New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals

Stuehr, Dennis J.; Dai, Yue; Biswas, Pranjal; Sweeny, Elizabeth A.; Ghosh, Arnab

doi:10.1515/hsz-2022-0197

Cited by 14 publications

(15 citation statements)

References 82 publications

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“…This process typically requires heme delivery by GAPDH and participation by cell chaperone Hsp90, which is usually bound to the apo‐heme proteins. [ 32 ] A negative impact of NO on heme protein maturation was first reported in 1996 as evidenced by NO blocking heme incorporation into the NO synthase enzyme itself as it was being expressed in cells and began generating NO. [ 33 ] Subsequently, NO was found to inhibit cell heme incorporation into a variety of heme proteins including hemoglobin α and β, catalase, and cytochrome P450s.…”

Section: No Is a Regulator Of Heme Allocation In Biologymentioning

confidence: 99%

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution

et al. 2023

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A natural heme deficiency that exists in cells outside of the circulation broadly compromises the heme contents and functions of heme proteins in cells and tissues. Recently, we found that the signaling molecule, nitric oxide (NO), can trigger or repress the deployment of intracellular heme in a concentration-dependent hormetic manner. This uncovers a new role for NO and sets the stage for it to shape numerous biological processes by controlling heme deployment and consequent heme protein functions in biology.

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Section: No Is a Regulator Of Heme Allocation In Biologymentioning

confidence: 99%

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution

et al. 2023

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“…GAPDH has various functions in biology including its chaperoning mitochondrially-generated heme for allocation in mammalian cells (13). The TC-hGAPDH reporter construct described here behaved like native GAPDH regarding its physical, heme binding, and enzymatic properties, and thus upon its FlAsH labeling could serve as a probe to report on GAPDH heme binding in living mammalian cells in real time using a conventional 96-well fluorescent plate reader.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, the glycolytic enzyme GAPDH was shown to fulfill this role by binding mitochondrial heme and enabling its delivery to diverse targets including hemoglobin α, β, and γ, myoglobin, tryptophan dioxygenase (TDO), indoleamine dioxygenase 1 (IDO1), soluble guanylyl cyclase (sGC), NO synthases, and heme oxygenase 2 (6)(7)(8)(9)(10)(11)(12). In most cases, the heme insertions into these proteins also required the cell chaperone Hsp90 and its ATPase activity (13).…”

Section: Introductionmentioning

confidence: 99%

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO

Biswas,

Palazzo,

Schlanger

et al. 2024

Preprint

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Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.

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“…The target heme proteins are present in cells in their heme-free states and are typically are in complex with the cell chaperone Hsp90, which drives their heme insertions in an ATP-dependent manner, possibly with assistance from co-chaperone proteins 13,35 . Hsp90 then dissociates from the heme-replete mature proteins, allowing their biological function 18 . Regarding TANGO2, it had recently been proposed to act as a heme chaperone in cells or to aid in heme transfer from heme-enriched compartments, perhaps by acting independent of other heme-transporting proteins 30,31 .…”

Section: Discussionmentioning

confidence: 99%

Heme allocation in eukaryotic cells relies on mitochondrial heme export through FLVCR1b to cytosolic GAPDH

Jayaram,

Sivaram,

Biswas

et al. 2024

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Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondrial heme reaches GAPDH. Experiments involving a human cell line and a novel GAPDH reporter construct whose heme binding in live cells can be followed by fluorescence revealed that the mitochondrial transmembrane protein FLVCR1b exclusively transfers mitochondrial heme to GAPDH through a direct protein-protein interaction that rises and falls as heme transfers. In the absence of FLVCR1b, neither GAPDH nor downstream hemeproteins received any mitochondrial heme. Cell expression of TANGO2 was also required, and we found it interacts with FLVCR1b to likely support its heme exporting function. Finally, we show that purified GAPDH interacts with FLVCR1b in isolated mitochondria and triggers heme transfer to GAPDH and its downstream delivery to two client proteins. Identifying FLVCR1b as the sole heme source for GAPDH completes the path by which heme is exported from mitochondria, transported, and delivered into protein targets within eukaryotic cells.

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New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals

Cited by 14 publications

References 82 publications

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution

A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO

Heme allocation in eukaryotic cells relies on mitochondrial heme export through FLVCR1b to cytosolic GAPDH

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