Biology subjects

Voutsinos, V.

Publications and source records attributed to Voutsinos, V..

2 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↗