VHL inhibitor binding increases intracellular level of VHL

The vonHippel Lindau (VHL) protein is a tumour suppressor protein frequently mutated in the VHL disease, which functions as substrate recognition subunit of a Cul2 E3 ubiquitin ligase (CRL2VHL). CRL2VHL plays an important role in oxygen sensing, by binding and targeting Hypoxia Inducible Factor-alpha subunits (HIF-alpha) for ubiquitination and degradation. VHL is also commonly hijacked by heterobifunctional degrader molecules known as proteolysis-targeting chimeras (PROTACs). In previous work we reported the structure-based design and functional characterisation of VHL inhibitors (VH032 and VH298) that induce the HIF response in cells. Here, we use unbiased quantitative mass spectrometry to identify the proteomic changes elicited by the VHL inhibitor and compare this to hypoxia or broad-spectrum prolyl-hydroxylase domain (PHD) enzyme inhibitor IOX2. Our results demonstrate the VHL inhibitor selectively activates the HIF response that vastly overlaps with hypoxia- and IOX2-induced proteomic changes. Interestingly, VHL inhibitors were found to selectively upregulate a single protein, which is VHL itself. Our analysis revealed that this occurs via protein stabilisation of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn to reduced levels of HIF-1 protein. Our results demonstrate the high specificity of VHL inhibitors and suggest that use of these inhibitors would not produce overtly side effects due to prolonged HIF stabilisation. They also exemplify the concept that small-molecule binding induced protein stabilisation can increase protein levels inside cells.

Effect of Pre-Fermentative Maceration and Fining Agents on Protein Stability, Macromolecular, and Phenolic Composition of Albariño White Wines: Comparative Efficiency of Chitosan, k-Carrageenan and Bentonite as Heat Stabilisers

In this work, the effect of pre-fermentative skin maceration (PFSM) on the chemical composition of the macromolecular fraction, polysaccharides and proteins, phenolic compounds, chromatic characteristics, and protein stability of Albariño monovarietal white wines was studied. PFSM increased the extraction of phenolic compounds and polysaccharides and reduced the extraction of pathogenesis-related proteins (PRPs). PFSM wine showed significantly higher protein instability. Sodium and calcium bentonites were used for protein stabilisation of wines obtained with PFSM (+PFSM) and without PFSM (−PFSM), and their efficiencies compared to fungal chitosan (FCH) and k-carrageenan. k-Carrageenan reduced the content of PRPs and the protein instability in both wines, and it was more efficient than sodium and calcium bentonites. FCH was unable to heat stabilise both wines, and PRPs levels remained unaltered. On the other hand, FCH decreased the levels of wine polysaccharides by 60%. Sodium and calcium bentonite also decreased the levels of wine polysaccharides although to a lower extent (16% to 59%). k-Carrageenan did not affect the wine polysaccharide levels. Overall, k-carrageenan is suitable for white wine protein stabilisation, having a more desirable impact on the wine macromolecular fraction than the other fining agents, reducing the levels of the wine PRPs without impacting polysaccharide composition.

Influence of the mannoproteins of different strains of Starmerella bacillaris used in single and sequential fermentations on foamability, tartaric and protein stabilities of wines

Aim: In this work, seven strains of Starmerella bacillaris were analysed for their ability to release polysaccharides during alcoholic fermentation (AF), both in single-strain and in sequential AF together with Saccharomyces cerevisiae.Methods and Results: A synthetic polysaccharide-free must was used to characterise the mannoproteins (MPs) released. The MPs were quantified, characterised in terms of carbohydrate composition, and tested to assess their ability to reduce protein and tartrate instabilities and their ability to affect the foaming properties of wine.Conclusions: All the tested strains in sequential AF increased the total MPs production. Moreover, the strains affected the MPs properties in different ways regarding tartaric and protein stabilities. The MPs released in sequential AF by some S. bacillaris strains showed a significant effect on protein stabilisation and tartaric stability. An effect on the foamability was found for MPs obtained in single-strain AFs of S. bacillaris.Significance and impact of the study: An improvement in wine stability can be achieved using the sequential AF.

Single molecule protein stabilisation translates to macromolecular mechanics of a protein network

Folded globular proteins are attractive building blocks for biomaterials as their robust structures carry out diverse biological functions. These biomaterials are ideal to study the translation of molecular properties to multi-molecular assemblies.

Protein conformation in pure and hydrated deep eutectic solvents

Deep eutectic solvents as media for protein stabilisation: conformation in the absence and presence of water.

Butane-1,2,3,4-tetraol-based amphiphilic stereoisomers for membrane protein study: importance of chirality in the linker region

Chirality variation in amphiphile architecture resulted in a significant difference in detergent efficacy for membrane protein stabilisation.