A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation

Abstract

Hsp90 is the ATP-consuming core component of a very abundant molecular chaperone machine that handles a substantial portion of the cytosolic proteome. Rather than one machine, it is in fact an ensemble of molecular machines, since most mammalian cells express two cytosolic isoforms of Hsp90 and a subset of up to 40 to 50 cochaperones and regulate their interactions and functions by a variety of posttranslational modifications. We demonstrate that the Hsp90 ensemble is fundamentally remodeled during muscle differentiation and that this remodeling is not just a consequence of muscle differentiation but possibly one of the drivers to accompany and to match the vast proteomic changes associated with this process. As myoblasts differentiate into myotubes, Hsp90␣ disappears and only Hsp90␤ remains, which is the only isoform capable of interacting with the novel musclespecific Hsp90 cochaperone Aarsd1L. Artificially maintaining Hsp90␣ or knocking down Aarsd1L expression interferes with the differentiation of C2C12 myotubes. During muscle differentiation, Aarsd1L replaces the more ubiquitous cochaperone p23 and in doing so dampens the activity of the glucocorticoid receptor, one of the Hsp90 clients relevant to muscle functions. This cochaperone switch protects muscle cells against the inhibitory effects of glucocorticoids and may contribute to preventing muscle wasting induced by excess glucocorticoids. H sp90 is a highly abundant molecular chaperone, including in nonstressed cells, which is required for the maturation, stability, activity, and/or degradation of a substantial portion of the proteome (1-5) (for a regularly updated list of Hsp90 interactors, see http://www.picard.ch/downloads/Hsp90interactors.pdf). To its clients, the Hsp90 dimer offers a moving platform whose complex choreography involves ATP hydrolysis, huge conformational changes, and dynamic interactions with a large cohort of cochaperones (3, 6-11). The cochaperones regulate and are part of the Hsp90 cycle (3,(8)(9)(10)(11), and some may act as client specificity factors (12, 13). With 40 to 50 cochaperones, far more have been reported (see http://www.picard.ch/downloads/Hsp90facts.pdf) than the ones that have been extensively characterized as interacting with Hsp90 as part of the core cycle. Moreover, Hsp90 and cochaperones are extensively regulated by posttranslational modifications (see, for example, references 14-17), and Hsp90 is present in excess over any individual cochaperone. Hence, the Hsp90 molecular chaperone machine constitutes in fact an ensemble of complexes. These complexes dynamically interchange and are fashioned by the cell specificity of signaling and cochaperone expression patterns.One of the most extensively characterized components of the Hsp90 core machinery is the cochaperone p23 (18-20), encoded by the PTGES3 gene in mammals. This small acidic protein binds and stabilizes the ATP-bound state of Hsp90 (19-23). p23 binding, which is disrupted by Hsp90 inhibitors competing with ATP and by hyperacetylation of Hsp90, promotes a ...

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

“…Likewise, the association patterns of cochaperones Aha1 and CDC37 closely follow the hsp90 association pattern, and thus are consistent with their binding to hsp90 to regulate its ATP hydrolysis, which in turn regulates its conformational cycling and function (54,55), presumably to help hsp90 drive heme insertion into apo-Mb. Of note, we confirmed that the hsp90 cochaperone p23 is poorly expressed in myoblasts during differentiation, whereas the homolog cochaperone Aarsd1 is strongly expressed (41), and in our case is found associated with the hsp90-apo-Mb complex, consistent with Aarsd1 taking the place of p23 and its being needed for myoblast differentiation (41). A model for hsp90/cochaperone assisted Mb maturation that is consistent with the results to date is presented in Fig.…”

“…However, when the hsp90 inhibitors were chronically present, or when cell hsp90b expression was knocked down, it arrested the C2C12 cell differentiation into myotubes and led to a loss of muscle-specific protein marker expression. These effects are consistent with chronic hsp90 inhibition causing significant deviations in the myoblast gene expression profile during the 4-d differentiation (41).…”

“…during a 4-d differentiation period (Supplemental Fig. S4A), confirming a previous report (41), we focused on knockdown of hsp90b. Unfortunately, even a partial knockdown of hsp90b expression prevented differentiation by the C2C12 cells as assessed by several factors and also prevented cell accumulation of Mb (Supplemental Fig.…”

“…In addition, Aarsd1 associated with Mb in a pattern that was similar to the hsp90 association (Fig. 5D, E), consistent with the Aarsd1-hsp90 complex being essential for muscle differentiation (41).…”

“…We also compared the expression levels of p23 and its homolog alanyl-tRNA synthetase domain containing 1 (Aarsd1) cochaperone because they typically play similar roles but are reported to be expressed and utilized differently in myoblasts (41). We saw an initial boost and then a loss in p23 expression during the cell differentiation period, as compared to a consistent expression of Aarsd1 (Fig.…”