Genome-Wide Identification and Interaction Analysis of Turbot Heat Shock Protein 40 and 70 Families Suggest the Mechanism of Chaperone Proteins Involved in Immune Response after Bacterial Infection

Abstract

Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

Keywords: Aeromonas salmonicida; heat shock protein; molecular docking; turbot.

Figure 1

The characteristics and domains of Hsp40 and Hsp70 members in S. maximus. (A) Protein sequence length distribution; AA, amino acid; (B) the protein molecular weight distribution; (C) distribution of conserved domains and gene structure on the Hsp40; (D) distribution of conserved domains and gene structure on the Hsp70.

Figure 1

The characteristics and domains of Hsp40 and Hsp70 members in S. maximus. (A) Protein sequence length distribution; AA, amino acid; (B) the protein molecular weight distribution; (C) distribution of conserved domains and gene structure on the Hsp40; (D) distribution of conserved domains and gene structure on the Hsp70.

Figure 2

The genome positions of Hsp40 and Hsp70 members in S. maximus. The Hsp40 genes and the Hsp70 genes are labeled in red and blue respectively.

Figure 3

Conserved motif information for Hsp40 and Hsp70 proteins in S. maximus, and each colored rectangular box represents a motif. (A) Motif information in turbot Hsp40 family subfamily A; (B) motif information in turbot Hsp40 family subfamily B; (C) motif information in turbot Hsp40 family subfamily C; (D) motif information in turbot Hsp70 family.

Figure 4

The phylogenetic tree constructed from Hsp40 and Hsp70 proteins in S. maximus, C. semilaevis, O. mykiss, D. rerio, X. tropicalis, T. c. triunguis, G. gallus, M. musculus, and H. sapiens. Different colors indicate different groups. The genes for turbot, half-smooth tongue sole, and rainbow trout are labeled with red dots and black and gray triangles, respectively. (A) Phylogenetic tree of subfamily A of the Hsp40 family; (B) phylogenetic tree of subfamily B of the Hsp40 family; (C) phylogenetic tree of dnajc1~dnajc10 in subfamily C of the Hsp40 family; (D) phylogenetic tree of dnajc11~dnajc20 in subfamily C of the Hsp40 family; (E) phylogenetic tree of dnajc21~dnajc30 in subfamily C of the Hsp40 family; (F) phylogenetic tree of Hsp70 family.

Figure 5

Duplication event analysis for the Hsp40 and Hsp70 gene families in the turbot genome and the synteny analysis between turbot and other fishes. (A) Number of Hsp40 and Hsp70 genes in six fish species; (B) the paralogues of Hsp40 and Hsp70 in the turbot genome—the blue and red lines indicate the Hsp40 and Hsp70 families members, respectively; (C) sytenic analysis of Hsp40 genes between S. maximus, C. semilaevis and O. mykiss—the blue lines indicate the Hsp40 family members in different species; (D) sytenic analysis of Hsp70 genes between S. maximus, C. semilaevis, and O. mykiss—the violet lines indicate the Hsp70 family members in different species.

Figure 6

PPI network of the Hsp40 and Hsp70 proteins.

Figure 7

Expression analysis of differentially expressed genes of Hsp40 and Hsp70 families after infection with A. salmonicida in turbot.

Figure 8

Dnajb1b and Hspa1a protein molecular docking model. Surface diagram of the docking model and their interfacing residues between Dnajb1b and Hspa1a protein (Dnajb1b, yellow; Hspa1a, blue; hydrogen bond interaction, dotted line).

Figure 9

Dnajb1b and Hspa1a protein molecular docking model combined with motif analysis (Dnajb1b, yellow; Hspa1a, blue; hydrogen bonding interactions, dashed lines). (A) Molecular structure of the JD of Dnajb1b interacting with the NBD of Hspa1a; (B) molecular structure of the GF-rich regions of Dnajb1b interacting with the NBD of Hspa1a; (C) molecular structure of the CTD of Dnajb1b interacting with the NBD of Hspa1a; (D) molecular structure of the CTD of Dnajb1b interacting with both the SBD and the CTD of Hspa1a; (E) domain interactions between Dnajb1b and Hspa1a and the motif where the interactions amino acid sites are located.