Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle—when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

Heme regulatory motifs (HRMs) are found in a variety of proteins with diverse biological functions. In heme oxygenase-2 (HO2), heme binds to the HRMs and is readily transferred to the catalytic site in the core of the protein. To further define this heme transfer mechanism, we evaluated the ability of GAPDH, a known heme chaperone, to transfer heme to the HRMs and/or the catalytic core of HO2. Our results indicate GAPDH and HO2 form a complex in vitro. We have followed heme insertion at both sites by fluorescence quenching in HEK293 cells with HO2 reporter constructs. Upon mutation of residues essential for heme binding at each site in our reporter construct, we found that HO2 binds heme at the core and the HRMs in live cells and that heme delivery to HO2 is dependent on the presence of GAPDH that is competent for heme binding. In sum, GAPDH is involved in heme delivery to HO2 but, surprisingly, not to a specific site on HO2. Our results thus emphasize the importance of heme binding to both the core and the HRMs and the interplay of HO2 with the heme pool via GAPDH to maintain cellular heme homeostasis.

Coenzyme Q (CoQ, aka ubiquinone) is a key component of the mitochondrial electron transport chain (ETC) and membrane-incorporated antioxidant. CoQ10 deficiencies encompass a heterogeneous spectrum of clinical phenotypes and can be caused by hereditary mutations in the biosynthesis pathway or result from pharmacological interventions such as HMG-CoA Reductase inhibitors, and statins, which are widely used to treat hypercholesterolemia and prevent cardiovascular disease. How CoQ deficiency affects individual tissues and cell types, particularly mitochondrial-rich ones such as brown adipose tissue (BAT), has remained poorly understood. Here we show that pharmacological and genetic models of BAT CoQ deficiency show altered respiration that can only in part be explained by classical roles of CoQ in the respiration chain. Instead, we found that CoQ strongly impacts brown and beige adipocyte respiration via the regulation of uncoupling protein 1 (UCP1) expression. CoQ deficiency in BAT robustly decreases UCP1 protein levels and uncoupled respiration unexpectedly, resulting in increased inner mitochondrial membrane potential and decreased ADP/ATP ratios. Suppressed UCP1 expression was also observed in a BAT-specific in vivo model of CoQ deficiency and resulted in enhanced cold sensitivity. These findings demonstrate an as yet unappreciated role of CoQ in the transcriptional regulation of key thermogenic genes and functions.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted upon eating to lower blood sugar. GLP-1 and GIP were found to induce the secretion of substance P (SP) from cultured sensory nerves. SP enhances insulin secretion. Mesenteric lymphatic fluid (MLF) also stimulates sensory neurons in a diet-dependent manner. These studies identify new actions of GLP-1 and GIP as incretins and suggest a mechanism for sensory nerves to respond to diet through MLF.

Coenzyme Q (CoQ) is an essential component of mitochondrial respiration1 and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations but mechanistic consequences of CoQ deficiency in specific tissues such as BAT remain poorly understood. Here we show that pharmacological or genetic CoQ deficiency (50-75% reduction) in BAT leads to accumulation of cytosolic mitochondrial RNAs (mtRNAs) and activation of the eIF2α kinase PKR resulting in the induction of the integrated stress response (ISR) and suppression of UCP1 expression in an ATF4-dependent fashion. Surprisingly, despite diminished UCP1 levels, BAT CoQ deficiency increases whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high fat diets (HFD). This mitohormesis like effect of BAT CoQ insufficiency is dependent on the ATF4-FGF21 axis in BAT revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.