Mapping the degradation pathway of a disease-linked aspartoacylase variant

Abstract

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.

Prediction of quality-control degradation signals in yeast proteins

“…Our simple, but accurate prediction algorithm to identify degrons from sequence may help understand the complex relationship between sequence variation and abundance. We and others have previously shown that many disease-causing missense variants are thermodynamically destabilized (Abildgaard et al, 2019; Casadio et al, 2011; Gersing et al, 2021; Kampmeyer et al, 2022; Matreyek et al, 2018; Pey et al, 2003; Stein et al, 2019; Yue et al, 2005). The results presented here provide a key missing part of the puzzle, namely a quantitative model for which exposed sequences are PQC targets, and we hope our work may help develop quantitative models that combine protein stability and recognition via the PQC.…”

HSP70-binding motifs function as protein quality control degrons

“…For Ubr1, a direct interaction with Hsp70 has been observed (Summers et al, 2013), indicating that some PQC E3s may delegate substrate recognition to the chaperones. Similarly, the human CHIP E3 directly interacts with Hsp70 and Hsp90 chaperones to ubiquitylate bound protein substrates (Höhfeld and Jentsch, 1997), while the chaperone NEFs BAG1 and Hsp110 mediate the release of bound substrates at the 26S proteasome (Abildgaard et al, 2020; Gersing et al, 2021; Höhfeld and Jentsch, 1997; Kandasamy and Andréasson, 2018; Kriegenburg et al, 2014). The broad substrate specificity of Hsp70 and its involvement in protein degradation suggest that it plays a fundamental and direct role in targeting misfolded proteins to the UPS via binding their short PQC degrons.…”

Increasing Aspartoacylase in the Central Amygdala: The Common Mechanism of Gastroprotective Effects of Monoamine-Based Antidepressants Against Stress

“…ASPA is predominantly expressed in mature OLs where it functions as a homodimer cleave NAA, releasing acetate that can be used for myelin lipid synthesis (Baslow, et al, 1999;Moore, et al, 2003;Jolly, et al, 2016). The lack of ASPA activity results in the accumulation of NAA and is tightly linked to the Canavan disease, which is a fatal neurodegenerative disorder genetically linked to polymorphisms in the ASPA gene such as substitution C152W (Gersing, et al, 2021;Madhavarao, et al, 2005). OLs' list of known functions extends beyond axon myelination and now includes the direct modulation of neuronal function (de Hoz and Simons 2015; Jha and Morrison 2020).…”