Protein sidekick exhibits dual roles in stress granule assembly and disassembly

ASPL regulates the assembly and disassembly of SGs in cells. (A) HeLa cells were transfected with a plasmid expressing GFP-ASPL and stained. Images demonstrate colocalization of GFP-ASPL with SG markers. (B) Percentage of HeLa cells containing SG-like constructs in cells expressing the indicated constructs. n > 100 cells from three biological replicates. ***P ≤ 0.001 by analysis of variance (ANOVA) with Tukey test. (C and D) U2OS cells transfected with non-targeted small interfering RNA (siRNA) (siCtrl) or siASPL were subjected to 60 min HS treatment at 43°C or 500 μM SA treatment for 60 min and then stained for SG markers. (C) Merged images. (D) Ratio of SG area to cytoplasmic area for each antibody. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001 according to Student’s t test. Credit: Scientific advances (2025). DOI: 10.1126/sciadv.ady3735

Stress granules are droplet-like protein centers that temporarily protect fragile RNA from cellular stresses such as toxins. VCP is a protein essential for breaking down stress granules and has been linked to neurodegenerative diseases. However, VCP has a protein partner, ASPL, whose role has been unclear until now.

Scientists at St. Jude Children’s Research Hospital discovered that ASPL regulates stress granule disassembly by facilitating the phosphorylation of VCP. They also found that ASPL facilitates stress granule assembly independently of VCP by stabilizing interactions between stress granule core proteins.

The results, published in Scientific advancesprovide key insights into the link between stress granules and neurodegenerative diseases.

Stress granules are carefully regulated, forming only during stress, such as heat or infection, and disassembling once the stress has resolved. Mutations in proteins such as VCP can derail this cycle, leading to abnormal protein accumulation, causing a condition called multisystem proteinopathy.

“VCP mutations are found in patients with multisystem proteinopathy, which includes amyotrophic lateral sclerosis, frontotemporal dementia, and Paget’s disease of bone”said corresponding author Mondira Kundu, MD, Ph.D., Department of Cellular and Molecular Biology at St. Jude. “Although VCP has multiple functions, its ability to disassemble RNA-protein assemblies, such as stress granules, is impaired by pathogenic mutations.”

ASPL, VCP and ULK work together to break down stress granules

After discovering that VCP and ASPL interact with ULK, another protein involved in regulating stress granule disassembly, Kundu and his team decided to explore this relationship further.

“ULK regulates stress granule dynamics, so we wondered whether ASPL was also involved through its interaction with ULK or VCP”Kundu said.

The team discovered that ASPL was necessary for ULK to perform its function, namely phosphorylating VCP (adding a phosphate group). Phosphorylation triggers VCP to remove the key stress granule protein G3BP from stress granules, causing them to rupture. Without VCP phosphorylation, stress granules persist.

These results are consistent with what occurs in multisystem proteinopathy.

“Some pathogenic VCP mutants do not interact as well with ASPL and ULK and also exhibit defects in stress granule disassembly”explained Kundu. “Restoring VCP phosphorylation restores disassembly, suggesting that these mutants decrease VCP binding to ASPL and subsequent phosphorylation by ULK.”

ASPL goes solo to help assemble stress granules

Surprisingly, the researchers also observed that overexpression of ASPL strongly increased the formation of stress granules. Deletion of ASPL resulted in smaller, slower-forming stress granules. This suggests that ASPL plays a role in stress granule assembly. But this function of ASPL did not involve VCP, so the researchers sought to understand how ASPL could promote stress granule formation on its own, independent of VCP.

“We created cell lines that express a version of ASPL that cannot bind VCP, and found that these cells had no problems with granule assembly but did a poor job with disassembly”Kundu said. “This showed us that ASPL promoted stress granule assembly and that the interaction of ASPL with VCP was important for efficient disassembly.”

The researchers discovered that this mechanism was linked to G3BP.

“Removal of ASPL reduced interactions between stress granule components, thereby reducing the likelihood of stress granule formation”explained Kundu. “Fluorescence recovery experiments suggest that ASPL alters the interactions of G3BP with other proteins, making the network less stable without ASPL.”

These findings add nuance to scientists’ understanding of stress granule regulation, expanding on the checks and balances that govern the process and highlighting potential pathways to disease.

“A key question is whether disruption of ASPL-VCP interaction, particularly via ASPL, can mimic multisystem proteinopathy”Kundu said. “This is the biggest missing link right now, and it’s a link we’re exploring.”