Coevolution analysis of RegX3.

<p>Contact map of coevolved residues in OmpR/PhoB family. Axes are residue numbers. Grey spots represent contacts within the same monomer, while black spots are predicted inter-monomeric contacts. The blue spots highlight those coevolved pairs in dimeric interface listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133389#pone.0133389.t001" target="_blank">Table 1</a>.</p

Conformational Dynamics of Response Regulator RegX3 from <i>Mycobacterium tuberculosis</i>

<div><p>Two-component signal transduction systems (TCS) are vital for adaptive responses to various environmental stresses in bacteria, fungi and even plants. A TCS typically comprises of a sensor histidine kinase (SK) with its cognate response regulator (RR), which often has two domains—N terminal receiver domain (RD) and C terminal effector domain (ED). The histidine kinase phosphorylates the RD to activate the ED by promoting dimerization. However, despite significant progress on structural studies, how RR transmits activation signal from RD to ED remains elusive. Here we analyzed active to inactive transition process of OmpR/PhoB family using an active conformation of RegX3 from <i>Mycobacterium tuberculosis</i> as a model system by computational approaches. An inactive state of RegX3 generated from 150 ns molecular dynamic simulation has rotameric conformations of Thr⁷⁹ and Tyr⁹⁸ that are generally conserved in inactive RRs. Arg⁸¹ in loop β4α4 acts synergistically with loop β1α1 to change its interaction partners during active to inactive transition, potentially leading to the N-terminal movement of RegX3 helix α1. Global conformational dynamics of RegX3 is mainly dependent on α4β5 region, in particular seven ‘hot-spot’ residues (Tyr⁹⁸ to Ser¹⁰⁴), adjacent to which several coevolved residues at dimeric interface, including Ile⁷⁶-Asp⁹⁶, Asp⁹⁷-Arg¹¹¹ and Glu²⁴-Arg¹¹³ pairs, are critical for signal transduction. Taken together, our computational analyses suggest a molecular linkage between Asp phosphorylation, proximal loops and α4β5α5 dimeric interface during RR active to inactive state transition, which is not often evidently defined from static crystal structures.</p></div

Coevolved residues of dimer interface of OmpR/PhoB family calculated by GREMLIN: Listed are the interacting residues with their respective numberings and topology.

<p>Italicized pairs indicate coevolved inter-monomeric contacts of RegX3 but intra-monomeric contacts in other OmpR/PhoB family members. Bold representation highlights those dimeric interactions conserved in all OmpR/PhoB family members. Contact pair with asterisk symbol satisfies all available active RR structures in this family but not RegX3.</p

Normal model analysis for conformational dependent regions of RegX3.

<p>(A) Overlap score of all non-zero low frequency modes between active and inactive state structures. (B) Overlap score of non-zero ENM modes based on the contribution of pocket residues of α4β5α5 face to the overall changes of full-length RegX3. (C) Contribution fraction (Y-axis) of residues to low frequency NMA modes in pocket residue analysis. (D) Key structural region with the nature of individual residues contributes to conformational changes during active to inactive transition of RegX3. ED is colored in grey and RD in blue. Helix α4 is colored in green and hot spot residues are in sphere models. The direction of an arrow represents the hinge motion of blue (ED) towards black (RD).</p

Dynamics of β1α1, β3α3 and β4α4 loops.

<p>(A-C) rmsd deviation plots of β1α1, β3α3 and β4α4 loops of RegX3 during 150 ns simulation. X-axis is time step and Y-axis indicates rmsd deviations. Interaction networks of these loops are explicated in active state (D) and inactive state (E). Residues are shown in ball and sticks. Brown and green spheres indicate La³⁺ ion and water molecule, respectively. All interactions are highlighted in dashed lines.</p

Coevolved residues in dimeric interface of OmpR/PhoB family.

<p>(A) Sequence conservation of the α4β5α5 dimeric interface of RegX3, PhoB, TorR and YycF in OmpR/PhoB family. Residues are numbered according to RegX3 and colored based on chemistry: acidic in red, basic in blue, hydrophobic in black, polar in green and neutral in purple. Coevolved pairs are under-dotted with the same colors. (B-E) Active state structures of RegX3 (2OQR), TorR (1ZGZ), PhoB (1ZES) and YycF (1NXP) in OmpR/PhoB family with coevolved residues in dimeric interface highlighted in sticks. It is noteworthy that RegX3 forms a complete dimeric structure with swapped domains that are colored in light and dark greys.</p

Data_Sheet_1_Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.PDF

IntroductionDamage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells.MethodsCells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining.ResultsOur results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression.DiscussionOur findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.</p

Data_Sheet_3_Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.PDF

IntroductionDamage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells.MethodsCells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining.ResultsOur results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression.DiscussionOur findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.</p

Image_1_Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.TIF

IntroductionDamage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells.MethodsCells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining.ResultsOur results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression.DiscussionOur findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.</p

Data_Sheet_2_Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.PDF

IntroductionDamage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells.MethodsCells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining.ResultsOur results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression.DiscussionOur findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.</p