heme complex and GAPDH-hemeprotein complexes by crystallography and single particle EM; (iv) utilize a GAPDH-biolD2-HA fusion protein to biotinylate intermediate or middleman proteins that associate with GAPDH during heme transfers; (v) deploy GAPDH surface charge mutants to independently probe GAPDH-target binding and role in heme transfers; (vi) perform heme transfer experiments with purified GAPDH & target proteins; (vii) screen for GAPDH-dependent heme deliveries to cell heme exporters (FLVCR1a & ABCG2), cytochrome P450’s (CYP2D6 & CYP3A4), two peroxidases (LPO & EPO), an NADPH oxidase (NOX5), and heme oxygenases 1 & 2. AIM 2. What controls GAPDH heme acquisition & deliveries? Cell & molecular mechanisms that control GAPDH heme acquisition and release are unknown. We will: (i) determine if mitochondrial heme exporter FLVCR1b and/or ER heme exporter PGRMC2 is a heme source for GAPDH; (ii) test if GAPDH heme acquisition involves direct interaction with the exporters; (iii) investigate if GAPDH-heme level in cells is regulated by cell heme exporters FLVCR1a & ABCG2; (iv) examine if post-translational GAPDH modification, chaperone hsp90, or azole drugs control GAPDH heme acquisition and delivery.