DsHsp90 is involved in the early response of Dunaliella salina to environmental stress

Abstract

Heat shock protein 90 (Hsp90) is a molecular chaperone highly conserved across the species from prokaryotes to eukaryotes. Hsp90 is essential for cell viability under all growth conditions and is proposed to act as a hub of the signaling network and protein homeostasis of the eukaryotic cells. By interacting with various client proteins, Hsp90 is involved in diverse physiological processes such as signal transduction, cell mobility, heat shock response and osmotic stress response. In this research, we cloned the dshsp90 gene encoding a polypeptide composed of 696 amino acids from the halotolerant unicellular green algae Dunaliella salina. Sequence alignment indicated that DsHsp90 belonged to the cytosolic Hsp90A family. Further biophysical and biochemical studies of the recombinant protein revealed that DsHsp90 possessed ATPase activity and existed as a dimer with similar percentages of secondary structures to those well-studied Hsp90As. Analysis of the nucleotide sequence of the cloned genomic DNA fragment indicated that dshsp90 contained 21 exons interrupted by 20 introns, which is much more complicated than the other plant hsp90 genes. The promoter region of dshsp90 contained putative cis-acting stress responsive elements and binding sites of transcriptional factors that respond to heat shock and salt stress. Further experimental research confirmed that dshsp90 was upregulated quickly by heat and salt shock in the D. salina cells. These findings suggested that dshsp90 might serve as a component of the early response system of the D. salina cells against environmental stresses.

Keywords: Dunaliella salina; Hsp90; gene structure; haloadaption; heat shock; osmotic stress; structural feature.

Figure 1

Phylogenetic tree analysis of Hsp90s. The sequences used for analysis are: Arabidopsis thaliana HSP81-1 (NP_200076.1), Arabidopsis thaliana HSP81-2 (NP_200414.1), Arabidopsis thaliana HSP81-3 (NP_200412.1), Arabidopsis thaliana HtpG (NP_200411.1), Arabidopsis thaliana endoplasmin-like protein (SHD) (NP_194150.1), Arabidopsis thaliana Chaperone protein htpG family protein (CR88) (NP_178487.1), Arabidopsis thaliana HSP89.1 (NP_187434.2), Cenchrus americanus HSP90 (ADP89126.1), Chara braunii HSP90 (BAH97107.1), Chlamydomonas reinhardtii HSP90A (XP_001695264.1), Chlamydomonas reinhardtii HSP90B (EDP06860.1), Chlamydomonas reinhardtii HSP90C (AAU10511.1), Dunaliella salina HSP90 (AFK31312.1), Glycine max HSP90-1 (NP_001236612.1), Hordeum vulgare HSP90 (AAP87284.1), Oryza sativa HSP90 (BAA90487.1), Solanum lycopersicum HSP90 (AAD30456.1), Strongylocentrotus purpuratus HSP gp96 (NP_999808.1), Triticum aestivum HSP90 (AEK01109.1), Volvox carteri f. nagariensis HSP90 (XP_002947115.1), Zea mays HSP (NP_001170480.1), Blastocladiella emersonii HSP90B (ABU45371.1), Danio rerio HSP90β (NP_571385.2), Ostreococcus lucimarinus HSP90 (ABP00103.1), Rattus norvegicus HSP90β (NP_001004082.3), Homo sapiens HSP90 α (AAH68474.1), Kryptolebias marmoratus HSP90β (AEM65181.1), Oryza sativa HSP90C2 (XP_483065.1).

Figure 2

Sequence alignment of Hsp90s. The sequences used for analysis are from D. salina Hsp90 (JQ735968), C. reinhardtii Hsp90A (XP_001695264.1), V. carteri Hsp90 (XP_002947115.1), C. braunii Hsp90 (BAK08741.1), O. sativa Hsp90 (NP_001063500.1), A. thaliana HtpG (NP_200411.1). Five signature sequences (I–V) characteristic of the cytosolic Hsp90A family according to Gupta [23] are highlighted by underlining. The C-terminal MEEVD sequence, which is also a characteristic motif of Hsp90A members, is highlighted by a box.

Figure 3

Distribution of exons and introns in the genomic DNA of hsp90s. The sequences used for analysis are from D. salina (JQ735969), A. thaliana (NM_124982.2), C. reinhardtii (XM_001695212.1), G. max (XM_003533929.1), Micromonas (XM_002499681.1), O. lucimarinus (XM_001419901.1), P. patens (XM_001777362.1), P. trichocarpa (XM_002305227.1), S. moellendorffii (XM_002981360.1), V. carteri (XM_002947069.1).

Figure 4

Sequence of the promoter region of dshsp90. The three Alfin1 putative binding sites with the motif of GTGGNG or GNGGTG are highlighted in red.

Figure 5

Biophysical and biochemical characterization of DsHsp90. (A) SEC profile of the recombinant DsHsp90 using a superdex G200 10/30 column. The apparent molecular weight was calculated from the standard curve obtained from the standard markers provided by GE Healthcare; (B) Far-UV CD spectrum of DsHsp90. The percentages of the secondary structures were determined using the CONTINLL, SELCON3 and CDSSTR algorithms within the CDPro analytical software [37]. The results are presented as the average ± standard error calculated from the values of the three methods; (C) ATPase activity of DsHsp90. The enzyme assay was conducted in the presence or absence of 5 mM ATP or 20 μg DsHsp90; (D) Predicted three-dimensional structure of DsHsp90 by SWISS-MODEL using the crystal structure of yeast Hsp90 (PDB ID: 2CG9) as the template structure. ND, MD and CD are the N-terminal domain, middle domain and C-terminal domain, respectively.

Figure 6

Relative fold increase of the expression level of dshsp90 and dsgpd transcripts during stress evaluated by quantitative RT-PCR. (A) Time-course study of the transcript level of dshsp90 after heat shock. Real-time quantitative PCR was carried out using the total RNA extracted from the D. salina cells after heat treatment at 37 °C for 0–120 min; (B) Time-course study of the transcript levels of dshsp90 and dsgpd after salt shock from 2 M to 4 M NaCl for 0–24 h. The expression level of ds18S was used as an internal control.