Biology subjects

Clausen, L.

Publications and source records attributed to Clausen, L..

4 recordsLinked to original sources

bioRxiv2023

Deep mutational scanning reveals a tight correlation between protein degradation and toxicity of thousands of non-native aspartoacylase protein variants

When the structural stability of a protein is compromised, the protein may form non-native interactions with other cell proteins and thus becomes a hazard to the cell. To mitigate this danger, destabilized proteins are targeted by the cellular protein quality control (PQC) network, which either corrects the folding defect or targets the protein for degradation. However, the details of how the protein folding and degradation systems collaborate to combat potentially toxic non-native proteins are unknown. To address this issue, we performed systematic studies on destabilized variants of the cytosolic aspartoacylase, ASPA, where loss-of-function variants are linked to Canavans disease, an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. Using Variant Abundance by Massively Parallel sequencing (VAMP-seq), we determined the abundance of 6152 out of the 6260 ([~]98%) possible single-site missense and nonsense ASPA variants in cultured human cells. The majority of the low abundance ASPA variants are degraded through the ubiquitin-proteasome system (UPS) and become toxic upon prolonged expression. Variant cellular abundance data correlates with predicted thermodynamic stability, evolutionary conservation, and separates most known disease-linked variants from benign variants. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in ASPA are located in buried regions, and reveals that the wild-type ASPA C-terminal region functions as a degron. Collectively, our data can be used to interpret Canavans disease variants and also offer mechanistic insight into how ASPA missense variants are targeted by the PQC system. These are essential steps towards future implementation of precision medicine for Canavans disease.

molecular biology↗

bioRxiv2023

A mutational atlas for Parkin proteostasis

The delicate balance of protein homeostasis can be disturbed by mutations that affect folding and stability of the encoded protein. More than half of disease-causing missense variants are thought to lead to protein degradation, but determining which and the molecular mechanisms involved remain enigmatic. To examine these issues, we selected the ubiquitin-protein ligase Parkin, where known missense variants result in an autosomal recessive, early onset Parkinsonism. We used the variant abundance by massively parallel sequencing (VAMP-seq) approach to quantify the abundance of Parkin missense variants in cultured human cells. The resulting mutational map, covering 9219 out of the 9300 possible single-site amino acid substitutions and nonsense Parkin variants, show that most low abundance variants are located within the structured domains of the protein, while the flexible linker regions are more tolerant. The vast majority of low abundance Parkin variants are degraded through the ubiquitin-proteasome system and are stabilized at a lowered temperature. The cellular abundance data correlate with thermodynamic stability, evolutionary conservation, and show that half of the known disease-linked variants are found at low abundance. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in Parkin largely overlap with regions that are buried, and highly sensitive to mutations. An exposed degron region proximal to the so-called "activation element" is enhanced by substitutions to hydrophobic residues and destroyed by introduction of hydrophilic residues. The data provide examples of how missense variants may cause degradation either via destabilization of the native protein, or by introducing local signals for degradation. Combined with the computational methods based on Parkin structure and conservation, our abundance map sheds light on the mechanisms that cause loss of function, and points to variants where function potentially can be restored.

biochemistry↗

bioRxiv2020

Evolutionarily conserved chaperone-mediated proteasomal degradation of a disease-linked aspartoacylase variant

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 examine the stability and degradation pathway of ASPA C152W. 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 prevents ASPA from reaching its stable native conformation, presumably by impacting on de novo folding. 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 human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. We propose that Hsp110 is a potential therapeutic target for misfolding ASPA variants that trigger Canavan disease due to excessive degradation.

biochemistry↗

bioRxiv2020

Folliculin variants linked to Birt-Hogg-Dube syndrome are targeted for proteasomal degradation

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dube (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present computational and experimental studies showing that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant ({Delta}E510) forms perinuclear protein aggregates. A yeast-based screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature to 29 {degrees}C led to a stabilization of two FLCN missense proteins, and for one variant (R362C), FLCN function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization of certain disease-linked variants may hold therapeutic potential.

molecular biology↗