Abstract
Ydj1, a class B J-protein (Hsp40) in yeast, has two zinc finger domains in each monomer and belongs to an important co-chaperone family that plays crucial roles in cells, such as recognizing and binding partially folded proteins and assisting the Hsp70 chaperone family in protein folding. Yeast cells with ydj1 deletion were less efficient at coping with zinc stress than wild-type cells, and site-directed mutagenesis studies that impair or delete the zinc finger region have confirmed the importance of this region to the function of Ydj1; however, little is known about whether the presence of zinc is critical for the function of the protein. To gain insights into the effect of zinc on the structure and function of Ydj1 without having to modify its primary structure, a method was developed and optimized to quantify and remove the zinc from the protein. Recombinant Ydj1 was produced and purified, and its zinc content was determined by ICP-MS. The result showed that two zinc atoms were bound per monomer of protein, a good indicator that all sites were saturated. To optimize the removal of the bound zinc, variations on chelating agent (EDTA, EGTA, 1,10-phenanthroline), chelator concentration, reaction time, pH, and temperature were tested. These procedures had no effect on the overall secondary structure of the protein, since no significant changes in the circular dichroism spectrum were observed. The most significant removal (91 ± 2%, n = 3) of zinc was achieved using 1,10-phenanthroline (1 × 10−3 mol L−1) at 37°C with a pH 8.5 for 24 h. Zinc removal affected the stability of the protein, as observed by a thermal-induced unfolding assay showing that the temperature at the middle of the transition (Tm) decreased from 63 ± 1°C to 60 ± 1°C after Zn extraction. In addition, the effect on the ability of Ydj1 to protect a model protein (luciferase) against aggregation was completely abolished after the Zn removal procedure. The main conclusion is that zinc plays an important role in the stability and activity of Ydj1. Additionally, the results highlight the medical importance of chaperones, as altered zinc homeostasis is implicated in many diseases, such as neurodegenerative disorders.
Highlights
There is mounting evidence that the homeostasis of metals has an important medicinal role, since the disturbance of this equilibrium appears to be related to the occurrence of severe diseases and to aging (Larbi et al, 2006; Susuki et al, 2008; Montes et al, 2014)
One of the several strategies that cells use to deal with stress is the expression of a family of proteins known as chaperones and heat shock proteins (Hsps) (Tiroli-Cepeda and Ramos, 2011)
The proposed methodology in this study provides significant Zn extraction from the Ydj1 co-chaperone, presenting good measures of merit, such as significant precision and accuracy, as well as good limits of detection and quantification
Summary
There is mounting evidence that the homeostasis of metals has an important medicinal role, since the disturbance of this equilibrium appears to be related to the occurrence of severe diseases and to aging (Larbi et al, 2006; Susuki et al, 2008; Montes et al, 2014). Molecular chaperones and Hsps are expressed to cope with protein aggregation, a phenomenon that results from the crowded environment inside the cell (Wong and Houry, 2004; Tiroli-Cepeda and Ramos, 2011; Lindberg et al, 2015). These proteins help to maintain homeostasis during regular and stressful situations and may be divided into foldases (Hsp and Hsp, for instance) and holders, which bind partially unfolded proteins to protect against aggregation (e.g., Hsp40s and small Hsps). Cys-201 and Cys143 in ZBDI and Cys162 and Cys185 in ZBDII (Figure 1) participate in the tetrahedral coordination of zinc (Fan et al, 2003)
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