Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity

Abstract

Proper muscle development and function depends on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination of in vivo C. elegans experiments and in vitro biophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in the in vivo setting and that these conserved regions may be involved in UNC-45 protein stability/ regulation via post translational modifications, protein-protein interactions, or some other unknown mechanism.

UNC-45 has a crucial role in maintaining muscle sarcomeres during aging in Caenorhabditis elegans

“…When allowed to develop at 25⁰C, this strain has lower steady state levels of both thick filament isoforms of myosin heavy chain, MHC A and MHC B, and fewer assembled thick filaments (Landsverk et al, 2007;Miller et al, 1983), similar to aged worms. We have shown, using our own antibody to UNC-45, that this mutant does in fact have significantly reduced levels of UNC-45 when grown at 25⁰C (Moncrief et al, 2021). For our purposes, we allowed the animals to develop normally at 15⁰C and transferred them to the restrictive temperature of 25⁰C only after muscle maturity had been reached (referred to as day 0 of adulthood).…”

“…The double mutant unc-45(e286); hsp-90(p673) was generated by genetic crossing. GB319 is the 2X outcrossed derivative of PHX789 ( unc-45(syb789)) which is a CRISPR-generated strain that expresses UNC-45-mNeonGreen and was described previously (Moncrief et al, 2021). The strain, PHX501 ( hsp-90(syb501)) , is a CRISPR-generated strain which expresses HSP-90 with a C-terminal mKate2 tag.…”

“…The generation of rabbit polyclonal antibodies to the C-terminal 120 residues of UNC-45 was described previously (Moncrief et al, 2021). A 149 residue region (aa 553-702) at the C-terminus of HSP-90 (“HSP-90 antigen”)(Figure S4A) was expressed in E. coli as a GST fusion protein and sent to Noble Life Sciences for antibody production in rats.…”

“…We used quantities of extracts and dilutions of antibodies that would place us into the linear range of detection by ECL and exposure to film. The following antibodies and dilutions were used: rabbit anti-UNC-45 (Moncrief et al, 2021) at 1:5,000; rat anti-HSP-90 at 1:2,500; mouse monoclonal 5-8 (Miller et al, 1983) for MHC B at 1:40,000; mouse monoclonal 5-6 ascites (Miller et al, 1983) for MHC A at 1:5,000; Phospho-(Ser/Thr) Kinase Substrate Antibody Sampler Kit (cell signaling) at 1:1,000; Phospho-Tyrosine (4G10, cell signaling) mouse mAb at 1:1,1000. The quantitation of steady-state levels of protein was performed as described in Miller et al (2009) (Miller et al, 2009).…”