Hsp104 facilitates the endoplasmic‐reticulum–associated degradation of disease‐associated and aggregation‐prone substrates

Abstract

Misfolded proteins in the endoplasmic reticulum (ER) are selected for ER-associated degradation (ERAD). More than 60 disease-associated proteins are substrates for the ERAD pathway due to the presence of missense or nonsense mutations. In yeast, the Hsp104 molecular chaperone disaggregates detergent-insoluble ERAD substrates, but the spectrum of diseaseassociated ERAD substrates that may be aggregation prone is unknown. To determine if Hsp104 recognizes aggregation-prone ERAD substrates associated with human diseases, we developed yeast expression systems for a hydrophobic lipid-binding protein, apolipoprotein B (ApoB), along with a chimeric protein harboring a nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator (CFTR) into which disease-causing mutations were introduced. We discovered that Hsp104 facilitates the degradation of ER-associated ApoB as well as a truncated CFTR chimera in which a premature stop codon corresponds to a disease-causing mutation. Chimeras containing a wild-type version of the CFTR domain or a different mutation were stable and thus Hsp104 independent. We also discovered that the detergent solubility of the unstable chimera was lower than the stable chimeras, and Hsp104 helped retrotranslocate the unstable chimera from the ER, consistent with disaggregase activity. To determine why the truncated chimera was unstable, we next performed molecular dynamics simulations and noted significant unraveling of the CFTR nucleotide-binding domain. Because human cells lack Hsp104, Additional Supporting Information may be found in the online version of this article.Lynley M. Doonan, Christopher J. Guerriero, and G. Michael Preston contributed equally to this work.Significance statement: Misfolded proteins can aggregate, which impairs cellular homeostasis and can give rise to disease. Although yeast express an enzyme that dissolves aggregates, humans lack this protein. We show that human aggregation-prone proteins associated with the endoplasmic reticulum are substrates for the yeast disaggregase. These data suggest that a human analog of the yeast disaggregase exists or that another mechanism removes aggregates associated with the endoplasmic reticulum.these data indicate that an alternate disaggregase or mechanism facilitates the removal of aggregation-prone, disease-causing ERAD substrates in their native environments.

Protein quality control in the secretory pathway

“…Chaperone holdases are especially critical for retrotranslocated membrane proteins since they contain hydrophobic transmembrane domains and are consequently aggregation prone. Since yeast lack Bag6, Hsp104 facilitates the solubilization and retrotranslocation of aggregationprone ERAD substrates (Preston et al, 2018;Doonan et al, 2019).…”

Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome

“…There was an increase in the abundance of Ssa1 and Ssa2, two Hsp70 family members in both mutants: ~1.5x increase in pup2ts and a ~2x increase in rpn5-ts. Ssa1/2 have been shown to play a role in delivering misfolded protein to the proteasome during ER-associated degradation (ERAD) (85) Additionally, Hsp104, another chaperone linked to ERAD, was seen to increase in protein abundance 1.7x and 1.4x in pup2-ts and rpn5-ts respectively (86). The upregulation of cellular chaperones could be a compensation mechanism for defects in 26S proteasome function and could help explain why these cells are able to survive at permissive temperature despite altered proteasomal activity.…”

DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy

“…CFTR is a 1480 amino acid anion channel containing two membrane-spanning domains (MSD1 and MSD2), two nucleotide-binding domains (NBD1 and NBD2), and a disordered regulatory R-domain. CFTR folding and misfolding is the topic of intense study, which has identified disease-causing mutations that cause arrested intermediates to accumulate in globally misfolded and aggregated states (Doonan et al, 2019). The F508del mutation is located in NBD1, so F508del-NBD1 is unstable and fails to correctly assemble with NBD2 and the cytoplasmic loops (CL) that lie in between CFTR's 12 transmembrane spans.…”