Background: Yeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts. Results: We characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L^-1 CuSO₄ in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts. Conclusions: Following evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L^-1 CuSO₄ and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper-binding proteome. However, copper elicits different physiological and molecular reactions in yeasts with different backgrounds.

Intrinsically disordered proteins (IDPs) are unable to adopt a unique 3D structure under physiological conditions and thus exist as highly dynamic conformational ensembles. IDPs are ubiquitous and widely spread in the protein realm. In the last decade, compelling experimental evidence has been gathered, pointing to the ability of IDPs and intrinsically disordered regions (IDRs) to undergo liquid–liquid phase separation (LLPS), a phenomenon driving the formation of membrane-less organelles (MLOs). These biological condensates play a critical role in the spatio-temporal organization of the cell, where they exert a multitude of key biological functions, ranging from transcriptional regulation and silencing to control of signal transduction networks. After introducing IDPs and LLPS, we herein survey available data on LLPS by IDPs/IDRs of viral origin and discuss their functional implications. We distinguish LLPS associated with viral replication and trafficking of viral components, from the LLPS-mediated interference of viruses with host cell functions. We discuss emerging evidence on the ability of plant virus proteins to interfere with the regulation of MLOs of the host and propose that bacteriophages can interfere with bacterial LLPS, as well. We conclude by discussing how LLPS could be targeted to treat phase separation-associated diseases, including viral infections.

The secondary structure of lipase 1 from Candida rugosa, a model system for large monomeric enzymes, has been studied by FTIR (Fourier-transform infrared) spectroscopy in water and 2H2O. The secondary structure content, determined by the analysis of the amide I band absorption through second derivative and curve fitting procedures, is in agreement with that estimated by X-ray data and predicts, in addition, the existence of two classes of α-helices. We have also investigated the enzyme stability and aggregation at high temperature by following the protein unfolding. The thermal stability determined by FTIR is in excellent agreement with the temperature dependence of the lipase activity. Furthermore, new insights on the glycosylation of the recombinant protein produced in Pichia pastoris and on its heterogeneity related to different fermentation batches were obtained by the analysis of the IR absorption in the 1200−900 cm−1 carbohydrate region. A drastic reduction of the intensity of this band was found after enzymic deglycosylation of the protein. To confirm that the FTIR absorption in the 1200–900 cm−1 region depends on the carbohydrate content and glycoform distribution, we performed an MS analysis of the protein sugar moieties. Glycosidic structures of the high mannose type were found, with mannoses ranging from 8 to 25 residues.

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate‐binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain‐length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.