The Myoblast C2C12 Transfected with Mutant Valosin-Containing Protein Exhibits Delayed Stress Granule Resolution on Oxidative Stress

Abstract

Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shockdactivated stress responses evident by T-intracellular antigen-1epositive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1epositive granules than wildtype in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCP R155H -knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2a unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration. (Am J Pathol 2016 http://dx

Active protein neddylation or ubiquitylation is dispensable for stress granule dynamics

“…The idea that SGs themselves are initially cytoprotective but only become cytotoxic upon recruitment of misfolded proteins during chronic UPS dysfunction is intriguing. The observations that the autophagy system, the proteasome, and the unfoldase p97/VCP are needed to clear these aberrant SGs are consistent with this notion 18, 19, 23, 48 . Future studies are needed to examine of the role of ubiquitin and ubiquitin pathway enzymes in regulating the dynamics and biophysical properties of these aberrant SGs.…”

Coupling of translation quality control and mRNA targeting to stress granules

“…Using single mRNA imaging in live cells 15,16 , we took an unbiased approach to determine if defects in the UPS perturb mRNA translation and partitioning into SGs during acute stress. We observe ribosomes stall on mRNAs during arsenite stress, and the release of transcripts from stalled ribosomes for their partitioning into SGs requires the activities of valosin-containing protein (VCP) and the proteasome, which is in contrast to previous work showing VCP primarily affected SG disassembly 11,13,14,17 .…”

“…Using single mRNA imaging in live cells 15,16 , we took an unbiased approach to determine if defects in the UPS perturb mRNA translation and partitioning into SGs during acute stress. We observe ribosomes stall on mRNAs during arsenite stress, and the release of transcripts from stalled ribosomes for their partitioning into SGs requires the activities of valosin-containing protein (VCP) and the proteasome, which is in contrast to previous work showing VCP primarily affected SG disassembly 11,13,14,17 .Moreover, members of a specialized complex in the UPS that targets aberrant nascent proteins for decay upon ribosome stalling, referred to as ribosome-associated quality control complex (RQC) 18 , are also required for mRNA release from ribosomes and partitioning into SGs. VCP alleles that increase segregase activity and cause neurodegeneration and inclusion body myopathies 5,6,19,20 increase mRNA recruitment to SGs, suggesting aberrant mRNA localization to SGs in disease contexts.…”

“…Intriguingly, VCP and the proteasome directly interact during arsenite stress 39 , suggesting the possibility that the functions and substrates of RQC factors are dynamically regulated in response to specific stress conditions. This study suggests VCP affects mRNAs in SGs by acting on ribosome-mRNA complexes, in addition to mediating SG assembly and disassembly [11][12][13][14]17 . One implication of this study is that SGs could serve as storage depots for mRNAs that are modified in response to stress and undergo processing by the saRQC.…”

Coupling of translation quality control and mRNA targeting to stress granules

“…However, inhibition of VCP or proteasome activity perturbs SG formation in response to acute stress ( Seguin et al, 2014 ). In addition, VCP inhibition can cause constitutive SGs and limits SG disassembly ( Buchan et al, 2013 ; Rodriguez-Ortiz et al, 2016 ; Turakhiya et al, 2018 ; Wang et al, 2019 ). Thus, SG disassembly requires the activities of VCP and the proteasome, potentially acting either by removing misfolded or aberrant proteins from SGs for proteasomal decay ( Turakhiya et al, 2018 ) or by limiting granulophagy, the degradation of SGs via autophagy ( Buchan et al, 2013 ).…”