Abstract
The HtrA protein family represents an important class of serine proteases that are widely distributed across taxa. These evolutionarily conserved proteins are crucial for survival and function as monitors of protein synthesis during various stresses. Here, we performed gene expression analysis of the entire set of putative serine protease genes in Halothece sp. PCC7418 under salt stress conditions. The gene-encoding HtrA2 (H3553) was highly upregulated. This gene was cloned and functionally characterized, and its sub-cellular localization was determined. The recombinant H3553 protein (rH3553) displayed a pH optimum of 8.0, remained stable at 45 °C, and its proteolytic activity was not affected by salts. H3553 completely degraded the unfolded model protein, β-casein. In contrast, the folded model substrates (lysozyme or BSA) were not degraded by rH3553. Denaturation of BSA at a high temperature significantly increased its degradation by rH3553. H3553 was detected in the soluble protein fraction as well as the plasma membrane and thylakoid membrane fractions. Interestingly, the majority of H3553 was present in the plasma membrane under salt and heat stress conditions. Thus, H3553 resides in multiple sub-cellular locations and its localization drastically changes after exposure to stresses. Taken together, H3553 underpins protein quality-control process and is involved in the response and adaptation to salinity and heat stresses.
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Acknowledgements
T.P. thanks the Development and Promotion of Science and Technology Talented Project (DPST) for her Ph.D. Scholarship. This research was funded by Institute for Fermentation (grant number: G-2019-3-002) (to H.K.) and the Graduate School of Chulalongkorn University to commemorate the 72nd anniversary of his Majesty King Bhumibol Adulyadej, and the 90th Anniversary of the Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment) (grant number: GCUGR1125623033D) (to T.P. and R.W.S.), respectively.
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Supplementary file1 (PDF 1043 kb) Table S1 Primers used in this study. Figure S1 Gene expression analysis of 30 serine protease genes from Halothece 7418 grown under high salinity stress. Cells were collected at 0, 6 and 24 hours after exposure to salt stress (2.0 M NaCl). The total RNA was extracted and used as a template for RT-PCR analysis as described in the Materials and Methods section. The PCR products were subjected to electrophoresis and the relative amounts of the DNA fragments were quantitated using Image LabTM 3.0 software. The AprnpB gene was used as an internal control. The values at time zero for each gene were set to 1. The data are represented as the mean ± SEM of three independent experiments. The asterisks indicate significant differences from the value at time zero (one-sample t-test, p<0.05) Figure S2 Phylogenetic analysis of 30 serine protease paralogs from Halothece 7418. The amino acid sequences of the serine protease paralogs were retrieved from the KEGG database. The tree was generated with the maximum-likelihood method using the FastTree v2.1.8 software. The bars represent evolutionary distance. The scale bar comprises 0.5 expected changes per amino acid site (0.5 substitutions/site). Bootstrap probabilities are shown at the nodes. Figure S3 Alignment of the amino acid sequences of three HtrA2 proteins from Halothece 7418 (H3553, H2358, and H0395). The sequences were aligned using ClustalW (https://www.genome.jp/tools-bin/clustalw). (A) the catalytic triad of H3553 (Histidine:H140, aspartic acid:D170, and serine:S247) is shown by highlighted green. (B) predicted Trypsin and PDZ domains for H3553 H3553, H2358, and H0395 were obtained via SMART (https://smart.embl.de/). Figure S4 The effect of inhibitors on rH3553 activity. Proteolytic activity was measured using β-casein as a substrate. The activity value in the absence of inhibitor was set to 100%. Each value represents an average of three independent measurements. The data are represented as the mean ± SD of three independent experiments. Figure S5 Complex formation of H3553 in vivo. Total soluble protein extracts from control and salt acclimation conditions were subjected to gel filtration chromatography followed by Western blotting using anti-rH3553 antibody. The asterisks indicate the peaks of standard proteins and their native molecular size. Figure S6 Adsorption spectra of Halothece 7418 extracts after fractionation by sucrose gradient ultracentrifugation. (A) Sucrose gradient fractions. (B) plasma membrane fraction and (C) thylakoid membrane fraction
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Patipong, T., Hibino, T., Kageyama, H. et al. The evolutionarily conserved HtrA is associated with stress tolerance and protein homeostasis in the halotolerant cyanobacterium Halothece sp. PCC7418. Extremophiles 24, 377–389 (2020). https://doi.org/10.1007/s00792-020-01162-4
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DOI: https://doi.org/10.1007/s00792-020-01162-4