UBB Knockout Cell Line (HEK293)

UBB, a ubiquitin variant protein resulting from a frameshift in the ubiquitin-B gene, is a pathological hallmark of Alzheimer disease (AD). At the cellular level, UBB disrupts the ubiquitin-proteasome system while inducing autophagy. Notably, UBB itself is secreted via autophagosome-like vesicles. Here, we demonstrate that UBB can be removed from the cell by degradative and secretory autophagy. Sequestosome 1 (SQSTM1)/p62 functions as a pivotal ubiquitin receptor for UBB, recognizing its ubiquitin domain and facilitating loading into autophagosomes. Oligomerization of SQSTM1/p62 was critical to isolate UBB in bodies preventing its aggregation. Intriguingly, both gain- and loss-of-function SQSTM1/p62 suppressed UBB secretion, causing intracellular retention: SQSTM1/p62 knockout led to UBB accumulation in insoluble aggregates, while its overexpression promoted the formation of p62-UBB bodies. We further identified distinct roles for SNARE-mediated membrane fusion in secretory autophagy of UBB. Specifically, the R-SNARE SEC22B and the Q-SNAREs Syntaxin-4 and SNAP23 participated in UBB exocytosis. Disruption of SEC22B impaired the fusion of UBB-containing autophagosomes with the plasma membrane, reducing UBB secretion without affecting its intracellular turnover. Inhibition of lysosomes partially stabilized UBB indicating that degradation and secretion are complementary processes that determine the fate of UBB. This study elucidates the dual roles of autophagy in managing neurotoxic proteins, highlighting SQSTM1/p62 as a key mediator of UBB trafficking and secretion. Although ubiquitin typically acts as a degradation signal, our findings reveal a rare instance of a ubiquitin-related protein driving secretory autophagy. These findings advance our understanding of cellular mechanisms underlying the clearance of misfolded proteins in neurodegenerative diseases.