Proteome-wide identification of HSP70/HSC70 chaperone clients in human cells

Abstract

The 70 kDa heat shock protein (HSP70) family of chaperones are the front line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here, we demonstrate quantitative identification of HSP70 and 71 kDa heat shock cognate (HSC70) clients using a ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely nonoverlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides, but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase 1 (SOD1) mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitinactivated interaction trap (UBAIT) fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.

(Dis)Solving the problem of aberrant protein states

“…In addition to interacting with co-chaperones, Hsp70 and Hsc70 exhibited a preference for newly translated proteins and ‘orphan’ proteins, which are monomeric proteins that require binding to other proteins for stability and function. Such a preference confirmed that the Hsp70 family plays an essential role in nascent polypeptide folding and protein complex formation ( Ryu et al, 2020 ). Intriguingly, expression of a misfolded protein such as ALS-linked SOD1 shifted the interactome for Hsp70 and Hsc70, with Hsc70 showing more significant engagement with disordered proteins upon SOD1 expression ( Ryu et al, 2020 ).…”

“…Such a preference confirmed that the Hsp70 family plays an essential role in nascent polypeptide folding and protein complex formation ( Ryu et al, 2020 ). Intriguingly, expression of a misfolded protein such as ALS-linked SOD1 shifted the interactome for Hsp70 and Hsc70, with Hsc70 showing more significant engagement with disordered proteins upon SOD1 expression ( Ryu et al, 2020 ). Thus, Hsp70 and Hsc70 are general folding chaperones and also function as part of a disaggregase system to reverse protein aggregation.…”

“…To achieve a global understanding of the Hsp70 interactome, Ryu and colleagues used proximity-based approaches to catalog the interaction partners for Hsp70 and Hsc70 (also known as HSPA8), a constitutively expressed Hsp70 family member, in human cells ( Ryu et al, 2020 ). In addition to interacting with co-chaperones, Hsp70 and Hsc70 exhibited a preference for newly translated proteins and ‘orphan’ proteins, which are monomeric proteins that require binding to other proteins for stability and function.…”

Co-Chaperones in Targeting and Delivery of Misfolded Proteins to the 26S Proteasome

“…In general HSP70-type chaperones have a similar structure and allosteric cycle, and thus show wide functional redundancy [87]. In contrast, a recent proteome-wide study by Ryu, et al shows that there is essentially no substrate specificity overlap between the HSP70 and HSC70 families, except that they share newly synthesized proteins as common substrates [88].…”

Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome

“…HSP70s (HSPAs) are among the most ubiquitous chaperones, and they have been shown to play a key role in maintaining protein homeostasis in virtually all domains of life (Gupta and Singh 1994; Hunt and Morimoto 1985; Lindquist and Craig 1988). Upon cross-referencing the NIA and aggregating fractions against a recently generated client database of HSPA8 (HSC70; constitutively active form of HSP70) and HSPA1A (constitutively active and stress-inducible HSP70) (Ryu et al 2020), we find that HSPA8 and HSPA1A clients are enriched among aggregating proteins (Figure 5C). We also mined the BioGRID human protein-protein interaction database using the complete KEGG dataset of (co)chaperones (168 entries).…”