Ubiquitin-proteasome system (UPS) dysfunction is associated with the pathology of a wide range of human diseases, including myopathies and muscular atrophy. However, the mechanistic understanding of specific components of the regulation of protein turnover during development and disease progression in skeletal muscle is unclear. Mutations in KLHL40, an E3 ubiquitin ligase cullin3 (CUL3) substrate-specific adapter protein, result in severe congenital nemaline myopathy, but the events that initiate the pathology and the mechanism through which it becomes pervasive remain poorly understood. To characterize the KLHL40-regulated ubiquitin-modified proteome during skeletal muscle development and disease onset, we used global, quantitative mass spectrometry-based ubiquitylome and global proteome analyses of klhl40a mutant zebrafish during disease progression. Global proteomics during skeletal muscle development revealed extensive remodeling of functional modules linked with sarcomere formation, energy, biosynthetic metabolic processes, and vesicle trafficking. Combined analysis of klh40 mutant muscle proteome and ubiquitylome identified thin filament proteins, metabolic enzymes, and ER-Golgi vesicle trafficking pathway proteins regulated by ubiquitylation during muscle development. Our studies identified a role for KLHL40 as a regulator of ER-Golgi anterograde trafficking through ubiquitin-mediated protein degradation of secretion-associated Ras-related GTPase1a (Sar1a). In KLHL40 deficient muscle, defects in ER exit site vesicle formation and downstream transport of extracellular cargo proteins result in structural and functional abnormalities. Our work reveals that the muscle proteome is dynamically fine-tuned by ubiquitylation to regulate skeletal muscle development and uncovers new disease mechanisms for therapeutic development in patients.
The data is publicly available via Sequence Read Archive (SRA) (Accession Number: PRJNA861969) and MassIVE (http://massive.ucsd.edu) and are accessible at ftp://MSV000090018@massive.ucsd.edu.
RNA-seq in KLHL40 KO zebrafish muscleSRA, PRJNA861969.
- Vandana A Gupta
- Vandana A Gupta
- Philip A Cole
- Philip A Cole
- Vandana A Gupta
- Pierre M Jean-Beltran
- Gianina Ravenscroft
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Zebrafish were maintained and bred using standard methods as described (Westerfield,2000). All experiments and procedures were approved by the Institutional Animal Care and Use Committee at Brigham and Women's Hospital. (2016000304).
Human subjects: Human Research Ethics Committee of the University of Western Australia (RA/4/20/1008). Written informed consent was provided by all families.
- Meir Aridor
© 2023, Mansur et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2-dependent and PPM1H phosphatase-reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain, and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.
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