Sarcopenia, the decline in skeletal muscle mass and function without any underlying disease, is a major contributor to physical disability, poor quality of life, and death among the elderly (Cruz-Jentoft et al., 2014). 40%-50% of individuals over 80 years of age suffer from this loss of muscle mass and function (Barbosa-Silva, Bielemann,

PIX proteins are guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42, and are known to have numerous functions in various cell types. Here, we show that a PIX protein has an important function in muscle. From a genetic screen in C. elegans, we found that pix-1 is required for the assembly of integrin adhesion complexes (IACs) at borders between muscle cells, and is required for locomotion of the animal. A pix-1 null mutant has a reduced level of activated Rac in muscle. PIX-1 localizes to IACs at muscle cell boundaries, M-lines and dense bodies. Mutations in genes encoding proteins at known steps of the PIX signaling pathway show defects at muscle cell boundaries. A missense mutation in a highly conserved residue in the RacGEF domain results in normal levels of PIX-1 protein, but a reduced level of activated Rac in muscle, and abnormal IACs at muscle cell boundaries.

SUMMARY Cysteine cathepsins play roles during development and disease beyond their function in lysosomal protein turnover. Here, we leverage a fluorescent activity-based probe (ABP), BMV109, to track cysteine cathepsins in normal and diseased zebrafish embryos. Using this probe in a model of mucolipidosis II, we show that loss of carbohydrate-dependent lysosomal sorting alters the activity of several cathepsin proteases. The data support a pathogenic mechanism where TGF-β signals enhance the proteolytic processing of pro-Ctsk by modulating the expression of chondroitin 4-sulfate (C4-S). In MLII, elevated C4-S corresponds with TGF-β-mediated increases in chst11 expression. Inhibiting chst11 impairs the proteolytic activation of Ctsk and alleviates the MLII phenotypes. These findings uncover a regulatory loop between TGF-β signaling and Ctsk activation that is altered in the context of lysosomal disease. This work highlights the power of ABPs to identify mechanisms underlying pathogenic development in living animals.

UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. We have previously demonstrated that its UCS domain is responsible for the chaperone-like properties of UNC-45B. In order to better understand the chaperoning function of the UCS domain we engineered mutations designed to: i) disrupt chaperone-client interactions by removing and altering the structure of the putative client-interacting loop and ii) disrupt chaperone-client interactions by changing highly conserved residues in the putative client-binding groove. We tested the effect of these mutations by using a novel combination of complementary biophysical (circular dichroism, intrinsic tryptophan fluorescence, chaperone activity, and SAXS) and in vivo tools (C. elegans sarcomere structure). Removing the client-holding loop had a pronounced effect on the secondary structure, thermal stability, solution conformation and chaperone function of the UCS domain. These results are consistent with previous in vivo findings that this mutation neither rescue the defect in C. elegans sarcomere organization nor bind to myosin. We found that mutating several conserved residues in the client-binding groove do not affect UCS domain secondary structure or structural stability but reduced its chaperoning activity. We found that these groove mutations also significantly altered the structure and organization of the worm sarcomeres. We also tested the effect of R805W, a mutation distant from the client-binding region. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic effect on the structure and organization of the worm sarcomeres. In humans, the R805W mutation segregates with human congenital/infantile cataract, indicating a crucial role of R805 in UCS domain stability and/or client interaction. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin/chaperone interactions in mechanistic detail.

Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab -deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-β–related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.