Myoglobin maturation is driven by the hsp90 chaperone machinery and by soluble guanylyl cyclase

Abstract

Myoglobin (Mb) maturation involves heme incorporation as a final step. We investigated a role for heat shock protein (hsp) 90 in Mb maturation in C2C12 skeletal muscle myoblasts and cell lines. We found the following: 1) Hsp90 directly interacts preferentially with heme‐free Mb both in purified form and in cells. 2) Hsp90 drives heme insertion into apoprotein‐Mb in an ATP‐dependent process. 3) During differentiation of C2C12 myoblasts into myotubes, the apo‐Mb‐hsp90 complex associates with 5 cell cochaperons, Hsp70, activator of hsp90 ATPase protein 1 (Aha1), alanyl‐tRNA synthetase domain containing 1 (Aarsd1), cell division cycle 37 (Cdc37), and stress induced phosphoprotein 1 (STIP1) in a pattern that is consistent with their enabling Mb maturation. 4) Mb heme insertion was significantly increased in cells that had a functional soluble guanylyl cyclase (sGC)‐cGMP signaling pathway and was diminished upon small interfering RNA knockdown of sGCβ1 or upon overexpression of a phosphodiesterase to prevent cGMP buildup. Together, our findings suggest that hsp90 works in concert with cochaperons (Hsp70, Aha1, Aarsd1, STIP1, and Cdc37) and an active sGC‐cGMP signaling pathway to promote heme insertion into immature apo‐Mb, and thus generate functional Mb during muscle myotube formation. This fills gaps in our understanding and suggests new ways to potentially control these processes.—Ghosh, A., Dai, Y., Biswas, P., Stuehr, D. J. Myoglobin maturation is driven by the hsp90 chaperone machinery and by soluble guanylyl cyclase. FASEB J. 33, 9885–9896 (2019). http://www.fasebj.org

Maturation, inactivation, and recovery mechanisms of soluble guanylyl cyclase

“…On a broader level, it is interesting to ask if sGC is just a one-off example of these behaviors or is instead herald of a more general condition in which cells and tissues naturally build up overlooked but significant levels of heme proteins in their heme-free immature forms. Indeed, evidence shows that this does in fact occur for other hemeproteins including indoleamine or tryptophan dioxygenases, both when they are expressed naturally in liver ( 128 ) or are expressed in cultured cells ( 129 ), NO synthases expressed in cultured cells ( 130 ), and for myoglobin and hemoglobin β and γ in cells of both erythrocytic and nonerythrocytic lineage ( 59 , 60 ). Notably, in a majority of these cases the apo-hemeprotein subpopulations were found associated with Hsp90, just like for apo-sGCβ.…”

“…Hsp90 can also govern the chaperoned client's interactions with protein partners. Apo-sGCβ is one of several heme proteins that associate with Hsp90 in their heme-free forms in mammalian cells and that require Hsp90 for their maturation, the others being NO synthases ( 58 ), hemoglobins β and γ ( 59 ), and myoglobin ( 60 ). Interestingly, the Hsp90-sGC association was first discovered in the context of its stabilizing sGC and increasing cGMP synthesis ( 61 , 62 , 63 ).…”

Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones

“…Short exposure (30 min) to the Hsp90 inhibitor radicicol has been shown to decrease NOX5 superoxide production [ 52 ], while longer exposure (12 h) leads to proteosomal degradation of NOX5 [ 67 ]. Hsp90 has been shown to be essential for heme insertion into a number of soluble heme proteins [ [47] , [48] , [49] , [50] , [51] ]. To determine if Hsp90 inhibition would affect the heme-dependent increases in NOX5 superoxide production we first confirmed that the Hsp90 inhibitor radicicol inhibited superoxide production by NOX5 in a concentration dependent manner ( Supp.…”

“…Due to the important role Hsp90 plays in heme insertion into soluble heme proteins [ [47] , [48] , [49] , [50] , [51] ], we hypothesized that Hsp90 may also be facilitating heme insertion into NOX5, and therefore that the suppression of NOX5 activity with radicicol treatment may be due to an inability of NOX5 to bind the provided heme. However, when we obtained difference spectra of cells incubated with heme with and without radicicol treatment we found that Hsp90 inhibition did not suppress the increase in the NOX5 specific heme peak ( Fig.…”

“…Recent work in our lab and others has shown that Hsp90 is directly involved in heme insertion into a number of heme proteins such as soluble guanylate cyclase (sGC) [ 47 , 48 ], inducible nitric oxide (NO) synthase [ 49 ], hemoglobin β and γ [ 50 ], and myoglobin [ 51 ]. Whether this reliance on Hsp90 is conserved for integral membrane heme proteins like the NOX family remains unclear.…”