Genomewide identification and analysis of heat‐shock proteins 70/110 to reveal their potential functions in Chinese soft‐shelled turtle Pelodiscus sinensis

Abstract Heat‐shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft‐shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved “heat shock protein 70” domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue‐specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B‐L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.


| INTRODUC TI ON
Heat-shock proteins (Hsps), which were first reported in Drosophila, are ubiquitously found in bacteria, plants, and animals (Arya, Mallik, & Lakhotia, 2007). When organisms are exposed to extreme temperatures or external stresses such as disease, toxins, and hypoxia, Hsps can be synthesized as stress proteins and accumulate to respond to various environmental insults (Gupta, Sharma, Mishra, Mishra, & Chowdhuri, 2010;Srivastava, 2002).
The intrinsic activities and allosteric coupling of NBD and SBD are associated with the functions of Hsp70 (Bertelsen et al., 2009).
Specifically, Hsp110, which exhibits a longer C-terminal extension, shares the same domain organization and exhibits highly similar crystal structures to Hsp70, which reveals the close relationships of the Hsp110 and Hsp70 protein families (Dragovic, Broadley, Shomura, Bracher, & Hartl, 2006 By functioning as a molecular chaperone in the folding, denaturation, degradation, and inhibition of proteins and controlling regulatory proteins, Hsp70 plays essential roles in heat adaptation and protection against stresses in diverse species (Murphy, 2013). Extensive evidence has suggested that Hsp70 not only exhibits ATP-dependent chaperoning function but is also a negative apoptosis-inducing factor (AIF) in response to a wide range of stimuli (Goloudina, Demidov, & Garrido, 2012;Jiang et al., 2011;Sabirzhanov, Stoica, Hanscom, Piao, & Faden, 2012). In rodent models, overexpression of Hsp70 provides a survival advantage to tumor cells because Hsp70 can interact with multiple components of the apoptotic machinery (Jäättelä, 1995). Conversely, it has been reported that Hsp70 knockdown leads to decreased cell proliferation and facilitates the induction of apoptosis in multiple cancer cell models (Kotoglou et al., 2009;Zhang et al., 2013). Indeed, Hsp70 can block apoptosis by binding to apoptosis protease-activating factor 1 (Apaf1), thereby preventing the recruitment of procaspase-9 to the apoptosome (Beere et al., 2000). Similarly, Hsp70 regulates the important apoptotic mediator Bax and prevents Bax from translocating to mitochondria, which is necessary for the disruption of the mitochondrial membrane (Stankiewicz, Lachapelle, Foo, Radicioni, & Mosser, 2005). The regulatory roles of Hsp70 in apoptosis are also due to the effects of Hsp70 on stress-induced kinases, including SAPK/JNK, p38, and apoptosis signal-regulating kinase (Park et al., 2002;Park, Lee, Huh, Seo, & Choi, 2001).
Additionally, Hsp70 can inhibit caspase-independent apoptosis by directly interacting with AIF and cathepsins (Jesper et al., 2004;Ravagnan et al., 2001). Despite considerable research advances, the antiapoptotic mechanism of Hsp70 is still controversial, especially in nonmodel animals. In recent studies, many genes encoding Hsp70 have been identified and characterized from nonmodel animals such as amphibians, insects, crustaceans, mollusks, and fishes (Luan et al., 2010;Simoncelli, Morosi, Rosa, Pascolini, & Fagotti, 2010;Song et al., 2016;Wang et al., 2019;Wang, Wu, Jian, & Lu, 2009), enriching knowledge of the phylogenetic relationships and biological functions of Hsp70. However, few studies have focused on the genomewide identification and functional analysis of the Hsp70 gene family in turtles.
Chinese soft-shelled turtle (Pelodiscus sinensis), a reptile, presents important economic value and is widely distributed in Asian countries such as China, Japan, and Korea. P. sinensis is an ectothermic aquaculture species with a specific evolutionary role linking ectothermic anamniotic animals (fishes and amphibians) and endothermic amniotic animals (birds and mammals) (Zimmerman, Vogel, & Bowden, 2010) and can thus be used as a potential animal model to study the evolution of critical genes or species (Liu, Chu, et al., 2016). The body temperature of P. sinensis is dependent on the ambient temperature, similar to other ectotherms, resulting in typical hibernation patterns in midwinter (Chen et al., 2015). Previous studies have revealed distinct seasonal apoptosis in the testis of P. sinensis on the basis of morphological and molecular evidence (Liu et al., 2017). Furthermore, it is well known that Hsp70/110 presents the obligatory function of responding to adverse external stimuli, especially heat shock, and exhibits survival-promoting effects and suppression of apoptosis (Gao  et al., 2014). However, the relationship between apoptosis and stressrelated Hsp70/110 is poorly understood in P. sinensis. Fortunately, the public genomic sequences and RNA-seq data of P. sinensis (Liu et al., 2017;Wang et al.,2013) can provide rich resources for the identification, phylogenetic analysis and functional exploration of PsHsp70/110 genes in P. sinensis.
The primary goals of this study were to systematically identify candidate PsHSP70/110 family genes based on the whole-genome sequence of P. sinensis and to analyze their classification, conserved structures, and phylogenetic relationships. Furthermore, we focused on the mRNA and protein expression of PsHSP70/110 genes to investigate their putative roles in germ cell apoptosis following seasonal temperature variation. These results will provide useful information for the exploration of PsHSP70/110 functions in P. sinensis and facilitate further investigation of Hsp70/110 family genes in turtles.

| Animals
In this study, all sample procedures and animal care were con-

| Phylogenetic analysis and alignment of Hsp70/110 genes in different species
To detect the phylogenetic relationship of

| Quantitative real-time PCR analysis
Quantitative real-time PCR (qRT-PCR) analysis was performed according to previous reports (Liu et al., 2017;. Total RNA was extracted using TRIzol reagent (Life Technologies).
cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen). Primer sequences for gene expression analysis were designed using Beacon Designer software (Premier Biosoft International). The relative expression levels of genes were normalized to β-actin and analyzed using the 2 −ΔΔCT method (Livak & Schmittgen, 2001).

| Interaction network and functional enrichment of PsHSP70/110 proteins
The protein sequences of PsHSP70/110 genes were used for analyzing protein-protein associations. The interaction relationships among PsHSP70/110 genes from P. sinensis were determined by using online STRING software (http://string-db.org).
The interaction networks were constructed by setting the minimum required interaction score to ≥0.7. The functional enrichments (P-value ≤ 0.05) in the interaction network were analyzed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp/kegg/pathw ay.html), Pfam, and InterProScan databases.

| Prediction of 3D protein structures
The 3D structures of PsHSP70/110 proteins were predicted for further analysis of protein functions. The 3D protein structures were generated by a homology modeling method using the known homologous structure as a template. The Phyre2 server (Kelley, Mezulis, Yates, Wass, & Sternberg, 2015) was used for homology modeling, secondary structure prediction, and domain analysis. The comparison of 3D protein structures was performed by using PyMOL Viewer software. The prediction of binding sites in proteins was performed using similar structures on the 3DLigandSite server (Wass, Kelley, & Sternberg, 2010).
TA B L E 2 The sequence identity of PsHSP70/110 proteins in Pelodiscus sinensis compared with each other

| Data analysis
The data were expressed as the means ± SEM. One-way ANOVA was performed using SPSS 16.0 software to assess the differences in gene expression levels. The level of significance was set at a p-value < 0.05.

| Identification and analysis of Hsp70/110 genes in P. sinensis
In this study, a genomewide search against the P. sinensis genome sequences generated 18 candidate genes belonging to the HSP70/110 family, including 17 Hsp70 genes and one Hsp110 gene (Table 1)   The lists of PsHSPA4-homologous genes constituted the second largest subfamily. Relatively, few genes belonged to the other subfamilies ( Figure 2b). Notably, PsHSPA2 homologs shared the closest phylogenetic relationships with PsHSPA8 homologs, which was consistent with the high similarity between PsHSPA2 and PsHSPA8 in their amino acid sequences (Table 2). HSPA2 and HSPA8 genes were assigned to the same subfamily as HSPA2/8. Compared with the phylogenetic analysis of PsHSP70/110 shown in Figure 1a, the detailed classification shown in Figure 2b was performed to indicate the phylogenetic relationships among PsHSP70/110 genes and the homologous genes in other species.

| Dynamic expression of PsHSP70/110 genes during spermatogenesis in P. sinensis
The

| Protein expression of PsHSPA1B-L, PsHSPA2, and PsHSPA8 in the testis of P. sinensis during spermatogenesis
The protein expression analyses of PsHSPA1B-L, PsHSPA2, and PsHSPA8 by IHC in different months (April, July, and October) showed that the positive reactions for the PsHSPA1B-L, PsHSPA2, and PsHSPA8 proteins in the testis of P. sinensis were similar within a given month (Figure 7). Weak immunostaining of the three proteins was observed in April, whereas intense immunostaining was observed in July. In addition, the testis displayed moderate immunoreactivity of the three proteins in October. No staining was detected in the negative control sections. The results of IHC analysis were coincident with the mRNA expression variations of PsHSPA1B-L, PsHSPA2, and PsHSPA8 determined by qRT-PCR analysis in response to seasonal temperature changes.

| Characterization and protein structure analysis of PsHSPA1B-L, PsHSPA2, and PsHSPA8 proteins
An overview of the analysis between PsHSPA1B-L, PsHSPA2, and PsHSPA8 proteins revealed that they exhibited high amino acid identity (Table 2) and similar conserved protein domains ( Figure 1) and that they were enriched in the same KEGG pathway of MAPK signaling (Table 5). Prediction of the 3D protein structures of PsHSPA1B-L, PsHSPA2, and PsHSPA8 showed that the three proteins presented very similar structures and binding sites, especially between PsHSPA2 and PsHSPA8 (Figure 8a; Figure S2; Table 6). More detailed phylogenetic analysis showed that PsHSPA1B-L, PsHSPA2, and PsHSPA8 shared the closest relationships with their corresponding homologous genes from western painted turtle (Chrysemys picta bellii), green sea turtle (C. mydas), and three-toed box turtle (Terrapene mexicana triunguis) (Figure 8b-d). Moreover, the mRNA and protein expression patterns of PsHSPA1B-L, PsHSPA2, and PsHSPA8 genes were similar in the testis of P. sinensis ( Figure 5; Figure 7), which further validated the putative parallel functions of the three genes in certain biological processes. Furthermore, the PsAPAF1 (ENSPSIG00000011998) protein of P. sinensis, a potential interacting protein of PsHSP70/110 (Beere et al., 2000), was selected and subjected to interaction analysis. The predicted protein interaction revealed that PsAPAF1 was associated with the PsHSPA1B-L, PsHSPA2, and PsHSPA8 proteins via both binding and catalysis relationships ( Figure S2), which implied F I G U R E 6 Interaction networks of PsHSP70/110 proteins. The catalysis, reaction, and binding relationships are indicated with purple, black, and blue lines, respectively. The green arrows represent the positive interaction relationship that PsHSPA1B-L, PsHSPA2, and PsHSPA8 may play similar roles and interact with the PsAPAF1 gene in P. sinensis.

| Overview of PsHSP70/110 genes in P. sinensis
The central biological roles of Hsp70/110 in various biological and physiological processes are attributed to their chaperone activity and their structurally and functionally conservative properties in evolution (Lindquist & Craig, 2003). Considerable evidence has demonstrated that the protein structure and conserved domains of Hsp70/110 proteins play roles in modulating multiple cellular processes induced by a wide variety of stimuli (Gupta et al., 2010).
In the present study, protein structure analysis showed that 18

| Conservation of PsHSP70/110 family genes in evolution
Hsp70 is the most conserved heat-shock stress protein in evolution, and its intrinsic functions and conserved domains are responsible for its conservation across species (Kiang & Tsokos, 1998;Murphy, 2013). In this study, a genomewide search identified 18 PsHSP70/110 family members in P. sinensis, which is comparable to the number of Hsp70/110 genes in other species. Phylogenetic analysis and classification of Hsp70 proteins from 13 selected animals indicated that some members of this group, such as HSPA12 genes, were ubiquitous in these species. Specifically, PsHSPA1B-L, PsHSPA2, and PsHSPA8 showed the closest phylogenetic relationships with homologs from C. picta bellii, C. mydas, and T. mexicana triunguis, which is concordant with the evolutionary relationships among these species. In addition, a similar number of Hsp70 family genes were found between P. sinensis and other species, suggesting the evolutionary conservation of Hsp70 among different species (Song et al., 2016;Wang et al., 2019).
Remarkably, HSPA2, HSPA8, and their homologous genes were assigned to the same subfamily of HSPA2/8 and exhibited the closest phylogenetic relationships, which is consistent with previous reports (Song et al., 2016). In general, comparative analysis can provide a foundation for a better understanding of the evolutionary relationships of PsHSP70/110.
In agreement with a previous report by 2016), the family of PsHSP70 and PsHSP110 proteins. In addition to the difference of one domain between the peptide-binding domain and the C-terminal region, Hsp110 exhibits high homology and a similar crystal structure to Hsp70 (Polier, Dragovic, Hartl, & Bracher, 2008), although Hsp110 is a divergent Hsp70 family member. In this study, one PsHSP110 was discovered, which was named PsHSPH1 in P. sinensis. On the basis of its structure and sequence, PsHSP110 was included in the subfamily of PsHSP70, and these proteins were studied and discussed together. Sequence alignment showed that PsHSPH1 shared less than 40% identity with most PsHSP70 proteins. Interestingly, PsHSPH1 presented higher sequence identities with PsHSPA4L, PsHSPA4-X1, and PsHSPA4-X2, and these four members were classified into a closer subgroup by phylogenetic analysis, with similar conserved domains. The observations suggested that PsHSP70/110 family genes with close phylogenetic relationships and similar protein structures may present similar potential roles in P. sinensis.

| Characterization of PsHSP70/110 gene expression and putative roles
The induction and accumulation of Hsp70/110 are tightly associated with a range of environmental and physical stresses (Gupta et al., 2010;Srivastava, 2002). In this study, tissue-specific expression analysis revealed that most PsHSP70/110 genes were constitutively expressed in different tissues of P. sinensis, which suggested that PsHSP70/110 genes may be important for organismic homeostasis. Notably, several genes, such as PsHSPA1A-L, PsHSPA1B-L, PsHSPA2, PsHSPA5, PsHSPA8, and PsHSPA9, exhibited specific high expression in the testis and oviduct of P. sinensis. Importantly, the high expression of the PsHSPA2 gene in the P. sinensis testis was consistent with previous reports that showed a high level of HSPA2 in the human testis (Daugaard et al., 2007;Son et al., 1999;Su et al., 2004), implying a potential special role of PsHSPA2 in the germ cells of P. sinensis. Substantial experimental evidence has revealed that Hsp70 is one of the positive necessary factors for tumor cell survival and, on the contrary, that Hsp70 negatively modulates apoptosis (Goloudina et al., 2012;Rérole et al., 2011). In response to stressful conditions and during diverse developmental processes, apoptosis, which is essential for tissue homeostasis, is conspicuous in multicellular organisms (Meier, Finch, & Evan, 2000). In seasonally breeding species, apoptosis is responsible for testicular atrophy during seasonal reproductive regression (Young & Nelson, 2001).
Seasonal spermatogenesis is characteristic of temperate and boreal reptilian species, including P. sinensis (Gribbins, 2011;Liu et al., 2017 (Chang & Karin, 2001;Lee et al., 2006;Taylor, Zheng, Liu, & Thompson, 2013). Apaf1, one of the important components of the p53 signaling pathway, can mediate apoptosis through its association with procaspase-9 (Zou, Li, Liu, & Wang, 1999). Other evidence has strongly suggested that the event of Hsp70 binding to Apaf1 seems to eliminate the oligomerization of Apaf1 and procaspase-9 and then suppresses apoptosis (Beere & Green, 2001;Beere et al., 2000;Saleh, Srinivasula, Balkir, Robbins, & Alnemri, 2000). As expected, we found protein interactions of PsHSPA1B-L, PsHSPA2, PsHSPA8, and PsAPAF1 with binding relationships. Moreover, IHC analysis showed that the protein expression of PsHSPA1B-L, PsHSPA2, and PsHSPA8 exhibited significant upregulation in July and October. More importantly, a distinct decrease in apoptosis in the testis of P. sinensis was detected in October (MT-3) (Liu et al., 2017). These observations indicated that the high protein levels of PsHSPA1B-L, PsHSPA2, and PsHSPA8 as well as the interaction with PsAPAF1 may favor the inhibition of apoptosis during spermatogenesis in P. sinensis. In addition, in the current study, PsHSPA8, also known as PsHsc70, encoding the predominant cognate member of the PsHSP70 family, was systematically characterized in P. sinensis for the first time to the authors' knowledge.
Hsc70 is the cochaperone of the antiapoptotic modulator BAG1, and BAG1 can bind to its ATPase domains to modulate chaperone activities and influence apoptotic responses (Song, Takeda, & Morimoto, 2001;Stuart et al., 1998). Taken together, these findings reveal the potential functions of critical PsHSP70/110 proteins in regulating apoptosis and associated with spermatogenesis in P. sinensis.

| CON CLUS ION
In this study, a total of 18 PsHSP70/110 family genes were identified and comprehensively analyzed in P. sinensis. All the PsHSP70/110 proteins contained the conserved NBD domain, which represents a typical