Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function

Erv1 is an FAD-dependent sulphydryl oxidase of the ERV/ALR sub-family, and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp95, no experimental study has been reported. In this study, we investigated the structural and functional roles of individual Trp residues of Erv1. Six single Trp-to-Phe yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from E. coli. Their effects on folding, FAD-binding, and Erv1 activity were characterised. Our results showed that Erv1W95F has the strongest effect on the stability and function of Erv1, and followed by Erv1W183F. Erv1W95F results in a decrease of the Tm of Erv1 by 23°C, a significant loss of the oxidase activity, and thus causing cell growth defects at both 30°C and 37°C. Erv1W183F induces changes in the oligomerisation state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whilst the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp157 mutant. Finally, computational analysis indicates that Trp95 plays a key role in stabilising the isoalloxazine ring to interact with Cys133. Taken together, this study provided important insights into the molecular mechanism of how sulfhydryl oxidases use FAD in catalyzing disulfide bond formation

Cytosolic Fe-S cluster protein maturation and iron regulation are independent of the mitochondrial Erv1/Mia40 import system

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways

Keratin-based particles for protection and restoration of hair properties

Objective Human hair is an element with unquestionable relevance in society both for women and men. Therefore, it is of great importance to develop new cosmetic products for hair care capable to restore and improve hair's characteristics. Here we explore the potential of keratinbased particles in the protection and recovery of hair mechanical properties and thermal stability. Methods Keratinbased particles were obtained by high pressure homogenization (HPH) using keratin and silk fibroin. The particles were characterized regarding size, superficial charge and polydispersity index. Their safety to cells was assessed using human skin keratinocytes. Virgin and overbleached Asian hair were treated with eight keratinbased formulations. The effect of particles on hair's mechanical properties was evaluated in terms of stiffness and tensile strength. The impact of treatments in hair thermal performance was studied using differential scanning calorimetry (DSC). Results Keratinbased particles were capable to recover and/or improve the mechanical properties of virgin and overbleached hair. Virgin hair treated with K80SF20P particles presented an improvement of the mechanical properties around 40%. An increase in keratin helix denaturation enthalpy and in surface smoothness for both types of hair was also verified after treatment. These particles demonstrated stability over time and proved to be safe when tested in human keratinocytes. Conclusion The keratinbased particles here presented have the potential to be incorporated in the development of new and effective hair care cosmetic formulations.This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020 (POCI01-0145-FEDER-006684) and BioTecNorte operation (NORTE-010145-FEDER-000004) and Nanotechnology Based Functional Solutions (NORTE-01-0145- FEDER-000019) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. We also acknowledge the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER007569) funded by national funds through Fundação para a Ciência e a Tecnologia (FCT) and by the ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI). Artur Ribeiro and Ana Tinoco thank FCT for funding the scholarships with the references SFRH/BPD/98388/2013 and SFRH/BD/ 114035/2015, respectively.info:eu-repo/semantics/publishedVersio