Bhat, P. D. | Search bioRxiv

bioRxiv2024

Age-dependent Changes in a Chaperone Complex in the Mouse Heart

The exquisitely organized sarcomere, the unit of contraction of striated muscle, is a stable structure with slow turnover of its components. The myosin chaperone UNC-45 and its binding partners, Hsp90 and Hsp70, are required for the initial folding of the myosin head domain and the assembly of myosin into thick filaments. There is increasing evidence that the UNC-45 system has an important role during aging to preserve sarcomere organization. Its decline may be a key factor in sarcopenia. Unlike skeletal muscle, the UNC-45 system in cardiac muscle in aging heart has not been examined extensively. Here we show that Unc45b and Hsp70 are localized to sarcomeric Z-discs in the mouse heart. We further show that during aging, there is a decline in the levels of myosin heavy chain, Unc-45b and Hsp70, but not Hsp90. While the decrease in Unc45b appears to be at the mRNA level, the decrease in the levels of myosin and Hsp70 were not at the mRNA but at the protein level. We have reported that in skeletal muscle, there is a decline in both Unc45b and Hsp90, and here we show that there is no such decline of Hsp70 in skeletal muscle. Hsp70 levels also did not decline with age in the brain or the liver. This heart-specific decrease of Hsp70 through its function as an Unc45b/Hsp70 complex might account for the age-dependent worsening of cardiomyopathies, and through Hsp70s multiple Unc-45b-independent functions, affect the folding and assembly of many other proteins in the aging heart.

cell biology↗

bioRxiv2020

A Region of UNC-89 (obscurin) Lying Between Two Protein Kinase Domains is a Highly Elastic Spring Required for Proper Sarcomere Assembly

In C. elegans, unc-89 encodes a set of giant multi-domain proteins (up 8,081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, high proline content and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals express normal levels of giant internally deleted UNC-89 proteins, and yet show severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generated in-frame deletion to contain an HA tag at the N-terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake. HighlightsO_LIThe giant muscle proteins UNC-89 and its mammalian homologs have an [~]700 aa non-domain region lying between two protein kinase domains C_LIO_LIBy single molecule force spectroscopy UNC-89 non-domain region is an elastic random coil C_LIO_LINematodes lacking this non-domain region have disorganized sarcomeres and reduced whole animal locomotion C_LIO_LIUNC-89 non-domain region is required for proper assembly of A-bands from thick filaments C_LI

cell biology↗