Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1

Cells that experience high levels of oxidative stress respond with the induction of antioxidant proteins through the activation of the transcription factor Nrf2. Nrf2 is negatively regulated by Keap1 which binds to Nrf2 to facilitate its ubiquitination and ensuing proteasomal degradation under basal conditions. Here, we study protein folding and misfolding in Nrf2 and Keap1 in yeast, mammalian cells, and purified proteins under oxidative stress conditions. Both Nrf2 and Keap1 are susceptible to protein misfolding and inclusion formation upon oxidative stress. We propose that the intrinsically disordered regions within Nrf2 and the high cysteine content of Keap1 contribute to their oxidation and the ensuing misfolding. Our work reveals previously unexplored aspects of Nrf2 and Keap1 regulation and dysregulation by oxidation-induced protein misfolding.

Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles

Polyethylene glycol (PEG) surface conjugations are widely employed to render passivating properties to nanoparticles in biological applications. The benefits of surface passivation by PEG are reduced protein adsorption, diminished non-specific interactions, and improvement in pharmacokinetics. However, the limitations of PEG passivation remain an active area of research, and recent examples from the literature demonstrate how PEG passivation can fail. Here, we study the adsorption amount of biomolecules to PEGylated gold nanoparticles (AuNPs), focusing on how different protein properties influence binding. The AuNPs are PEGylated with three different sizes of conjugated PEG chains, and we examine interactions with proteins of different sizes, charges, and surface cysteine content. The experiments are carried out in vitro at physiologically relevant timescales to obtain the adsorption amounts and rates of each biomolecule on AuNP-PEGs of varying compositions. Our findings are relevant in understanding how protein size and the surface cysteine content affect binding, and our work reveals that cysteine residues can dramatically increase adsorption rates on PEGylated AuNPs. Moreover, shorter chain PEG molecules passivate the AuNP surface more effectively against all protein types.

Cysteine Proteome Reveals Response to Endogenous Oxidative Stress in Bacillus cereus

At the end of exponential growth, aerobic bacteria have to cope with the accumulation of endogenous reactive oxygen species (ROS). One of the main targets of these ROS is cysteine residues in proteins. This study uses liquid chromatography coupled to high-resolution tandem mass spectrometry to detect significant changes in protein abundance and thiol status for cysteine-containing proteins from Bacillus cereus during aerobic exponential growth. The proteomic profiles of cultures at early-, middle-, and late-exponential growth phases reveals that (i) enrichment in proteins dedicated to fighting ROS as growth progressed, (ii) a decrease in both overall proteome cysteine content and thiol proteome redox status, and (iii) changes to the reduced thiol status of some key proteins, such as the transition state transcriptional regulator AbrB. Taken together, our data indicate that growth under oxic conditions requires increased allocation of protein resources to attenuate the negative effects of ROS. Our data also provide a strong basis to understand the response mechanisms used by B. cereus to deal with endogenous oxidative stress.

Clinical relevance of genotype–phenotype correlations beyond vascular events in a cohort study of 1500 Marfan syndrome patients with FBN1 pathogenic variants

Purpose Marfan syndrome (MFS) is a connective tissue disorder in which several systems are affected with great phenotypic variability. Although known to be associated with pathogenic variants in the FBN1 gene, few genotype–phenotype correlations have been found in proband studies only. Methods In 1,575 consecutive MFS probands and relatives from the most comprehensive database worldwide, we established survival curves and sought genotype–phenotype correlations. Results A risk chart could be established with clinical and genetic data. Premature termination codon variants were not only associated with a shorter life expectancy and a high lifelong risk of aortic event, but also with the highest risk of severe scoliosis and a lower risk for ectopia lentis (EL) surgery. In-frame variants could be subdivided according to their impact on the cysteine content of fibrillin-1 with a global higher severity for cysteine loss variants and the highest frequency of EL surgery for cysteine addition variants. Conclusion This study shows that FBN1 genotype–phenotype correlations exist for both aortic and extra-aortic features. It can be used for optimal risk stratification of patients with a great importance for genetic counseling and personalized medicine. This also provides additional data for the overall understanding of the role of fibrillin-1 in various organs.

The features of metabolic transformations of homocysteine and cysteine in rats’ hepatocytes under the nutritional imbalance

The work is devoted to the study of the homocysteine concentration in blood plasma and the activity of the enzymes of cysteine catabolic transformations γ-glutamate-cysteine ligase and cysteine dioxygenase in rats’ hepatocytes under the conditions of consumption of excessive sucrose content on the background of alimentary protein deprivation. It was found, that the consumption of a high-sucrose diet in animals is accompanied by the increase of homocysteine concentration in plasma by 57% compared to control, whereas the excess of sucrose under the conditions of protein deficiency leads to hypohomocysteinemia (the content of homocysteine decreased by 43% compared to control). The increased plasma homocysteine levels in excess-consuming sucrose can be considered as a prognostic marker of functional disorders of the transsulfuration pathway in the liver and used in the diagnosis of hepatopathologies. Since, the cysteine content in liver cells increased under the conditions of high-sucrose diet consumption, the increasing γ-GCS, a key enzyme of glutathione synthesis, is probably associated with the maintenance of intercellular glutathione stores. Our data of the occurrence hypohomocysteinemia under the conditions of the consumption of excessive amounts of sucrose on the background of dietary protein deficiency highlight gaps in the understanding of the correlation between the metabolic processes of methionine, homocysteine and cysteine in the liver. Under the excessive consumption of sucrose on the background of protein deficiency, the exogenous protein deficiency can be considered as a key factor in reducing the γ-GCS activity (36%) and the cysteine content, because not only the amount of this amino acid is reduced but also its synthesis is disrupted. At the same time, the maximum increase in cysteine dioxygenase activity in rats’ hepatocytes under the conditions of high-sucrose/low-protein diet consumption on the background of decreased γ-GCS activity indicates the utilization of excess cysteine with the formation of taurine and sulfates.

Improved Hardness and Thermal Stability of Nanocrystalline Nickel Electrodeposited with the Addition of Cysteine

Experiments were conducted for the study of the effect of cysteine addition on the microstructure of nanocrystalline Ni films electrodeposited from a nickel sulfate-based bath. Furthermore, the thermal stability of the nanostructure of Ni layers processed with cysteine addition was also investigated. It was found that with increasing cysteine content in the bath, the grain size decreased, while the dislocation density and the twin fault probability increased. Simultaneously, the hardness increased due to cysteine addition through various effects. Saturation in the microstructure and hardness was achieved at cysteine contents of 0.3–0.4 g/L. Moreover, the texture changed from (220) to (200) with increasing the concentration of cysteine. The hardness of the Ni films processed with the addition of 0.4 g/L cysteine (∼6800 MPa) was higher than the values obtained for other additives in the literature (<6000 MPa). This hardness was further enhanced to ∼8400 MPa when the Ni film was heated up to 500 K. It was revealed that the hardness remained as high as 6000 MPa even after heating up to 750 K, while for other additives, the hardness decreased below 3000 MPa at the same temperature.

The evolution of insect metallothioneins

Metallothioneins (MTs) are a family of cysteine-rich metal-binding proteins that are important in the chelating and detoxification of toxic heavy metals. Until now, the short length and the low sequence complexity of MTs have hindered the inference of robust phylogenies, hampering the study of their evolution. To address this longstanding question, we applied an iterative BLAST search pipeline that allowed us to build a unique dataset of more than 300 MT sequences in insects. By combining phylogenetics and synteny analysis, we reconstructed the evolutionary history of MTs in insects. We show that the MT content in insects has been shaped by lineage-specific tandem duplications from a single ancestral MT. Strikingly, we also uncovered a sixth MT, MtnF, in the model organism Drosophila melanogaster . MtnF evolves faster than other MTs and is characterized by a non-canonical length and higher cysteine content. Our methodological framework not only paves the way for future studies on heavy metal detoxification but can also allow us to identify other previously unidentified genes and other low complexity genomic features.

Comparative genomics analyses of lifestyle transitions at the origin of an invasive fungal pathogen in the genus Cryphonectria

Emerging fungal pathogens are a threat to forest and agroecosystems, as well as animal and human health. How pathogens evolve from non-pathogenic ancestors is still poorly understood making the prediction of future outbreaks challenging. Most pathogens have evolved lifestyle adaptations, which were enabled by specific changes in the gene content of the species. Hence, understanding transitions in the functions encoded by genomes gives valuable insight into the evolution of pathogenicity. Here, we studied lifestyle evolution in the genus Cryphonectria, including the prominent invasive pathogen C. parasitica, the causal agent of chestnut blight on Castanea species. We assembled and compared the genomes of pathogenic and putatively non-pathogenic Cryphonectria species, as well as sister group pathogens in the family Cryphonectriaceae (Diaporthales, Ascomycetes) to investigate the evolution of genome size and gene content. We found a striking loss of genes associated with carbohydrate metabolism (CAZymes) in C. parasitica compared to other Cryphonectriaceae. Despite substantial CAZyme gene loss, experimental data suggests that C. parasitica has retained wood colonization abilities shared with other Cryphonectria species. Putative effectors substantially varied in number, cysteine content and protein length among species. In contrast, secondary metabolite gene clusters show a high degree of conservation within the genus. Overall, our results underpin the recent lifestyle transition of C. parasitica towards a more pathogenic lifestyle. Our findings suggest that a CAZyme loss may have promoted pathogenicity of C. parasitica on chestnuts. Analyzing gene complements underlying key nutrition modes can facilitate the detection of species with the potential to emerge as pathogens.

The Evolution of Insect Metallothioneins

ABSTRACTMetallothioneins (MTs) are a family of cysteine-rich metal-binding proteins that are important in the chelating and detoxification of toxic heavy metals. Until now, the short length and the low sequence complexity of MTs has hindered the possibility of any phylogenetic reconstruction, hampering the study of their evolution. To answer this longstanding question, we developed an iterative BLAST search pipeline that allowed us to build a unique dataset of more than 300 MT sequences in insects. By combining phylogenetics and synteny analysis, we reconstructed the evolutionary history of MTs in insects. We show that the MT content in insects has been shaped by lineage-specific tandem duplications from a single ancestral MT. Strikingly, we also uncovered a sixth MT, MtnF, in the model organism Drosophila melanogaster. MtnF evolves faster than other MTs and is characterized by a non-canonical length and higher cysteine content. Our methodological framework not only paves the way for future studies on heavy metal detoxification but also can allow us to identify other previously unidentified genes and other low complexity genomic features.