The objective of the study was to understand changes occurring in the metabolism of fish when exposed to temperature stress. The study throws light on the differential expression of proteins under stress. During normal conditions the common proteins show fishes undergoing gluconeogenesis that only happens during the starvation conditions, when energy in the body is low and the fish uses stored energy. Differential expression of proteins show how fish adapts to environmental changes in fish through metabolism at cellular level. The 25 most relevant pathways sorted by p-value for proteins expressed in experimental fishes from 163 proteins expressed predominantly belonging to the Platelet endothelial cell adhesion molecule-1 (PECAM-1) interactions. (PECAM-1 is a member of the immunoglobulin (Ig) and is a key participant in the adhesion cascade leading to extravasation of leukocytes during the inflammatory process16 (Newman, P.J., 1997). The pathways differentially expressed were DCC mediated attractive signalling pathway, (DCC mediated attractive signalling pathway includes DCC and neogenin in vertebrates), regulation of KIT signalling (KIT is a cytokine receptor expressed on the surface of hematopoietic stem cells as well as other cell types. Altered forms of this receptor may be associated with some types of cancer  , Abasic sugar-phosphate removal via the single-nucleotide replacement pathway (Apurinic/apyrimidinic endonuclease 1 (APE1) acts as a reductive activator of many transcription factors (TFs) and is involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents by controlling levels of reactive oxygen and nitrogen species. Abasic sugar phosphate removal via the single nucleotide replacement pathway requires displacement of DNA glycosylase by apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), APEX1-mediated endonucleolytic cleavage at the 5' side of the base free deoxyribose residue, recruitment of DNA polymerase beta (POLB) to the apurinic/apyrimidinic (AP) site and excision of the abasic sugar phosphate (5'dRP) residue at the strand break. The two major activities of APEX1 multifunctional protein are DNA repair and redox regulation of transcriptional factors. It functions as a apurinic/apyrimidinic (AP) endodeoxyribonuclease in the DNA base excision repair (BER) pathway of DNA lesions induced by oxidative and alkylating agents and possesses a DNA 3' phosphodiesterase activity capable of removing lesions. May also play a role in the epigenetic regulation of gene expression by participating in DNA demethylation and plays a role in the protection from granzymes-mediated cellular repair leading to cell death. Also involved in the DNA cleavage step of class switch recombination (CSR). The following protein pathways were involved in stress regulation viz, EPHA-mediated growth cone collapse, ephrin signalling, regulation of gap junction activity, activated NTRK3 signals through PI3K, adrenaline signalling through Alpha-2 adrenergic receptor, metalloprotease DUBs, resolution of AP sites via the single nucleotide replacement pathway, signalling by NTRK3 (TRKC), fructose catabolism, NTRK2 activates RAC1, activated NTRK2 signals through FYN, eukaryotic translation elongation, reelin signalling pathway, molybdenum cofactor biosynthesis, Vitamin D (calciferol) metabolism, nuclear receptor transcription pathway, deubiquitination, Netrin-1 signaling, APEX1-Independent resolution of AP sites via the single nucleotide replacement pathway, regulation of granulopoiesis and synthesis of dolichyl-phosphate.
The 25 most relevant pathways for proteins expressed differentially in control fishes from 306 proteins expressed predominantly belong to be associated with the pathways associated with synthesis of PIPs at the ER membrane, regulation of TP53 expression, VEGF ligand-receptor interactions, , receptor dimerization, release of Hh-Np from the secreting cell, transcriptional activation of p53 responsive genes, transcriptional activation of cell cycle inhibitor p21, regulation of TP53 activity through methylation, synthesis of PE, oncogene induced senescence, synthesis of PIPs at the golgi membrane, FGFR1c and klotho ligand binding and activation, Beta Klotho-mediated ligand binding, Proton/ oligopeptide cotransporters, N-glycan trimming in ER and Calnexin/ Calreticulin cycle, transport of mature mRNA derived from an Intron-Containing Transcript, transport of mature transcript to cytoplasm, mRNA 3'-end processing, RNA Polymerase II transcription termination, Invadopodia formation, Zinc efflux and compartmentalization by the SLC30 family, synthesis of IPs in nucleus, MASTL facilitates mitotic progression, trafficking of myristoylated proteins to cilium, FGFR1b ligand binding and activation.
The 25 most relevant pathways sorted by p-value for proteins commonly expressed in control and experimental fishes from 275 proteins expressed predominantly belong to be associated with SEMA3A-Plexin repulsion signalling by inhibiting integrin adhesion, Sema3A PAK dependent Axon repulsion, CRMPs in Sema3A signalling, VEGFR2 mediated cell proliferation, crosslinking of collagen fibrils, PECAM1 interactions, metalloprotease DUBs, abasic sugar-phosphate removal via single-nucleotide replacement pathway, gluconeogenesis, unblocking of NMDA receptors, glutamate binding and activation, activation of Ca-permeable Kainate receptor, DCC mediated attractive signalling, ionotropic activity of kainate receptors, regulation of KIT signalling, RHO GTPases regulate CFTR trafficking, regulation of gap junction activity, activated NTRK3 signals through PI3K, adrenaline signalling through Alpha-2 adrenergic receptor, anchoring fibril formation, MET activates PTK2 signalling, resolution of AP sites via single-nucleotide replacement pathway, TFAP2 (AP-2) family regulates transcription of cell cycle factors, signalling by NTRK3 (TRKC), antagonism of activin by Follistatin neurophilin interactions with VEGF and VEGFR.
Semaphorins are extracellular signaling proteins characterized by a single cysteine-rich extracellular sema domain, function as axon guidance molecules, but it is now understood that semaphorins, are key regulators of morphology and Semaphorin signaling occurs predominantly through Plexin receptors and results in changes to the cytoskeletal and adhesive machinery that regulate cellular morphology. Sema3A, a prototypical semaphorin, acts as a chemorepellent or a chemoattractant for axons by activating a receptor complex comprising neuropilin-1 as the ligand-binding subunit and plexin-A1 as the signal-transducing subunit. FARP2 is a key molecule involved in the response of neuronal growth cones to class-3 semaphorins. The members of the collapsin response mediator protein (CRMP) family—five cytosolic phosphoproteins—are highly expressed throughout brain development. Moreover, the expression of CRMPs is altered in neurodegenerative diseases, and these proteins may be of key importance in the physiopathology of the adult nervous system . Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) are increased in majority species of cancers and suppress tumor progression by blocking VEGF/VEGFR2. Apatinib is a highly selective VEGFR2 antagonist which has inhibitive effect on antiapoptotic and cell growth in CCA. While, the effect of apatinib cell migration and invasion in CCA is still unknown[23 ]. PECAM-1/CD31 is a member of the immunoglobulin superfamily (IgSF) and has been implicated to mediate the adhesion and trans-endothelial migration of T-lymphocytes into the vascular wall, T cell activation and angiogenesis. For 275 Commonly Expressed Proteins (CEP), in experimental and control samples, gene ontology, enzyme class and pathway was analyzed by comparing proteins with curated zebrafish UniPort database with reviewed Swiss-Prot database. Similar analysis was performed for 163 Differentially Expressed Proteins (DEP) in experimental samples. The Molecular Function, Cellular Component and Biological process of 275 Commonly Expressed Proteins in control and experimental samples of muscle tissue exposed to temperature stress as depicted in Figure 4 shows more proteins involved in biological process. Enzymatic activity of 275 Commonly Expressed Proteins (CEP) in control and experimental samples of muscle tissue exposed to temperature stress is shown in Figure 5, depicting oxidoreductases, transferases, hydrolases and enzymes altering polypeptide conformation. Figure 6 shows Pathway analysis of 275 Commonly Expressed Proteins in control and experimental samples of muscle tissue exposed to temperature stress with major proteins involved in protein modification mainly ubiquitination and glycosylation. Figure 7 shows Gene Ontology of 581 proteins expressed only in Control Samples of Muscle tissue. Figure 8 shows Gene ontology of 163 proteins expressed in experimental samples of muscle tissue exposed to temperature stress. Figure 9 depicts enzymatic analysis of 163 proteins expressed in experimental muscle tissue samples exposed to temperature stress. Figure 10 depicts pathway analysis of 163 proteins expressed in experimental muscle tissue samples exposed to temperature stress
The major pathway of proteins expressed in response to high temperature stress was related to protein modifications glycosylation and ubiquitination. Ubiquitination is a multistep exzymatically catalyzed post-translational modification process that targets proteins for degradation and recycling . Ubiquitination is critical in almost every cellular process, has a role in modulating diverse cellular functions like cell proliferation and differentiation, autophagy, apoptosis, immune response, DNA repair, neural degeneration, myogenesis, and stress response as well as a major player in almost any disease or disorder[24,25,26,27 ,28]. The pathway analysis and enzymatic activity of differentially expressed proteins provide a glimpse of the strategy used by Clarias magur for adapting for developing resilience for climate change. Further quantitative study can be helpful for understanding micro level interaction of proteins. This study provides a glimpse into the adaptive strategy used by Clarias magur for adaptation for temperature stress.