Proteasome Inhibition Reduces NBR1 Protein Levels in mATG8-Deficient Cells Independently of Lysosomal Degradation and RAB27A

Abstract

Autophagy is a lysosome-dependent degradation process that involves autophagosome formation, typically mediated by mammalian ATG8 proteins (mATG8s). Autophagosomes can still form in their absence, suggesting alternative mechanisms. NBR1, a selective autophagy receptor, has been shown to compensate for autophagy defects. Here, we examined NBR1 regulation under proteotoxic stress in mATG8s-deficient HeLa cells. NBR1 levels were elevated in mATG8s knockout (KO) cells under basal conditions but decreased significantly after treatment with the proteasome inhibitor MG132. This reduction was not prevented by lysosomal inhibition with BafA1, indicating a non-lysosomal mechanism. Silencing of RAB27A reduced basal NBR1 levels, and the effects of MG132 were no longer observed, likely due to already diminished NBR1. Remaining NBR1 localized to puncta positive for ubiquitin and the ESCRT-0 component HRS, suggesting involvement of a ubiquitin-dependent endosomal pathway. Overall, our results suggest that under proteotoxic stress and impaired autophagy, cells activae alternative routes, potentially involving unconventional secretion, to regulate NBR1 levels. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/664987v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@10568bcorg.highwire.dtl.DTLVardef@1f3d53org.highwire.dtl.DTLVardef@f33508org.highwire.dtl.DTLVardef@11ec28e_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender https://BioRender.com/geoh3sz In the absence of mATG8s, NBR1 may be recruited to multivesicular bodies (MVBs) via HRS, for later degradation in lysosomes (left panel). Under proteasomal inhibition (right panel), NBR1 associated with MVBs (with ubiquitinated cargos) is downregulated, independent of lysosomes. Possibly, this downregulation is due to a secretion process, through a RAB27A-independent mechanism.