AtTPR10 Containing Multiple ANK and TPR Domains Exhibits Chaperone Activity and Heat-Shock Dependent Structural Switching Ho Byoung Chae 1 ,† , Su Bin Bae , Seol Ki Paeng , Chang Ho Kang , Joung Hun Park , Eun Seon Lee 1 and Sang Yeol Lee 1,* 1 1 1 1 1 Division of Applied Life Sciences (BK21+) and PMBBRC, Gyeongsang National University, Jinju 52828, Korea. Abstract Among the several tetratricopeptide (TPR) repeat-containing proteins encoded by the Arabidopsis thaliana genome, AtTPR10 exhibits an atypical structure with three TPR domain repeats at the C-terminus in addition to seven ankyrin
(ANK) domain repeats at the N-terminus. However, the function of AtTPR10 remains elusive. Here, we investigated the biochemical function of AtTPR10. Bioinformatic analysis revealed that AtTPR10 expression is highly enhanced by heat shock compared with the other abiotic stresses, suggesting that AtTPR10 functions as a molecular chaperone to protect intracellular proteins from thermal stresses. Under the heat shock treatment, the chaperone activity of AtTPR10 increased significantly; this was accompanied by a structural switch from the low molecular weight (LMW) protein to a high molecular weight
(HMW) complex. Analysis of two truncated fragments of AtTPR10 containing the TPR and ANK repeats showed that each domain exhibits a similar range of chaperone activity (approximately one-third of that of the native protein), suggesting that each domain cooperatively regulates the chaperone function of AtTPR10. Our results clearly demonstrate that AtTPR10 functions as a molecular chaperone in plants to protect intracellular targets from heat shock stress. METHODS 1. Determination of Holdase Chaperone Activity Holdase chaperone activity was measured using malate dehydrogenase (MDH;
Sigma-Aldrich) and citrate synthase (CS) as substrates. MDH and CS were incubated with AtTPR10, ANK-D, and TPR-D in 50 mM HEPES-KOH (pH 8.0) at 43 °C for 15 min, and the heat-induced thermal aggregation of both substrates was monitored using a DU800 spectrophotometer (Beckman, Brea, CA, USA) equipped with a thermostatic cell holder preheated to 43 °C, as described previously 2. Heat Stability Analysis of AtTPR10 To determine the heat stability of AtTPR10, 1 µg each of the recombinant MDH and AtTPR10 proteins was incubated at 23 °C, 50 °C, and 60 °C for 15 min. Proteins were then centrifuged at
13,000× g for 20 min. The supernatant and pellet fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% gel, followed by Coomassie brilliant blue staining. The solubility ratio of each protein was calculated by dividing the band density of the supernatant fraction with that of the pellet fraction by densitometer. RESULTS Figure 1. Heat shock-dependent changes in AtTPR10 structure and hydrophobicity. (a) Figure 2. Chaperone activity analysis and heat shock-dependent activity regulation of Structural changes in AtTPR10 in response to the heat
shock treatment. AtTPR10 was AtTPR10. (a) and (b) Analysis of the chaperone function of AtTPR10 using MDH (a) and incubated at 23 °C, 50 °C, and 60 °C for 30 min, centrifuged at 13,000× g for 15 min, and citrate synthase (CS) (b) as substrates. Thermal aggregation of MDH and CS was monitored analyzed by 10% native PAGE gel and silver staining. SM represents the size marker. (b) at 340 nm after a 15 min incubation with AtTPR10. In (a), 1.5 µM MDH was incubated at Size exclusion chromatography (SEC) analysis of AtTPR10 by HPLC. Recombinant purified 43 °C, either alone (○; control) or with 7.5 µM
GST (●), 4.5 µM 2-Cys Prx (◇), and 1.5 µM AtTPR10 was heat-treated, as described in (a), and 2 mg of each protein sample was (■), 4.5 µM (◆), and 7.5 µM (▲) AtTPR10 in 50 mM HEPES (pH 7.0). In (b), 1.2 µM CS was separated by SEC, based on the MW. (c) Heat-shock dependent changes in the surface incubated at 43 °C, either alone (○; control) or with 6 µM GST (●), 3.6 µM 2-Cys Prx (◇) hydrophobicity of AtTPR10. Recombinant AtTPR10 protein (20 µg) was incubated with 10 and 1.2 µM (■), 3.6 µM (◆), and 6 µM (▲) AtTPR10 in 50 mM HEPES (pH 7.0). (c) Heat- shock dependent chaperone activity of AtTPR10.
In this experiment, 1.5 µM MDH was µM bis-ANS at 23 °C (■), 50 °C (◆), and 60 °C (▲) for 20 min. Incubation of 10 µM bis- incubated either alone (○; control) or with 3 µM AtTPR10 at 23 °C (■), 50 °C (◆), and 60 °C ANS with no AtTPR10 protein served as a control (○). The fluorescence intensity of bis- (▲) for 15 min. ANS was measured at an excitation wavelength of 390 nm and emission spectra of 430–630 nm. Figure 4. Heat shock- dependent structural changes in AtTPR10, ANK-D, and TPR- D. (a)–(d) Analysis of heat shock–dependent structural changes by 10% native gel electrophoresis (a) and (c) and
SEC using HPLC (b) and (d) upon incubation at different Figure 3. Comparison of the chaperone activity and hydrophobicity of AtTPR10, ANK- temperatures (23 °C, 50 °C, and D, and TPR-D at different temperatures. (a) Relative chaperone activity of AtTPR10, 60 °C) for 30 min. ANK-D, and TPR-D at 23 °C, 50 °C, and 60 °C. (b) and (c) Hydrophobicity analysis of ANK- SM represents the size marker D (b) and TPR-D (c), along with that of AtTPR10, upon incubation with bis-ANS at 23 °C, (a) and (c). 50 °C, and 60 °C for 30 min. The fluorescence of bis-ANS was measured using a fluorometer, with excitation
at 390 nm and emission at 430–630 nm. The representative results are means of at least three independent experiments. REFERENCES 1. Schapire, A.L.; Valpuesta, V.; Botella, M.A. TPR Proteins in Plant Hormone Signaling. Plant Signal. Behav. 2006, 1, 229–230. 2. Yang, C.; Yu, Y.; Huang, J.; Meng, F.; Pang, J.; Zhao, Q.; Islam, A.; Xu, N.; Tian, Y.; Liu, J.; et al. Binding of the Magnaporthe oryzae Chitinase MoChia1 by a Rice Tetratricopeptide Repeat Protein Allows Free Chitin to Trigger Immune Responses. Plant Cell 2019, 31, 172–188. 3. Jang, H.H.; Lee, K.O.; Chi, Y.H.; Jung, B.G.; Park, S.K.;
Park, J.H.; Lee, J.R.; Lee, S.S.; Moon, J.C.; Yun, J.W.; et al. Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function. Cell 2004, 117, 625–635. [S. Plant biology-1] AtTPR10 containing multiple ANK and TPR domains exhibits chaperone activity and heat-shock dependent structural switching Ho Byoung Chae¹˙#, Su Bin Bae¹, Seol Ki Paeng¹, Chang Ho Kang¹, Joung Hun Park¹, Eun Seon Lee¹, Sang Yeol Lee¹˙* ¹Division of Applied Life Sciences (BK21+) and PMBBRC, Gyeongsang National University, Jinju 52828, Korea Among
the several tetratricopeptide (TPR) repeat-containing proteins encoded by the Arabidopsis thaliana genome, AtTPR10 exhibits an atypical structure with three TPR domain repeats at the C-terminus in addition to seven ankyrin (ANK) domain repeats at the N-terminus. However, the function of AtTPR10 remains elusive. Here, we investigated the biochemical function of AtTPR10. Bioinformatic analysis revealed that AtTPR10 expression is highly enhanced by heat shock compared with the other abiotic stresses, suggesting that AtTPR10 functions as a molecular chaperone to protect intracellular proteins from
thermal stresses. Under the heat shock treatment, the chaperone activity of AtTPR10 increased significantly; this was accompanied by a structural switch from the low molecular weight (LMW) protein to a high molecular weight (HMW) complex. Analysis of two truncated fragments of AtTPR10 containing the TPR and ANK repeats showed that each domain exhibits a similar range of chaperone activity (approximately one-third of that of the native protein), suggesting that each domain cooperatively regulates the chaperone function of AtTPR10. Our results clearly demonstrate that AtTPR10 functions as a
molecular chaperone in plants to protect intracellular targets from heat shock stress. Chaperone function of Arabidopsis NPR1 Su Bin Bae , Ho Byoung Chae , Seol Ki Paeng , Yong Hun Chi , Seong Dong Wi , Kieu Anh Thi Phan and Sang Yeol Lee 1,* 1 1 1,† 1 1 1 1 The Next-Generation Biogreen Program (SSAC,No.2011),Division of Applied Life Sciences (BK21 Plus), Gyeongsang National University, 501 Jinjudaero, Jinju52828, Korea ABSTRACT Among the defense systems, the NPR1 playing a key role in a plant systemic acquired immune responses has been shown to have multiple functions. The molecular structure
of NPR1 has two domains, BTB/POZ and ANK repeat, that are involved in protein–protein interactions. Despite the function of its SA- induced defense activity in nucleus, the biochemical property of its cytosolic oligomers has not been elucidated. Based on the results that the reversible structural change of redox proteins is a typical property of molecular chaperones, we investigated the biochemical characteristics of NPR1. From the study, the recombinant NPR1 functions as a protein chaperone to protect plants from heat stress through its structural switching by its oligomer form. Under heat-
induced condition, the NPR1 protein prevents from aggregation of substrate. And the structural change was regulated upon the redox changes, such as DTT treatment dissociated its structure to monomer and reduced its chaperone activity, suggesting that the heat-induced chaperone activity of NPR1 is dependent on its redox status. In summary, the cytosolic NPR1 oligomer performs the important function of molecular chaperone to protect plants from heat stress that can be applied to the preparation of heat shock-tolerant useful crops. METHODS 1. Plasmid cloning and purification of recombinant AtNPR1
in E. coli AtNPR1 was cloned from an Arabidopsis cDNA library by PCR and AtNPR1 constructs were subcloned into the pET28a binary vector and transformed into the E. coli BL21 (DE3). Cells were cultured until A600~0.6 and then recombinant 6 × His tagged proteins were cultured with 1 mM isopropyl-β-d- thiogalactopyranoside (IPTG) for another 12 h at 18 °C. The 6×tagged AtNPR1 proteins underwent dialysis with 50 mM HEPES–KOH (pH 8.0) bufer solution. Purity of the 6 × His tagged AtNPR1 proteins (2 µg) was tested by SDS-PAGE gel. And different concentrations of DTT were added to protein samples to
check the structural changes of AtNPR1 from oligomer to monomeric forms. 2. Measurement of chaperone activity The holdase chaperone activity was measured using malate dehydrogenase (MDH, Sigma-Aldrich). Thermal-induced aggregation of MDH was incubated by additional different concentrations of AtNPR1 in 50 mM HEPES–KOH (pH 8.0) at 43 ℃ for 15 min. Aggregated substrates were monitored using thermo- controllable DU800 spectrophotometer (Beckman, USA). RESULTS Fig. 2 Hydrophobicity changes of AtNPR1 and its chaperone activity. a Hydrophobicity plot of AtNPR1. The hydrophobicity score was predicted
by the bioinformatic database, website of https ://web.expasy.org/prot-scale /. b Hydrophobicity changes of AtNPR1 measured by incubating variable amounts of AtNPR1 with 10 μM bis-ANS. The fluorescence of bis-ANS was measured under the wavelength; excitation, 390 nm and emission, 430–630 nm. c Measurement of the chaperone activity of AtNPR1 using MDH as a substrate under the various molar ratios of AtNPR1 to MDH in 1:1 ~ 3:1. Head-induced denaturation of MDH was measured at Fig. 1 Nucleotide and deduced amino acid sequences of AtNPR1 in Arabidopsis. 340 nm after a 15 min incubation of the
reaction mixture. 2 μM MDH was incubated with different a Nucleotide and amino acid sequences of AtNPR1. The domains of BTB/POZ domain (blue), C- concentrations of AtNPR1 proteins in HEPES–KOH (pH 8.0) buffer. 2-Cys Prx and GST proteins terminal ankyrin (ANK) repeat domain (orange), and C-terminus nuclear-located sequence (NLS, were used as positive and negative controls, respectively. d Structural changes of AtNPR1 protein green) were indicated, respectively. Asterisk indicates the stop codon. The number represents the after heat treatment. 2 μg of recombinant AtNPR1 proteins was treated by
various heat shock po
