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  • Articles  (3)
  • Journal of Biological Chemistry  (3)
  • 43
  • 1
    Publication Date: 2018-09-15
    Description: Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 2
    Publication Date: 2018-10-06
    Description: Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu+ movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu+ permeation through membranes or its mechanism of action. Here, we identify three members of a Cu+ importer family from the thermophilic fungus Chaetomium thermophilum: Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu+ importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag+ transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo, with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu+ ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu+ ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu+ pools.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 3
    Publication Date: 2018-03-24
    Description: NO synthase (NOS) enzymes perform interdomain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain-docking model, we assessed the importance of cross-domain charge interactions in the FMN-to-heme electron transfer in neuronal NOS (nNOS). We reversed the charge of three residues (Glu-762, Glu-816, and Glu-819) that form an electronegative triad on the FMN domain and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, and Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a cross-domain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was probably muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that cross-domain charge pairing is required for the FMN-to-heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain-docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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