Transcriptomic responses to heat stress in rainbow trout Oncorhynchus mykiss head kidney

Fish and Shellfish Immunology 82 (2018) 32–40

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Transcriptomic responses to heat stress in rainbow trout Oncorhynchus mykiss head kidney

T

Jinqiang Huanga, Yongjuan Lia,b, Zhe Liua,∗, Yujun Kanga, Jianfu Wanga a b

College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, 730070, China College of Science, Gansu Agricultural University, Lanzhou, 730070, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Rainbow trout Heat stress RNA-seq Transcriptome

Rainbow trout (Oncorhynchus mykiss) are widely cultured throughout the word for commercial aquaculture. However, as a cold-water species, rainbow trout are highly susceptible to heat stress, which may cause pathological signs or diseases by alleviating the immune roles and then lead to mass mortality. Understanding the molecular mechanisms that occur in the rainbow trout in response to heat stress will be useful to decrease heat stress-related morbidity and mortality in trout aquaculture. In the present study, we conducted transcriptome analysis of head kidney tissue in rainbow trout under heat-stress (24 °C) and control (18 °C) conditions, to identify heat stress-induced genes and pathways. More than 281 million clean reads were generated from six head kidney libraries. Using an adjusted P-value of P < 0.05 as the threshold, a total of 443 differentially expressed genes (DEGs) were identified, including members of the HSP90, HSP70, HSP60, and HSP40 family and several cofactors or cochaperones. The RNA-seq results were confirmed by RT-qPCR. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs were performed. Many genes involved in maintaining homeostasis or adapting to stress and stimuli were highly induced in response to high temperature. The most significantly enriched pathway was “Protein processing in endoplasmic reticulum (ER)”, a quality control system that ensures correct protein folding or degradation of misfolded polypeptides by ER-associated degradation. Other signaling pathways involved in regulation of immune system and post-transcriptional regulation of spliceosome were also critical for thermal adaptation. These findings improve our understanding of the molecular mechanisms of heat stress responses and are useful to develop strategies for the improvement of rainbow trout survival rate during summer high-temperature period.

1. Introduction Accelerated climate change poses a significant threat to the environment and to biological organisms, including poikilothermic fish, for which water temperature is a critical environmental factor. Like other organisms, fish have a preferred temperature, and, when faced with changes in ambient temperature, fish undergo different aspects of physiological adaption, including changes in metabolism and growth rates of individual fish [1,2], and alterations in sociality and activity [3]. Fish are able to cope with daily and seasonal variations in water temperature or quality, but their health and survival are threatened when temperatures reach close to or beyond species-specific thermal tolerances [4]. As a result of global warming, mean water temperatures are increasing globally, according to a report of the Intergovernmental Panel on Climate Change [5]; this will inevitably cause challenges to farmed fish, especially cold-water species, in semi-open aquaculture.

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The alterations in physiology and behavior of fish in response to heat stress vastly differ, even among similar species or species with comparable geographic distribution. Elucidation of these changes at the transcriptome level would facilitate our understanding of the biological and physiological mechanisms by which fish adapt to thermal stress, or fail to respond to it. Transcriptomics has been used to study complex responses by organisms to the environment, interpret functional elements of the genome, and understand the biological processes that occur in natural populations. DNA microarray was the dominant method used for ecological transcriptomics studies for the past decade [6]. However, as mRNA sequencing using next-generation sequencing technologies (RNA-seq) becomes more affordable, RNA-seq has more often been the method of choice to investigate transcriptional response to environmental stress. Abundant RNA-seq reads can construct a complete transcriptome, provide a wealth of information on differential gene

Corresponding author. E-mail address: [email protected] (Z. Liu).

https://doi.org/10.1016/j.fsi.2018.08.002 Received 17 April 2018; Received in revised form 24 July 2018; Accepted 1 August 2018 Available online 02 August 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved.

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stress was performed by increasing the temperature at a relatively constant speed (increments of 1 °C per 24 h) from 18 °C to 24 °C. In a previous study, we show that 24 °C is a “key high-temperature point” that commits rainbow trout from adaptive regulation to injury, according to non-specific immune and metabolism parameters, [18]. Therefore, we selected 18 °C and 24 °C as control and heat stress treatment temperatures, respectively. The aim of this study was to identify kidney-specific genes and relevant pathways in response to heat stress in rainbow trout.

expression, and can be used to identify biological pathways involved in response to thermal stress. Fish are widely used as a model system for medical and genetic research; in addition to their ecological importance and economic value, a rapidly growing body of literature has emerged regarding identifying mechanisms of temperature acclimation in different fish species using RNA-seq [7]. These studies revealed that changes occur in the expression of many genes in response to heat stress, including heat shock protein (HSP) genes and immune-related genes; these are associated with either long-term or short-term heat stress responses. In the majority of species studied, HSP genes are upregulated, as is the case for hybrid catfish [8], crimson spotted rainbow fish [9], rainbow trout [10], redband trout [11], snow trout [12], halfsmooth tongue sole [13], Iberian freshwater fish [14], and genetically modified farmed tilapia [15]. However, HSP genes were downregulated after short-term heat exposure in Pagothenia borchgrevinki [16]; this suggested that a variety of heat coping mechanisms exist among different fish species. Immune related genes were upregulated in crimson spotted rainbow fish [9], snow trout [12], redband trout [11], and tubenose goby [17]. These results were consistent analyses using microarrays and qPCR. These studies also identified important regulatory pathways involved in the response to heat stress, including metabolism, protein folding and degradation, and immune response; modulation of these pathways were observed in a variety of fish species under different stress conditions, indicating that these biological pathways are critical for thermal adaptation. Rainbow trout (Oncorhynchus mykiss), a member of the Salmonidae family, is rapidly becoming an important fish in aquaculture, and is a biologically relevant model to study adaptation and response to heat stress. Farmed fish experience numerous stressors, especially thermal stress, when temperatures are elevated during the summer. Suitable water temperatures for rainbow trout range from 12 to 18 °C. In water at 21 °C, pathological changes were observed in liver tissue, and inflammation resulted from heat stress; moreover, the immune function of the trout was significantly reduced, and tissues were severely injured past 24 °C [18,19]. Previous reports have demonstrated that the primary gene expression changes in heat stress response include molecular chaperone genes (e.g., members of the HSP gene family) [20–23]. Responses to temperature change in rainbow trout has been evaluated using microarray [24], and microarray technology has been used to compare heat stress responses between different rainbow trout strains [25–27]. Under either acute or mild heat stress, many HSP genes were highly expressed in the high-temperature selected strain, or in fish challenged with higher temperatures; similar but distinct tissue-specific expression patterns were also observed, and the links of different temperature-dependent pathways and gene networks were revealed between different breeding lines. A recent study used RNA-seq to discover that several pathways are influenced by heat stress in response to heat stress, including protein metabolism, energy metabolism, and immune system function [9]. These results provide abundant insights and comparable datasets to study heat adaptation processes. It still remains of great interest to perform more comprehensive analyses of tissue-specific responses to heat stress in rainbow trout in order to elucidate the molecular mechanisms of thermal adaptation in fish. The head kidney is a crucial organ that contains cortisol-producing interrenal cells. Cortisol is the primary glucocorticoid that is released when teleost fish are exposed to a stressor, is the end product of activation of the hypothalamic-pituitary-interrenal axis, and has long-term effects on stress adaptation [28–31]. In a study by Li et al., serum cortisol was significantly upregulated in rainbow trout under conditions of heat stress (25 °C for 8 h) [21]. Although the head kidney is associated with the thermal response, the transcriptome data are very limited for stressed kidney tissue in rainbow trout. We previously reported on a heat stress response that is specific for the liver, a crucial metabolic organ [9]. In the present study, we determine transcriptome changes that occur in response to heat stress in rainbow trout head kidney. To simulate a natural aquatic environment, moderate heat

2. Materials and methods 2.1. Animals and ethics approval All methods used in this study were conducted according to the guiding principles of the Chinese Legislation on the Use and Care of Laboratory Animals. The animal protocol was approved by the institutional ethics committee of Gansu Agricultural University. Full-sib rainbow trout were purchased from a trout farm in Yongjing, Gansu Province, China. Fish with a mean weight 400 ± 10.5 g were transferred into a 3000 L aerated water tank and were cultured at 18 °C for seven days. Prior to the experiment, the fish were randomly divided into six groups in 300 L water tanks, and allowed to acclimate for another seven days. To simulate temperature conditions in natural environment, the water temperature in the heat stress groups was increased from 18 °C to 24 °C at a constant rate of 1 °C per 24 h using a temperature control system. After anesthetizing with a lethal dose of MS-222 (Sigma Aldrich Co., St. Louis, USA), the head kidneys were harvested from three female fishes from both the 18 °C control group and the 24 °C heat stress group. Tissues were immediately flash frozen in liquid nitrogen and stored at −80 °C for gene expression profiling analysis. 2.2. RNA isolation and library preparation Head kidney tissue samples were homogenized and total RNA was extracted using a TRIzol reagent, according to standard protocol (Invitrogen, Carlsbad, CA, USA). The purity of the isolated RNA was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and agarose gel electrophoresis. The quality and quantity of the extracted RNA were assessed using a Qubit 2.0 fluorometer (Life Technologies, CA, USA) and a Bioanalyzer 2100 System (Agilent Technologies, CA, USA). Six sequencing libraries were created by reverse-transcription from ∼3 μg of RNA from each sample using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® sequencing (NEB, USA), according to a published protocol [9]. In brief, mRNA was purified using poly-T oligoattached magnetic beads and broken into short fragments using divalent cations in NEBNext® First Strand Synthesis Reaction Buffer (5 × ). First-strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNase H−) and random hexamer primers, reaction buffer, RNase H, and DNA polymerase I. Remaining overhangs were converted into blunt ends by exonuclease/polymerase activities. Before hybridization, the 3′ends of the DNA fragments were mono-adenylated, and the NEBNext® hairpin loop adaptor was ligated. Next, the library fragments were purified using the AMPure XP System (Beckman Coulter, Beverly, USA) to select 150–200 bp cDNA fragments. The PCR products from enriched fragments with ligated sequencing adapters were purified using the AMPure XP System. An Agilent Bioanalyzer 2100 System was used to assess the library quality. According to the manufacturer's instructions, the cBot Cluster Generation System with the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, CA, USA) was used to cluster index-coded samples. Six library preparations of cluster generation were sequenced on the Illumina Hiseq™ 4000 platform to generate 150-bp paired-end raw reads. 33

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the mRNA transcripts were detected as FPKM > 1, and 3.55–4.32% was detected as FPKM > 60. The FPKM interval and the total genes in each library are shown in Table S2. Pearson's correlation coefficients (R2) for gene expression from the replicate samples were used to assess the accuracy and reliability of the results. The R2 values were all greater than 0.92 between the CHK1, CHK2, and CHK3 samples, as well as between the HHK1, HHK2, and HHK3 samples (Figure S1), confirming the robustness of the biological replicates and the reliability of the results.

2.3. Illumina sequencing, assembly, and differential gene expression analysis After removing reads representing adaptor contamination, reads with high poly-N (i.e. more than 10% unidentified nucleotides) and low-quality reads, the clean reads were screened against the rainbow trout reference genome and aligned to the reference genome using TopHat v2.0.1.2 [32]. The reference genome and gene model annotation files were downloaded from the genome website (https://www. genoscope.cns.fr/trout/) [33], and an index of the reference genome was created using Bowtie v2.2.3. The read numbers mapped to each gene were counted using HTSeq V0.6.1. Fragments per kilobase of exon per million mapped reads (FPKM) were used to estimate gene expression levels of each gene. Analysis of genes differentially expressed between the control and heat stress groups was performed using the DESeq R package 1.18.0; an adjusted P-value < 0.05 was used to identify significantly differentially expressed genes (DEGs) [34]. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) statistical enrichment analysis of DEGs was performed using the GO seq R package [35] and KOBAS software [36], respectively. The KEGG pathway annotations were generated using the KEGG database (http://www. genome.jp/kegg).

3.2. Differential gene expression in the rainbow trout head kidney transcriptome after exposure to heat stress In total, 39,352 genes were obtained from the CHK and HHK groups and mapped to the rainbow trout genome. A total of 36,271 genes were expressed in both groups, while 1534 genes were uniquely expressed in the CHK group, and 1547 genes were uniquely expressed the HHK group (Fig. 1A). With an adjusted P-value threshold of P < 0.05, a total of 443 DEGs were identified between the CHK and HHK groups. Of these 443 DEGs, 125 were novel genes, 236 genes were downregulated and 207 were upregulated (Fig. 1B). The expressions of DEGs from the six samples were clustered into two distinct groups by hierarchical clustering (Fig. 2). Among these DEGs, 11 genes (|log2 foldchange| > 4) were strongly significantly differentially expressed under heat stress, including Novel04973 (33.8-fold), XRCC6 (29.8-fold), HSP90AA (27.8-fold), DNAJB6 (24.5-fold), HSP90AB (21.6-fold), LEC (21.4-fold), DNAH1 (19.1-fold), GPR158 (18.9-fold), Novel03947 (16.8-fold), PVR131 (−24.9-fold), and SCN4AB (−28.9-fold). 83 genes (2 < |log2 fold-change| < 4) were mildly induced under heat stress, including one or several members of different HSP families. For example, HSP47A, HSP47B, HSP90-alpha, HSP70, HSC71, HSPA4, HSPA8, DNAJA1 and DNAJB4, these genes showed significant upregulation in the range of 14.8 (HSP47B) to 4.8-fold (HSPA8). CDC37 (4.8-fold) and AHSA1 (4.2-fold) were two cofactors as HSP90 co-chaperone, which were also mildly upregulated in head kidney of rainbow trout under heat stress condition. Other slightly temperature-regulated HSP-encoding genes included HSPA4L (3.5-fold), DNAJB1 (3.4-fold), HSPA4L (3.5-fold), HSPA5 (2.9-fold), DNAJA1 (2.8-fold), HSPD1 (2.5-fold), HSP90BA (2.5-fold) and HSP90BB (1.6-fold). Additional details of the DEGs are presented in Table S3.

2.4. Quantitative real-time (RT)-qPCR analysis RT-qPCR was performed on 13 DEGs selected from the RNA sequencing data according to potential functional importance. Primers were designed according to sequencing data of the rainbow trout head kidney transcriptome using Primer Premier 5.0 (Table S1). First-strand cDNA was synthesized using the PrimerScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China), according to the manufacturer's instructions. RT-qPCR was performed on of each sample in triplicate using SYBR® Premix Ex Taq (TaKaRa, Dalian, China) on a LightCycler® 480 Instrument II (Roche, Switzerland) in a 20 μL reaction volume. βactin was used as an internal control to normalize gene expression level, as β-actin expression showed no change in response to temperature in preliminary experiments [23]. The cycling parameters for the PCR amplification were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Amplification specificity was checked by melting curve analysis. The relative expression of target gene transcripts was calculated using the comparative Ct method (2-ΔΔCt), and subjected to statistical analysis with SPSS software (version 21).

3.3. GO enrichment and KEGG pathway analysis GO enrichment was performed to evaluate the biological and functional implications of the identified DEGs. DEGs were classified into three major functional categories (‘biological processes’, ‘molecular function’, and ‘cellular components’) and were enriched in 13 GO categories (Fig. 3 and Table S4). The 13 significantly enriched GO categories (corrected P < 0.05) included 12 in “biological process” and one in “molecular function”. The 12 enriched pathway sub-categories belonging to ‘biological processes’ included ‘protein folding’ (GO:0006457), ‘cell morphogenesis’ (GO:0000902), ‘cellular component morphogenesis’ (GO:0032989), ‘cellular developmental process’ (GO:0048869), ‘anatomical structure development’ (GO:0048856), ‘anatomical structure morphogenesis’ (GO:0009653), ‘cell redox homeostasis’ (GO:0045454), ‘cellular homeostasis’ (GO:0019725), ‘single-organism developmental process’ (GO:0044767), ‘developmental process’ (GO:0032502), ‘homeostatic process’ (GO:0042592), and ‘regulation of biological quality’ (GO:0065008). 6 (GO: 0048869, 0048856, 0045454, 0019725, 0032502 and 0065008) of 12 enriched categories were biological processes involved in the maintenance of an internal steady state, or in adapting to changes in environmental conditions. The enriched sub-category belonging to ‘molecular function’ was ‘unfolded protein binding’ (GO:0051082). Additionally, seven subcategories were enriched, representing more than 15 DEGs. The top 20 enriched pathways are shown in Fig. 4. Among these pathways, the

3. Results 3.1. RNA-seq analysis to determine rainbow trout head kidney transcriptome profiles Six cDNA libraries, including three from the control group (CHK1, CHK2, CHK3) and three from the heat-treated group (HHK1, HHK2, HHK3), were constructed and analyzed by high-throughput sequencing. An overview of the reads and quality filtering of the six libraries is presented in Table 1. A total of 290, 962, 188 raw reads were obtained, and were deposited to the National Center for Biotechnology Information under the accession number SRP137926. After trimming and filtering the raw reads, 281,663,426 clean reads were generated from the six libraries. From these six libraries, 33,322,323, 30,316,774, 28,433,976, 32,496,243, 32,403,321, and 31,186,205 reads were mapped to the rainbow trout genome, representing 65.73–68.09% of the clean reads from six samples, respectively (Table 1). The mapped reads represented slightly lower than 70% of the rainbow trout genome; nevertheless, it is established that analysis of DEGs based on the genome are more reliable than de novo transcriptome analysis. The FPKM were used to assess gene expression levels by calculating the read numbers mapped to each gene. In six samples, 62.65–65.4% of 34

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Table 1 Summary of sequencing data quality and the statistics of the transcriptome assemblies. Sample name

CHK1a

CHK2a

CHK3a

HHK1b

HHK2b

HHK3b

Raw reads Clean reads Clean bases Q20 (%) Q30 (%) GC content (%) Total mapped Uniquely mapped

51705292 50097732 7.51G 95.98 90.76 48.89 33322323 (66.51%) 32670072 (65.21%)

46131014 44760312 6.71G 96.02 90.8 48.9 30316774 (67.73%) 29716119 (66.39%)

44643742 43257030 6.49G 95.98 90.8 49.03 28433976 (65.73%) 27859251 (64.4%)

50792882 49109566 7.37G 95.89 90.66 49.49 32496243 (66.17%) 31775811 (64.7%)

50358754 48636710 7.3G 95.84 90.47 49.72 32403321 (66.62%) 31806483 (65.4%)

47330504 45802076 6.87G 96.06 90.89 50.07 31186205 (68.09%) 30534411 (66.67%)

a b

CHK1, CHK2, CHK3 indicate libraries derived from the head kidney tissue of control rainbow trout in three biological replicates. HHK1, HHK2, HHK3 indicate libraries derived from the head kidney tissue of heat-treated rainbow trout in three biological replicates.

Fig. 1. Comparative results of RNA-seq and differentially expressed gene distributions between the head kidney of control rainbow trout (CHK) and heat-treated rainbow trout (HHK). (A) Venn diagram showing genes expressed only in the CHK group (yellow circle), expressed only in the HHK group (light red circle), and common to both groups (intersection). (B) Volcano scatter plot of differentially expressed genes (CHK vs. HHK). Red points represent the genes upregulated with log2 (fold change) > 1 and adjusted P-value (padj) < 0.05, i.e. -log10 (padj) ≥ 1.3. Green points represent genes downregulated with log2 (fold change) < −1 and padj < 0.05, i.e. -log10 (padj) ≥ 1.3. Blue points represent genes with no significant differences. Fold change = normalized gene expression of the HHK group/ normalized gene expression of the CHK group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

significantly enriched pathways (corrected P < 0.05) included ‘genetic information processing’ (folding, sorting and degradation/protein processing in endoplasmic reticulum; transcription/spliceosome), ‘cellular processes’ (transport and catabolism-phagosome), and ‘organismal systems’ (immune system-NOD-like receptor signaling pathway). There was an overrepresentation of the KEGG pathway associated with ‘protein processing in the endoplasmic reticulum (ER)’, and 39 genes were enriched in this pathway (Table S5).

to rainbow trout cultivation and information about heat stress response in fishes has increased rapidly over the past few years, the molecular mechanism remains not clear on rainbow trout response to heat stress. In order to identify the kidney specific genes and its pathway in response to the mild heat stress from rainbow trout, the transcriptome profiles of rainbow trout head kidney were evaluated by RNA-seq. A total of 39,352 genes were obtained from six libraries and 443 DEGs were screened using a rigorous set of threshold. Many biological processes were significantly altered and some pathways like protein processing, regulation of immune system, phagosome & NLR pathway were found after mild heat stress treatment in the head kidney. These results can featured for the scientific community to find out the further more genes and it regulation for the pathways especially protein folding and chaperons.

3.4. Validation of RNA-seq data by RT-qPCR The expression profiles of 13 DEGs were evaluated by RT-qPCR and compared with the expression profiles from the RNA-seq analysis. The data from RT-qPCR analysis exhibited excellent agreement with the RNA-seq data (Fig. 5), and the Pearson's correlation coefficient of 0.91 between RNA-seq and RT-qPCR gene expression confirmed the reproducibility and reliability of the RNA-seq method.

4.1. Regulation of HSPs gene expression HSP genes play a central role in cellular homeostasis by facilitating a rapid increase in the synthesis of stress proteins in response to sudden adverse environmental changes [37]. One of the types of proteins most strongly induced by heat stress in head kidney of rainbow trout were the HSPs, which comprise an evolutionally conserved protein family, and are involved in the folding and unfolding of other proteins in major cellular compartments. In this present study, numerous HSP genes were upregulated after exposure to heat stress. These included five members of the HSP90 family, HSP90AA, HSP90AB, HSP90B1, HSP90BA, HSP90BB, and two cofactors, CDC37 and AHSA1, which were all upregulated in head kidney of rainbow trout under heat stress conditions. The HSP90 protein family assists in protein folding and stabilization, and is important in responding to stress and maintaining cellular

4. Discussion The rainbow trout, an “aquatic lab-rat”, is a very important teleost fish species for research, and it is the most widely cultivated cold freshwater fish species with an important economic value in the world. The rainbow trout is used in a variety of biological research areas, such as comparative physiology, molecular evolution, and stress response, since it has undergone multiple rounds of genome duplication [33]. In recent years, extreme weather has critical effects on physiology and behavior of aquatic animal. For example hotter summer than normal was considered as an important incentive for rainbow trout mortality syndrome in summer. Although high temperatures are a serious threat 35

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Fig. 2. Hierarchical clustering of differentially expressed (DE) mRNAs among the six sample libraries. Heatmap of the count data for DE mRNA libraries for the DEGs identified between the CHK and HHK groups. The DE mRNA heatmap represents the top 100 DEGs. 36

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Fig. 3. Gene ontology (GO) classifications of differentially expressed genes (DEGs) between the HHK and CHK groups.

kidney was examined to identify the major biological process and mechanisms involved in adaptation to higher temperatures. GO analysis revealed that a many biological processes were significantly altered. Consistent with other studies [8–10,46–48], exposure to increased temperatures was associated with the GO term related to ‘protein folding’ in rainbow trout. This ‘protein folding’ GO term is a process involved in assisting in assembly of single chain polypeptides or multisubunit complexes into the correct tertiary structure. The life process of protein included four stages: synthesizing, folding, assembling and degradation. Under stress conditions, disruption of any one stage may affect the function of protein, and even impact the normal function of whole cell. Given that increases in temperatures can cause protein denaturation, it is expected that protein-folding machinery would correspondingly increase along with temperature [8]. The upregulation of expression of genes with chaperoning activities belonging to the ‘protein folding’ GO term suggests that that protein-folding rates increase to maintain the function of whole cell under conditions of elevated temperatures. Moreover, the most significantly enriched KEGG pathway in the head kidney of rainbow trout in response to heat stress was ‘protein processing in the ER’, with 38 genes upregulated and 1 gene downregulated compared to control head kidney. This enriched pathway was also observed in large yellow croaker [41], amur carp [47], half-smooth tongue sole [13], and in the liver of rainbow trout [9] in response to heat stress. In the ER, proteins undergo strict quality control (QC) to ensure correct folding, or to facilitate degradation of misfolded polypeptides by a series of tightly regulated processes called ER-associated degradation (ERAD). The QC system include the lectin chaperones calnexin (CNX) and calreticulin (CRT), as well as the co-chaperone ERp57, a glycoprotein specific thiol-disulfide oxidoreductase; together these comprise the so-called ‘calreticulin/calnexin cycle’ [49]. Under conditions of heat stress, the expression of CALR, protein disulfide-

homeostasis [38]. CDC37 is a HSP90 co-chaperone that functions as an accessory factor for HSP90. In the absence of HSP90, CDC37 can function as a molecular chaperone alone, and can interact with other HSP90 co-chaperones [39]. AHSA1 is a HSP90 chaperone and stimulates the ATPase activity of HSP90 [40]. The upregulation of these genes is similar to results of heat stress studies in catfish [8], large yellow croaker [41], and the liver of rainbow trout [9]. In addition, six members of the HSP40 family were upregulated, including DNAJA1, DNAJB1, DNAJB4, DNAJB6, HSP47A, and HSP47B. Members of the HSP40 family are important for protein translation, translocation, folding, and unfolding, and can regulate the activity of HSP70 proteins by stimulating their ATPase activity and by stabilizing their interactions with substrate proteins [42]. HSP70 proteins act as chaperones of HSP40 proteins, which bind to HSP70 proteins through a conserved J domain. Seven members of the HSP70 family were upregulated in the rainbow trout head kidney in response to heat stress: HSP70, HSPA4, HSC71, HSPA8, HSPA4L, HSPA5, and HSPA9. HSP70 possesses general cytoprotective properties, and overexpression of HSP70 enhances anti-apoptotic activity against stressful and noxious stimuli [43,44]. The HSPD1 gene was upregulated in rainbow trout head kidney by heat stress. HSPD1 belongs to the HSP60 family, is localized inside mitochondria, and acts as a chaperone in concert with HSP10 to assist protein folding inside the mitochondria [45]. These HSP genes that were strongly induced by heat stress facilitate cytoprotection, anti-stress responses, and maintenance of protein homeostasis, and can protect the rainbow trout from heat stress. These data indicate the importance of transcriptional regulation of HSP genes during the heat stress response.

4.2. Adaptive regulation of protein processing The transcriptome of control and heat-treated rainbow trout head 37

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Fig. 4. Scatterplot of KEGG pathways enriched in the differentially expressed genes (DEGs) between the CHK and HHK groups. Rich factor is the ratio of the DEG number to the total gene number in a given pathway. The size and color of the dots represent the gene number and range of the q value, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

including HYOU1, HSPA5, and HSP90B1, were upregulated, indicating that the ratio of misfolded protein is increased when ambient water temperature increases from 18 °C to 24 °C. Because accumulation of misfolded proteins hampers ER function and can eventually lead to cell death, rapid removal of misfolded polypeptides by the ERAD pathway is a critical response. ERAD involves chaperones of the HSP family that act in concert with ubiquitin ligases [52]. In response to heat stress, the expression of genes from the HSP70, HSP90, and HSP40 families were upregulated in rainbow trout head kidney, likely to promote

isomerase A3 (PDIA3) and T-complex protein 1 subunit gamma (CCT3) are upregulated to promote correct protein folding. Under heat stress conditions, PDI may interact with CNX and can recover partial function of CNX, thereby promoting the recovery and maintenance of normal kidney function in rainbow trout [50]. The misfolded proteins in the ER lumen are recognized by luminal chaperones such as HSP40, BIP, GRP94, and NEF, which deliver misfolded proteins to dislocons at the ER membrane for ubiquitin-dependent degradation [51]. In the head kidney of rainbow trout, the gene expressions of luminal chaperones,

Fig. 5. Comparison of gene expression levels determined by RNA-seq and RT-qPCR methods. The relative expression values were normalized to β-actin gene expression. Error bars indicate standard deviation. Log2FC refers to the log2 fold-change between the HHK and CHK groups. 38

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temperature changes, the spliceosome was also the most enriched pathway in zebra fish [1], large yellow croaker [41], and amur carp [47]. Protein complexes containing PRP19, which is comprised of hPRP19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, play a key role during catalytic activation of the spliceosome. PRP19 and its related proteins are also major components of the spliceosome's catalytic RNP core [66]. The expression of HSP73-related genes, including HSC70, HSPA8, and HSC71 were upregulated in rainbow trout head kidney response to heat stress. In general, HSP70 mRNA expression is enhanced when heat stress represses global gene expression. Pretranslationally, HSP70 mRNA is excluded from stress granules and is preferentially translated when pre-mRNA splicing and global cap-dependent translation are repressed in response to heat stress. Posttranslationally, nascent HSP70 protein is thermodynamically stable at elevated temperatures, and intron-lacking HSP70 mRNA circumvents a host of decay pathways, allowing for the rapid commencement of chaperone activity after HSP70 synthesis to attenuate the heat shock response and protect against subsequent injury [67].

degradation of misfolded proteins and to maintain cell homeostasis. The hypoxia-induced gene, HYOU1, was also upregulated by heat stress in the ‘protein processing in the ER’ pathway, indicating that thermal and hypoxic tolerances are positively associated with each other [53], and suggesting that heat stressed rainbow trout suffer from impaired aerobic metabolism [54]. A possible link between temperature and low oxygen saturation has been reported in crucian carp [55], Atlantic salmon [56], and half-smooth tongue sole [13]. BAX, a gene downregulated by heat stress, is in the ‘protein processing in the ER’ pathway. Apoptosis, or programmed cell death, is critical for removal of dead cells, and is linked with various biological processes. Repression of apoptosis may lead to cancer and autoimmune diseases, while high cell death rates may increase ischemic conditions and promote neurodegeneration [57]. The downregulation of BAX expression in rainbow trout under heat stress may suggest that the trout transitioned from adaptive regulation to injury in response to heat stress [18]. 4.3. Immune response

5. Conclusions

The most overrepresented biological function in kidney tissue in response to environmental stress was ‘cellular immune response’ [27]. In the present study, two enriched pathways were involved in regulation of immune system: ‘phagosome pathway’ and ‘NOD-like receptor (NLR) signaling pathway’. Phagocytosis is a central mechanism in tissue remodeling, inflammation, and in defense against infectious agents. An early phagosome is formed when specific receptors on the phagocyte surface recognize ligands, which progressively acquire digestive characteristics. Lysosomes fuse with phagosomes during maturation, and release toxic products that kill and digest invading pathogenic microorganisms [58]. In the common carp, phagocytic rate and activity was significantly lower in elevated water temperatures [59], suggesting that increased susceptibility to disease may be a main cause for mortality in common carp during summer months [60]. NADPH oxidase, part of the phagosome pathway, plays a crucial role in host defense by producing superoxide anions to kill invading microbes [61]. Under heat stress conditions, the expression of genes involved in NADPH oxidase were significant downregulated in rainbow trout head kidney, implying that the immune function of the head kidney of rainbow trout was suppressed. The major histocompatibility complex (MHC) is the most important factor with respect to infection and autoimmunity in the vertebrate genome, and is crucial in adaptive and innate immunity [62]. MHC class I and class II molecules have similar fundamental roles in regulating immune responses, and can engage in cross-presentation of antigens [63]. The expressions of MHC-related genes were upregulated in head kidney of rainbow trout under heat stress, and may facilitate antigen processing and presentation when trout are exposed to high temperatures. Another immune-related pathway was the NLR signaling pathway. The genes involved in this pathway, including HSP90 family and its cochaperone suppressor of SGT1, are required for maintenance of NLRtype sensors [64]. NOD1, a prototypic NLR, can sense bacterial peptidoglycan fragments that escape from endosomal compartments in the cytosol, and drives activation of the NF-κB and MAPK pathways, cytokine production, and apoptosis. NALR3 can induce caspase-1 activation through the assembly of an inflammasome [65]. When the immune systems of rainbow trout are challenged with elevated water temperature, the upregulation of HSP90 and SGT1 may enhance defenses by inducing the activation of NOD1 and caspase-1.

Deep sequencing-based expression profiling identified heat stress responsive genes in rainbow trout head kidney is reported in this study. A large number of significantly differentially expressed genes were found in response to heat stress, including up-regulated genes encoding diverse HSPs; various biological reactions may be impacted, including portein folding and degradation, changes in the immune systems, posttranscriptional regulation of spliceosome. These results provide a better understanding of the molecular mechanisms regulating thermal response of rainbow trout, which will help to prevent and treat damage to fish caused by high water temperatures. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31460687). Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.fsi.2018.08.002 References [1] Y. Long, G. Song, J. Yan, X. He, Q. Li, Z. Cui, Transcriptomic characterization of cold acclimation in larval zebrafish, BMC Genom. 14 (2013) 612. [2] W. Zuo, M.E. Moses, G.B. West, C. Hou, J.H. Brown, A general model for effects of temperature on ectotherm ontogenetic growth and development, Proc. Biol. Sci. 279 (2012) 1840–1846. [3] T. Bartolini, S. Butail, M. Porfiri, Temperature influences sociality and activity of freshwater fish, Environ. Biol. Fish. 98 (2014) 825–832. [4] T.P. Hurst, Causes and consequences of winter mortality in fishes, J. Fish. Biol. 71 (2007) 315–345. [5] IPCC, T.F. Stocker, D. Qin, G.K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex, P.M. Midgley (Eds.), Summary for Policymakers in Climate Change: the Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge Univ. Press, 2013, pp. 3–29. [6] M. Alvarez, A.W. Schrey, C.L. Richards, Ten years of transcriptomics in wild populations: what have we learned about their ecology and evolution? Mol. Ecol. 24 (2015) 710–725. [7] R.A. Oomen, J.A. Hutchings, Transcriptomic responses to environmental change in fishes: insights from RNA sequencing, Facets 2 (2017) 610–641. [8] S. Liu, X. Wang, F. Sun, J. Zhang, J. Feng, H. Liu, K.V. Rajendran, L. Sun, Y. Zhang, Y. Jiang, E. Peatman, L. Kaltenboeck, H. Kucuktas, Z. Liu, RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding, and degradation in response to heat stress in catfish, Physiol. Genom. 45 (2013) 462–476. [9] Y. Li, J. Huang, Z. Liu, Y. Zhou, B. Xia, Y. Wang, Y. Kang, J. Wang, Transcriptome analysis provides insights into hepatic responses to moderate heat stress in the rainbow trout (Oncorhynchus mykiss), Gene 619 (2017) 1–9. [10] S. Smith, L. Bernatchez, L.B. Beheregaray, RNA-seq analysis reveals extensive

4.4. Post-transcriptional regulation The spliceosome is a dynamic assembly of proteins and RNAs that catalyzes the excision of intron sequences from nascent mRNAs, and various spliceosome forms (e.g. A-, Be, and C-complexes) have been identified. In rainbow trout, 11 genes responsive to heat stress were significantly enriched in the spliceosome pathway. In response to 39

Fish and Shellfish Immunology 82 (2018) 32–40

J. Huang et al.

[11] [12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29] [30] [31]

[32]

[33]

[34] [35] [36]

[37]

transcriptional plasticity to temperature stress in a freshwater fish species, BMC Genom. 14 (2013) 375. S.R. Narum, N.R. Campbell, Transcriptomic response to heat stress among ecologically divergent populations of redband trout, BMC Genom. 16 (2015) 103. A. Barat, P.K. Sahoo, R. Kumar, C. Goel, A.K. Singh, Transcriptional response to heat shock in liver of snow trout (Schizothorax richardsonii)–a vulnerable Himalayan Cyprinid fish, Funct. Integr. Genom. 16 (2016) 203–213. L. Guo, Y. Wang, S. Liang, G. Lin, S. Chen, G. Yang, Tissue-overlapping response of half-smooth tongue sole (Cynoglossus semilaevis) to thermostressing based on transcriptome profiles, Gene 581 (2016) 97–104. T.F. Jesus, A.R. Grosso, V.M. Almeida-Val, M.M. Coelho, Transcriptome profiling of two Iberian freshwater fish exposed to thermal stress, J. Therm. Biol. 55 (2016) 54–61. J. Qiang, W.J. Bao, F.Y. Tao, J. He, X.H. Li, P. Xu, L.Y. Sun, The expression profiles of miRNA-mRNA of early response in genetically improved farmed tilapia (Oreochromis niloticus) liver by acute heat stress, Sci. Rep. 7 (2017) 8705. T.J. Huth, S.P. Place, RNA-seq reveals a diminished acclimation response to the combined effects of ocean acidification and elevated seawater temperature in Pagothenia borchgrevinki, Mar. Genomics 28 (2016) 87–97. K.W. Wellband, D.D. Heath, Plasticity in gene transcription explains the differential performance of two invasive fish species, Evol. Appl. 10 (2017) 563–576. B.P. Xia, Z. Liu, Y.Z. Zhou, Y.J. Wang, J.Q. Huang, Y.J. Li, Y.J. Kang, J.F. Wang, X.X. Liu, Effects of chronic heat stress on part of serum non-specific immunity parameters in rainbow trout (Oncorhynchus mykiss), J. Agric. Biotechnol. 25 (2017) 1078–1085. Y. Zhou, Z. Liu, B. Xia, Y. Kang, Y. Wang, X. Liu, Effect of continuing heat stress on the liver tissue morphology of rainbow trout (Oncorhynchus mykiss), J Gansu Agric. Univ. 52 (2017) 1–5. N. Ojima, M. Yamashita, S. Watabe, Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells, Biochem. Biophys. Res. Commun. 329 (2005) 51–57. Z. Li, Z. Liu, Y.N. Wang, Y.J. Kang, J.F. Wang, H.N. Shi, J.Q. Huang, L. Jiang, Effects of heat stress on serum cortisol, alkaline phosphatase activity and heat shock protein 40 and 90β mRNA expression in rainbow trout (Oncorhynchus mykiss), Biologia 71 (2016) 109–115. N. Ojim, M. Yamashit, S. Watabe, Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress, Biochim. Biophys. Acta 1681 (2005) 99–106. N.H. Shi, Z. Liu, J.P. Zhang, Y.J. Kang, J.F. Wang, J.Q. Huang, M.W. Wang, Effect of heat stress and recovery on mRNA expression of heat-shock protein (Hsp60) in different tissues of rainbow trout (Oncorhynchus mykiss), Genet. Mol. Res. 14 (2015) 5280–5286. J.W. Lewis, T.S. Hori, M.L. Rise, P.J. Walsh, S. Currie, Transcriptome responses to heat stress in the nucleated red blood cells of the rainbow trout (Oncorhynchus mykiss), Physiol. Genom. 42 (2010) 361–373. A. Rebl, M. Verleih, J.M. Köbis, C. Kühn, K. Wimmers, B. Köllner, T. Goldammer, Transcriptome profiling of gill tissue in regionally bred and globally farmed rainbow trout strains reveals different strategies for coping with thermal stress, Mar. Biotechnol. 15 (2013) 445–460. E. Tan, S. Kinoshita, Y. Suzuki, T. Ineno, K. Tamaki, A. Kera, K. Muto, T. Yada, S. Kitamura, S. Asakawa, S. Watabe, Different gene expression profiles between normal and thermally selected strains of rainbow troutas revealed by comprehensive transcriptome analysis, Gene 576 (2016) 637–643. M. Verleih, A. Borchel, A. Krasnov, A. Rebl, T. Korytář, C. Kühn, T. Goldammer, Impact of thermal stress on kidney-specific gene expression in farmed regional and imported rainbow trout, Mar. Biotechnol. 17 (2015) 576–592. S. Winberg, A. Nilsson, P. Hylland, V. Söderstöm, G.E. Nilsson, Serotonin as a regulator of hypothalamic-pituitary-interrenal activity in teleost fish, Neurosci. Lett. 230 (1997) 113–116. B.A. Barton, Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids, Integr. Comp. Biol. 42 (2002) 517–525. N.R. Bury, A. Sturm, Evolution of the corticosteroid receptor signalling pathway in fish, Gen. Comp. Endocrinol. 153 (2007) 47–56. A. Rafacho, H. Ortsater, A. Nadal, I. Quesada, Glucocorticoid treatment and endocrine pancreas function: implications for glucose homeostasis, insulin resistance and diabetes, J. Endocrinol. 233 (2014) 49–62. C. Trapnell, A. Roberts, L. Goff, G. Pertea, D. Kim, D.R. Kelle, H. Pimentel, S.L. Salzberg, J.L. Rinn, L. Pachter, Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks, Nat. Protoc. 7 (2012) 562–578. C. Berthelot, F. Brunet, D. Chalopin, A. Juanchich, A. Bernard, B. Noël, et al., The rainbow trout genome provides novel insights into evolution after whole-genome duplication in vertebrates, Nat. Commun. 5 (2014) 3657. S. Anders, W. Huber, Differential expression analysis for sequence count data, Genome Biol. 11 (2010) 106. M.D. Young, M.J. Wakefield, G.K. Smyth, A. Oshlack, Gene ontology analysis for RNA-seq: accounting for selection bias, Genome Biol. 11 (2010) 14. X. Ma, T. Cai, J.G. Olyarchuk, L. Wei, Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary, Bioinformatics 21 (2005) 3787–3793. M. Yamashita, T. Yabu, N. Ojima, Stress protein HSP70 in fish, Aqua 3 (2010) 111–141.

[38] S.E. Jackson, Hsp90 structure and function, Top. Curr. Chem. 328 (2013) 155–240. [39] M. MacLean, D. Picard, Cdc37 goes beyond Hsp90 and kinases, Cell Stress Chaperones 8 (2003) 114–119. [40] J. Shao, L. Wang, C. Zhong, R. Qi, Y. Li, AHSA1 regulates proliferation, apoptosis, migration, and invasion of osteosarcoma, Biomed. Pharmacother. 77 (2016) 45–51. [41] B. Qian, L. Xue, Liver transcriptome sequencing and de novo annotation of the large yellow croaker (Larimichthy crocea) under heat and cold stress, Mar. Genomics 25 (2016) 95–102. [42] X.P. Qiu, Y.M. Shao, S. Miao, L. Wang, The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones, Cell. Mol. Life Sci. 63 (2006) 2560–2570. [43] M. Tranter, R.N. Helsley, W.R. Paulding, M. McGuinness, C. Brokamp, L. Haar, Y. Liu, X. Ren, W.K. Jones, Coordinated post-transcriptional regulation of Hsp70.3 gene expression by microRNA and alternative polyadenylation, J. Biol. Chem. 286 (2011) 29828–29837. [44] T. Kondo, T. Matsuda, M. Tashima, H. Umehara, N. Domae, K. Yokoyama, T. Uchiyama, T. Okazaki, Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance, J. Biol. Chem. 272 (1997) 18033–18037. [45] F. Cappello, E. Conway de Macario, A.J.L. Macario, HspD1 (heat shock 60kDa protein 1), Atlas Genet. Cytogenet. Oncol. Haematol. 19 (2015) 575–585. [46] N.L. Quinn, C.R. McGowan, G.A. Cooper, B.F. Koop, W.S. Davidson, Identification of genes associated with heat tolerance in Arctic charr exposed to acute thermal stress, Physiol. Genom. 43 (2011) 685–696. [47] L. Liang, Y. Chang, X. He, R. Tang, Transcriptome analysis to identify cold-responsive genes in Amur Carp (Cyprinus carpio), PLoS One 10 (2015) e0130526. [48] T.S. Hori, A.K. Gamperl, L.O. Afonso, S.C. Johnson, S. Hubert, J. Kimball, S. Bowman, M.L. Rise, Heat-shock responsive genes identified and validated in Atlantic cod (Gadus morhua) liver, head kidney and skeletal muscle using genomic techniques, BMC Genom. 11 (2010) 72. [49] G.S. Gupta, Lectins in Quality Control: Calnexin and Calreticulin, Springer Vienna, 2012, pp. 29–56. [50] H. Zhang, J. He, Y. Ji, A. Kato, Y. Song, The effect of calnexin deletion on the expression level of PDI in Saccharomyces cerevisiae under heat stress conditions, Cell. Mol. Biol. Lett. 13 (2008) 38–48. [51] R.K. Plemper, S. Böhmler, J. Bordallo, T. Sommer, D.H. Wolf, Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation, Nature 388 (1997) 891–895. [52] A.L. Goldberg, Protein degrada-tion and protection against misfolded or damaged proteins, Nature 426 (2003) 895–899. [53] H.O. Pörtner, M.A. Peck, Climate change effects on fishes and fisheries: towards a cause-and-effect understanding, J. Fish. Biol. 77 (2010) 1745–1779. [54] Z. Xu, T. Li, E. Li, K. Chen, Z. Ding, J.G. Qin, L. Chen, J. Ye, Comparative transcriptome analysis reveals molecular strategies of oriental river prawn Macrobrachium nipponense in response to acute and chronic nitrite stress, Fish Shellfish Immunol. 48 (2016) 254–265. [55] E. Rissanen, H. Tranberg, J. Sollid, G. Nilsson, M. Nikinmaa, Temperature regulates hypoxia-inducible factor-1 (HIF-1) in a poikilothermic vertebrate, crucian carp (Carassius carassius), J. Exp. Biol. 209 (2006) 994–1003. [56] P.A. Olsvik, V. Vikeså, K.K. Lie, E.M. Hevrøy, Transcriptional responses to temperature and low oxygen stress in Atlantic salmon studied with next-generation sequencing technology, BMC Genom. 14 (2013) 817. [57] P.E. Czabotar, G. Lessene, A. Strasser, J.M. Adams, Control of apoptosis by the BCL2 protein family: implications for physiology and therapy, Nat. Rev. Mol. Cell Biol. 15 (2014) 49–63. [58] A.A. Herskovits, V. Auerbuch, D.A. Portnoy, Bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system, PLoS Pathog. 3 (2007) e51. [59] V. Ouellet, F. Pierron, M. Mingelbier, M. Fournier, M. Fournier, P. Couture, Thermal stress effects on gene expression and phagocytosis in the common carp (Cyprinus Carpio): a better understanding of the summer 2001 st. Lawrence River Fish Kill, Open Fish Sci. J. 6 (2013) 99–106. [60] V. Ouellet, M. Mingelbier, A. St-Hilaire, J. Morin, Frequency analysis as a tool for assessing adverse conditions during a massive fish kill in the St. Lawrence River, Canada, Water Qual. Res. J. Can. 45 (2011) 47–57. [61] T.E. Decoursey, E. Ligeti, Regulation and termination of NADPH oxidase activity, Cell. Mol. Life Sci. 62 (2005) 2173–2193. [62] R. Horton, L. Wilming, V. Rand, B.C. Lovering, E.A. Bruford, V.K. Khodiyar, M.J. Lush, S. Povey, C.C. Talbot Jr., N.W. Wright, H.M. Wain, J. Trowsdale, A. Ziegler, S. Beck, Gene map of the extended human MHC, Nat. Rev. Genet. 5 (2004) 889–899. [63] J.M. Vyas, A.G. Van der Veen, H.L. Ploegh, The known unknowns of antigen processing and presentation, Nat. Rev. Immunol. 8 (2008) 607–618. [64] K. Shirasu, The HSP90-SGT1 chaperone complex for NLR immune sensors, Annu. Rev. Plant Biol. 60 (2009) 139–164. [65] M.H. Shaw, T. Reimer, Y.G. Kim, G. Nuñez, NOD-like receptors (NLRs): bona fide intracellular microbial sensors, Curr. Opin. Immunol. 20 (2008) 377–382. [66] E.J. Song, S.L. Werner, J. Neubauer, F. Stegmeier, J. Aspden, D. Rio, J.W. Harper, S.J. Elledge, M.W. Kirschner, M. Rape, The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome, Genes Dev. 24 (2010) 1434–1447. [67] J.T. Silver, E.G. Noble, Regulation of survival gene hsp70, Cell Stress Chaperones 17 (2012) 1–9.

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