Biology subjects

Voutsinos, V.

Publications and source records attributed to Voutsinos, V..

7 recordsLinked to original sources

bioRxiv2026

PolyA/polyQ-mediated conformational rewiring regulates DNA engagement and drives aggregation in the neuronal transcription factor Ascl1

Ascl1 is a pioneer transcription factor that drives neuronal fate decisions, yet the structural basis of its activity remains elusive. Besides the basic helix-loop-helix (bHLH) domain which dimerizes with other transcription factors and binds DNA, Ascl1 contains long low-complexity intrinsically disordered regions (IDRs), including a polyA/polyQ tract of unknown function. Here we use single-molecule FRET to map the conformational ensemble of full-length Ascl1 across monomeric, heterodimeric, and DNA-bound states. Monomeric Ascl1 is largely disordered but displays multivalent coupling between the N-terminal IDR and the bHLH domain, with the polyA/polyQ tract opposing bHLH compaction. E12 binding folds the bHLH domain and remodels the N-IDR, increasing dynamics across the N-IDR while suppressing them locally within the polyA/polyQ tract. Deleting the tract weakens nonspecific DNA binding by the Ascl1/E12 heterodimer without disrupting heterodimerization or high-affinity E-box binding, abolishes aggregation in vitro, and increases Ascl1 abundance in HEK293T cells. Thus, the polyA/polyQ tract acts as a regulatory module that promotes nonspecific DNA engagement while imposing a cost in solubility and cellular abundance.

biophysics↗

bioRxiv2026

Comprehensive Variant Effect Map of Parkin-Mediated Mitophagy in Parkinson's Disease

The development of Parkinsons disease (PD) has a substantial genetic basis. Variants in the PRKN gene account for roughly half of autosomal-recessive PD cases, yet most variants remain classified as variants of uncertain significance. PRKN encodes the E3 ubiquitin-protein ligase, Parkin, that plays a key role in mitochondrial quality control by initiating mitophagy. In this work, we introduce a multiplexed mitophagy assay and measure the activity of more than 99% of all possible single-amino acid substitution and nonsense variants of Parkin. We also demonstrate that misfolded, rapidly degraded variants autonomously trigger Parkin-independent mitophagy. The obtained activity landscape closely reflects known structural and functional aspects of Parkin while revealing new insights into specific functional effects across the protein. This dataset near-perfectly distinguishes pathogenic from benign variants and surpasses both computational predictors and abundance-based metrics in pathogenicity assessment. Finally, the data pinpoint hypomorphic variants that could be amenable to rescue in future personalized therapeutic approaches for PD.

genetics↗

bioRxiv2026

BAG6 and RNF126 are broadly involved in protein quality control of non-native missense protein variants

Protein quality control (PQC) degradation is a vigorous and selective system that limits the accumulation of harmful, non-native and aggregation-prone proteins. Specific PQC pathways have been described for misfolded ER proteins, aberrant translational products and mislocalized proteins. However, many components involved in PQC of structurally unstable cytosolic proteins are unknown despite such proteins being a common consequence of pathogenic coding variants. Here, we present results from a genome-wide CRISPR knockout screen to identify components involved in PQC of the Parkin R42P variant. We find that HSP90 and STIP1 stabilize Parkin, while the BAG6 chaperone and E3 ubiquitin ligase RNF126 are critical for PQC degradation. Variant abundance by massively parallel sequencing (VAMP-seq) reveal >1000 Parkin variants, including several variants linked to autosomal recessive juvenile Parkinsonism, are BAG6 targets. Pathogenic missense variants in phenylalanine hydroxylase (PAH) and the tumor suppressor FLCN are also stabilized in BAG6 and RNF126 knockout cells. We propose that BAG6 and RNF126 are broadly involved in PQC and may represent targets for the development of therapeutics for protein misfolding diseases.

biochemistry↗

bioRxiv2025

Comprehensive degron mapping in human transcription factors

Gene expression is regulated by the targeted degradation of transcription factors through the ubiquitin-proteasome system. Transcription factors destined for degradation are recognized by E3 ubiquitin-protein ligases through short motifs termed degrons, embedded within the sequence. In this study, we systematically map degrons in all 1,626 human transcription factors. We find thousands of both known and previously unidentified degrons and characterize their sequence properties. Degrons placed within exposed and intrinsically disordered regions regulate the cellular abundance of the transcription factors, while the most common somatic mutations that are linked to skin cutaneous melanoma lead to unfolding and exposure of a buried degron in zinc fingers. We present examples of compartment specific degrons and demonstrate that variant effects in transcription factors correlate with degron potency. Finally, we show that while >60% of all predicted transcriptional activation domains overlap with strong degrons, acidic residues within the remaining transactivating regions counter the degron potency.

biochemistry↗

bioRxiv2025

A complete map of human cytosolic degrons and their relevance for disease

Degrons are short protein segments that target proteins for degradation via the ubiquitin-proteasome system and thus ensure timely removal of signaling proteins and clearance of misfolded proteins from the intracellular space. Here, we describe a systematic screen for degrons in the human cytosol. We determine degron potency of >200,000 different 30-residue tiles from more than 5,000 cytosolic human proteins with 99.7% coverage. In total, 19.1% of the tiles function as strong degrons, 30.4% as intermediate degrons, while 50.5% did not display degron properties. The vast majority of the degrons are dependent on the E1 ubiquitin-activating enzyme and the proteasome but independent of autophagy. The results reveal both known and novel degron motifs, both internal as well as at the C-terminus. Mapping the degrons onto protein structures, predicted by AlphaFold2, revealed that most of the degrons are located in buried regions, indicating that they only become active upon unfolding or misfolding. Training of a machine learning model allowed us to probe the degron properties further and predict the cellular abundance of missense variants that operate by forming degrons in exposed and disordered protein regions, thus providing a mechanism of pathogenicity for germline coding variants at such positions.

biochemistry↗

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↗