Yokomizo, C. H. | bioRxiv Science

bioRxiv2021

Specific Adaptive Gene Expression of Smooth Muscle Cells Challenged by Organotelluranes.

Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/425411v4_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@52bb82org.highwire.dtl.DTLVardef@19476d9org.highwire.dtl.DTLVardef@6aa99org.highwire.dtl.DTLVardef@1aa605e_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2020

Investigation on the requirements for YbbN/CnoX displaying thiol-disulfide oxidoreductase and chaperone activities

YbbN/CnoX are proteins that display a Trx domain linked to a tetratricopeptide (TPR) domain, which are involved in protein-protein interactions and protein folding processes. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl (bleach). EcYbbN contains a SQHC motif within the Trx domain and displays no thiol-disulfide oxidoreductase activity. EcYbbN also presents a second Cys residue at Trx domain (Cys63) 24 residues away from SQHF motif that can form mixed disulfides with substrates. Here, we compared EcYbbN with two other YbbN proteins: from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). While EcYbbN displays two Cys residues along a SQHC[N24]C motif; XfYbbN and PaYbbN present two and three Cys residues in the CAPC[N24]V and CAPC[N24]C motifs, respectively. These three proteins are representatives of evolutionary conserved YbbN subfamilies. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (5,5'-dithiobis-(2-nitrobenzoic acid) = DTNB); and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that in vivo these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km=1.27 x 107 M-1.s-1), like the canonical XfTrx (XfTsnC). Furthermore, EcYbbN (as described before) and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function when the SQHC was replaced by CQHC. In contrast, the XfYbbN C40A mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. Finally, we generated a P. aeruginosa strain with the ybbN gene deleted, which did not present increased sensitivity to heat shock or to oxidants or to reductants. Altogether, our results suggest that different YbbN/CnoX proteins display distinct properties and activities, depending on the presence of the three conserved Cys residues. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/034579v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@6970f0org.highwire.dtl.DTLVardef@75da52org.highwire.dtl.DTLVardef@15067c9org.highwire.dtl.DTLVardef@1cdd97d_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- CXXC motif is required for the thiol-disulfide reductase activity of YbbN proteins. - XfYbbN and PaYbbN display thiol-disulfide oxidoreductase activity - The affinities of XfTrxR for XfYbbN and XfTsnC (canonical Trx) are comparable - XfYbbN and EcYbbN, but not PaYbbN, display holdase activity induced by hypochlorous acid - Engineering EcYbbN/CnoX by inserting a Cys residue in the SQHC motif resulted in a gain of function (thiol-disulfide oxidoreductase activity) and abolished the HOCl-induced holdase activity.

biochemistry↗