Protein homeostasis (proteostasis) is essential for the cell and is maintained by a highly conserved protein quality control (PQC) system, which triages newly synthesized, mislocalized and misfolded proteins. The ubiquitin-proteasome system (UPS), molecular chaperones, and co-chaperones are vital PQC elements that work together to facilitate degradation of misfolded and toxic protein species through the 26S proteasome. However, the underlying mechanisms are complex and remain partly unclear. Here, we provide an overview of the current knowledge on the co-chaperones that directly take part in targeting and delivery of PQC substrates for degradation. While J-domain proteins (JDPs) target substrates for the heat shock protein 70 (HSP70) chaperones, nucleotide-exchange factors (NEFs) deliver HSP70-bound substrates to the proteasome. So far, three NEFs have been established in proteasomal delivery: HSP110 and the ubiquitin-like (UBL) domain proteins BAG-1 and BAG-6, the latter acting as a chaperone itself and carrying its substrates directly to the proteasome. A better understanding of the individual delivery pathways will improve our ability to regulate the triage, and thus regulate the fate of aberrant proteins involved in cell stress and disease, examples of which are given throughout the review.

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Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.

Since life is completely dependent on water, it is difficult to gauge the impact of solvent change. To analyze the role of water as a solvent in biology, we replaced water with heavy water (D2O), and investigated the biological effects by a wide range of techniques, using the fission yeast Schizosaccharomyces pombe as model organism. We show that high concentrations of D2O lead to altered glucose metabolism, growth retardation, and inhibition of meiosis. However, mitosis and overall cell viability were only slightly affected. After prolonged incubation in D2O, cells displayed gross morphological changes, thickened cell walls as well as aberrant septa and cytoskeletal organization. RNA sequencing revealed that D2O causes a strong downregulation of most tRNAs and triggers activation of the general stress response pathway. Genetic screens identified several D2O sensitive mutants, while mutants compromised in the cell integrity pathway, including the protein kinase genes pmk1, mkh1, pek1 and pck2, that control cell wall biogenesis, were more tolerant to D2O. We speculate that D2O affects the phospholipid membrane or cell wall glycans causing an activation of the cell integrity pathway. In conclusion, the effects of solvent replacement are pleiotropic but the D2O-triggered activation of the cell integrity pathway and subsequent increased deposition of cell wall material and septation problems appear most critical for the cell growth defects..

Diabetes is a complex disease spanning from the heterogeneous etiology of type 1 and type 2 diabetes to monogenic diabetes. A common monogenic form of diabetes is glucokinase (GCK) maturity-onset diabetes of the young (GCK-MODY), which is caused by heterozygous inactivating variants in the gene encoding GCK. GCK is known as the pancreatic glucose sensor, as it regulates insulin secretion to maintain appropriate blood glucose levels. Accordingly, variants that alter GCK activity can cause hypo- and hyperglycemia, associated with hyperinsulinemic hypoglycemia (HH) and GCK-MODY, respectively, affecting up to 10 million people worldwide. Patients with GCK-MODY, in contrast to other people with diabetes, often do not require treatment but are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. To address this, we generated a comprehensive map of GCK variant activity in yeast. The activity map includes 97% of the possible missense and nonsense variants and correlates with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and evolutionary conservation analysis. Activity scores include both hyper- and hypoactive variants. We found that some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. As expected, hypoactive variants were concentrated at buried positions, near the active site, and at a partially surface-exposed region involved in GCK conformational dynamics. In conclusion, we provide a comprehensive assessment of GCK variant activity to facilitate variant interpretation and diagnosis, and we expand the mechanistic understanding of hyperactive variants to support development and refinement of drugs targeting GCK.