GAPDH is involved in the heme‐maturation of myoglobin and hemoglobin

Abstract

GAPDH, a heme chaperone, has been previously implicated in the incorporation of heme into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme‐maturation, we investigated the role of GAPDH in the maturation of this globin, as well as hemoglobins α, β, and γ. Utilizing cell culture systems, we found that overexpression of wild‐type GAPDH increased, whereas GAPDH mutants H53A and K227A decreased, the heme content of Mb and Hbα and Hbβ. Overexpression of wild‐type GAPDH fully recovered the heme‐maturation inhibition observed with the GAPDH mutants. Partial rescue was observed by overexpression of sGCβ1 but not by overexpression of a sGCΔβ1 deletion mutant, which is unable to bind the sGCα1 subunit required to form the active sGCα1β1 complex. Wild type and mutant GAPDH was found to be associated in a complex with each of the globins and Hsp90. GAPDH at endogenous levels was found to be associated with Mb in differentiating C2C12 myoblasts, and with Hbγ or Hbα in differentiating HiDEP‐1 erythroid progenitor cells. Knockdown of GAPDH in C2C12 cells suppressed Mb heme‐maturation. GAPDH knockdown in K562 erythroleukemia cells suppressed Hbα and Hbγ heme‐maturation as well as Hb dimerization. Globin heme incorporation was not only dependent on elevated sGCα1β1 heterodimer formation, but also influenced by iron provision and magnitude of expression of GAPDH, d‐aminolevulinic acid, and FLVCR1b. Together, our data support an important role for GAPDH in the maturation of myoglobin and γ, β, and α hemoglobins.

Hsp90 in Human Diseases: Molecular Mechanisms to Therapeutic Approaches

“…Given the novel role of Hsp90 in hemeprotein maturation, the effects of an overactive or a downregulated Hsp90 can both be deleterious to cellular homeostasis. This is now more evident from the fact that Hsp90 regulates the heme maturation of three key hemeproteins (iNOS, sGC and Hb) and our studies [21,59,60] now link the activation of sGC to the maturation of the globins (Mb/Hb), thereby contributing to the formation of novel NO-sGC-Globin axes and brings this pathway into a "new light" whose significance has thus far not been explored in cancer cells. Since Hsp90 is central to the heme maturation of iNOS, sGC and globin, its well defined role in cancer progression needs to be critically looked at cautiously with a fresh perspective so as to overcome potential deleterious effects of various Hsp90 inhibitors for better therapeutic intervention.…”

“…New research from our group has also identified Hsp90 and GAPDH to be major players in the process by which hemoglobin (Hb) and myoglobin (Mb) form and mature [21,59,60]. We found that Hsp90 chaperones hemoglobin (Hb) maturation in erythroid and non-erythroid cells (RAW, A549 cells) following a similar mechanism [21] (Figure 4).…”

“…We found that Hsp90 chaperones hemoglobin (Hb) maturation in erythroid and non-erythroid cells (RAW, A549 cells) following a similar mechanism [21] (Figure 4). While in erythroid cells, Hb-α and Hb-β/γ (-βadult/γfetal Hb) are independently chaperoned New research from our group has also identified Hsp90 and GAPDH to be major players in the process by which hemoglobin (Hb) and myoglobin (Mb) form and mature [21,59,60]. We found that Hsp90 chaperones hemoglobin (Hb) maturation in erythroid and non-erythroid cells (RAW, A549 cells) following a similar mechanism [21] (Figure 4).…”

“…We also found that muscle Myoglobin (Mb)/Hb not only requires Hsp90 and its co-chaperon machinery, but also needs an active sGC for its heme-maturation [59] (Figure 4). We recently established that in all these globin maturation events, GAPDH acts as a heme chaperon, allocating/providing mitochondria generated heme to the apo-Hsp90/AHSP-Hb α/β/γ /Mb complexes [60], where these globin heme maturations are influenced by iron provision, magnitude of expression of GAPDH, d-aminolevulinic acid plus FLVCR1b and that Hsp90 may be the ultimate heme donor to these globins [60]. Together these finding imply that Hsp90 and GAPDH play key roles in globin maturation and our current studies link the activation of sGC to the maturation of the globins (Mb/Hb), thereby contributing to the formation of novel NO-sGC-Globin axes.…”

“…Second, sGC/cGMP signaling may influence proliferation and/or differentiation of the tumor cells. Since our studies link globin expression to the NO-sGC axis [21,59,60], it provides additional ways to eliminate such globin expression. Inhibiting iNOS overexpression and the tumor inflammatory microenvironment, together with normalizing sGC/cGMP signaling, may be one of the favorable ways to reduce malignant tumors [91,92].…”