Objective: To characterize the polyglycine (polyG) interaction network and identify its degradation mechanisms.

Background: polyG translated from GGC repeat expansions in the 5′UTR of NOTCH2NLC is a key pathogenic component of neuronal intranuclear inclusion disease (NIID). polyG forms intranuclear aggregates and recruits p62 and ubiquitin, suggesting disruption of protein quality control systems.

Method: polyG constructs with different GGC repeat lengths were overexpressed in Neuro2A cells. Co-immunoprecipitation (Co-IP) combined with mass spectrometry identified interacting proteins. Gene Ontology enrichment and protein-protein interaction (PPI) network analyses were performed. Key interactors were validated by Western blotting. Proteasome inhibitor MG132 and autophagy inhibitor chloroquine (CQ) were used to assess degradation pathways. siRNA knockdown and cycloheximide (CHX) chase assays evaluated functional roles of key regulators. Immunofluorescence was used to visualize aggregate formation and chaperone colocalization.

Results: PPI analysis of polyG interactome identified a central chaperone/co-chaperone module. Co-IP confirmed enhanced interactions of polyG with the BAG6 complex (BAG6, GET4, UBL4A), chaperone-assisted selective autophagy (CASA) complex (HSC70, HSPB8, p62, BAG3, STUB1) and DNAJB1. MG132 increased polyG accumulation in soluble and insoluble fractions, accompanied by increased polyG-associated ubiquitin and strengthened binding of BAG3, BAG6, HSC70, DNAJB1, and HSPB8. In contrast, CQ caused only a modest increase in polyG levels but strengthened polyG-BAG3 interaction. Knockdown of DNAJB1, HSC70, or BAG6 significantly elevated polyG abundance, and CHX chase assays demonstrated reduced polyG degradation after BAG6 depletion, whereas BAG3 knockdown had only minor effects. DNAJB1, HSC70, BAG6, and BAG3 formed a peripheral layer surrounding polyG aggregates.

Conclusion: PolyG is primarily degraded through the proteasome. After recognition by the HSP-DNAJ chaperone system, the BAG6 complex likely facilitates transfer of polyG to the proteasome for degradation. When proteasomal capacity is limited, CASA-associated factors may redirect polyG to the autophagy-lysosome pathway. The formation of a chaperone-enriched shell around polyG aggregates reflects a coordinated cellular response to misfolded proteins. These findings provide mechanistic insight into polyG induced proteostasis disruption and the pathogenesis of NIID.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/degradation-mechanisms-of-polyg/