Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset
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This important work utilizes a model organism, zebrafish, to explore changes to the proteome and the role of KLHL40, a component of the ubiquitin-proteasome system, in the development of skeletal muscle disease. Using mass spectrometry, the authors demonstrate a major and selective role for proteome remodeling in development. They identify a specific role for KLHL40 deletion in regulating the expression of Sar1 - a key component of biosynthetic secretion, where the resulting elevated levels of Sar1 expression potentially lead to collagen secretion defects in the disease state. The findings are incomplete as further experimental characterization of the overall morphological changes and secretion defects, in particular ones derived from the deregulation of Sar1 levels, is required.
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Abstract
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.
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eLife assessment
This important work utilizes a model organism, zebrafish, to explore changes to the proteome and the role of KLHL40, a component of the ubiquitin-proteasome system, in the development of skeletal muscle disease. Using mass spectrometry, the authors demonstrate a major and selective role for proteome remodeling in development. They identify a specific role for KLHL40 deletion in regulating the expression of Sar1 - a key component of biosynthetic secretion, where the resulting elevated levels of Sar1 expression potentially lead to collagen secretion defects in the disease state. The findings are incomplete as further experimental characterization of the overall morphological changes and secretion defects, in particular ones derived from the deregulation of Sar1 levels, is required.
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Reviewer #1 (Public Review):
The manuscript by Mansur et al examines the roles of KLHL40, mutations which lead to the development of skeletal muscle disease (nemaline myopathy, NM). The authors use CRISPR-based gene editing in a model organism (zebrafish) to disrupt the two fish isoforms of KLHL40 (a and b) and examine the resulting phenotypes. The authors find that disease-like phenotypes develop in adulthood selectively with the deletion of the KLHL40a isoform. Phenotypes include reduced body size, reduced endurance, and reduced life span with cellular effects that include perturbations to sarcomere organization, perturbed morphology of secretory organelles and mitochondria, and defects in collagen secretion and ECM deposition. The system provided the advantage of following both development and pre-disease state (onset) allowing the …
Reviewer #1 (Public Review):
The manuscript by Mansur et al examines the roles of KLHL40, mutations which lead to the development of skeletal muscle disease (nemaline myopathy, NM). The authors use CRISPR-based gene editing in a model organism (zebrafish) to disrupt the two fish isoforms of KLHL40 (a and b) and examine the resulting phenotypes. The authors find that disease-like phenotypes develop in adulthood selectively with the deletion of the KLHL40a isoform. Phenotypes include reduced body size, reduced endurance, and reduced life span with cellular effects that include perturbations to sarcomere organization, perturbed morphology of secretory organelles and mitochondria, and defects in collagen secretion and ECM deposition. The system provided the advantage of following both development and pre-disease state (onset) allowing the authors to look at changes in translation but mainly in the proteome with a focus on ubiquitylation (followed by mass spectrometry). Selective changes to the proteomthe e in KLHL40a deletion mutant are evident in the pre-symptomatic stage. Pathway analysis suggests that mutant cells show selective increases in glycolytic and biosynthetic enzymes/pathways, perhaps, akin to a Warburg effect. Monitoring the correlation between loss of KLHL40a-dependent ubiquitylation and increased protein levels defined the small GTPase Sar1a as a direct target for KLHL40a-directed degradation. Sar1a interacts with KLHL40a and is ubiquitylated by Cul3-KLHL40 in cell-free and over-expression assays in mammalian cells. Overexpression of Sar1a in muscle leads to endoplasmic reticulum (ER) membrane tubulation and thickening of the Z-lines similar to ones showing in KHLH40a deletion and NM patients. Markedly elevated levels of Sar1a and defects in collagen secretion are also recorded in patients with KLHL40 mutations. These observations suggest that selective control of COPII coat protein Sar1a levels (and thus the activity of the COPII coat, which mediates biosynthetic secretion from the ER), perturbs collagen secretion and ECM deposition. Overall this comprehensive work delineates the roles of Cul3-KLHL40a in the development of NM and specifically in regulating secretion by controlling the levels of one component of the COPII coat. The work is very interesting yet requires additional experimental clarifications and analysis.
Strength
This is a very interesting study showing global developmental and disease onset-related changes to the proteome focusing on changes derived from KLHL40a deletion. The work demonstrates a key role of ubiquitylation and selective protein degradation in the development and muscle disease onset. The global proteome view identified changes to energy production modes and defined direct regulation of Sar1a levels by Cul3-KHLH40a ubiquitylation which regulates ECM secretion, providing a mechanistic explanation for the development of NM in patients with KLHL40 mutations. Furthermore, the study highlights an interesting mechanism in which the levels of an individual component of the COPII coat are controlled by degradation to regulate biosynthetic secretion from the ER.
Weaknesses
There are weaknesses in the analysis that would markedly benefit from added clarifications. The differential outcome with the deletion of klhl40 a and b requires explanation. Morphological observations, which are key to understanding the overall phenotypes of KLHL40a deletion should be developed to provide a better definition of effects on organelle morphology and in particular ones involved in secretion. Some of the transcriptome-proteome data are left unexplored, in particular a view of the unfolded protein response (UPR) within the data, which will complement the documented defects in protein secretion and provide intrinsic controls to the work. The findings on Sar1a and the role of controlled degradation in regulating COPII activities are highly interesting yet a more complete analysis of COPII components is missing. Information on Sar1b, previously implicated in selective effects on secretion, Sec23-Sec24 and ratio (where levels are regulated by ubiquitylation and de ubiquitylation), and outer layer COPII proteins Sec13 and in particular Sec31, which is by itself a target for Cul3-KLHL12 regulation during development and modifies selective biosynthetic secretion, is lacking. Added analysis can provide new perspectives on the potential broader implications and significance of this study.
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Reviewer #2 (Public Review):
Mansur et al highlight interesting aspects of KLHL40-mediated proteostatic mechanisms in secretion and skeletal muscle development in zebrafish. They propose that KLHL40-mediated ubiquitylation of functional modules in the muscle proteome, particularly membrane traffic components, regulates protein abundance to control development. The authors present solid evidence for the role of KLHL40-mediated ubiquitylation and degradation of the cellular proteome but would benefit from further supporting evidence for their direct consequences on protein secretion.
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Reviewer #3 (Public Review):
The manuscript is addressing the hypothesis that KLHL40, of which mutations lead to a nemaline myopathy, leads to aberrant processing/turnover via the UPS of specific proteins. The aberrant turnover of these specific proteins then leads to the disease phenotype.
The manuscript creates two fish models knocking out orthologs of KHL40 in fish and finds that KHLH40a is necessary for maintaining fish size.
A multi-omic approach identifies potential candidates that are KHLH40 targets, specifically, Sar1a. Overexpression of Sar1a leads to some phenotypic changes ultrastructurally that resemble khl40a knockout. In vitro studies suggest some co-regulation of KHL40a with sar1a but lack the methodologic rigor at this point to be convincing. In addition, whether Sar1a dysregulation leads to more global issues seen in …
Reviewer #3 (Public Review):
The manuscript is addressing the hypothesis that KLHL40, of which mutations lead to a nemaline myopathy, leads to aberrant processing/turnover via the UPS of specific proteins. The aberrant turnover of these specific proteins then leads to the disease phenotype.
The manuscript creates two fish models knocking out orthologs of KHL40 in fish and finds that KHLH40a is necessary for maintaining fish size.
A multi-omic approach identifies potential candidates that are KHLH40 targets, specifically, Sar1a. Overexpression of Sar1a leads to some phenotypic changes ultrastructurally that resemble khl40a knockout. In vitro studies suggest some co-regulation of KHL40a with sar1a but lack the methodologic rigor at this point to be convincing. In addition, whether Sar1a dysregulation leads to more global issues seen in patients and fish remains to be established.
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