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ALLERGY ALZHEIMER ANEMIA AUTOIMMUNE CANCER CVD OBESITY MUSCULOSKELETAL STEM CELLS

Electrophoresis.2016 Jul;37(12):1704­1717

Proteomic changes in the liver of Channa striatus in response to high temperature stress
Arabinda Mahanty1∗, Gopal Krishna Purohit2∗, Sudeshna Banerjee1, Dhanasekar Karunakaran1, Sasmita Mohanty2∗, Bimal Prasanna Mohanty1

1 ICAR–Central Inland Fisheries Research Institute, FREM Division, Barrackpore, Kolkata, India

2KIIT School of Biotechnology, KIIT University, Bhubaneswar, Odisha, India

Abstract

The present study was undertaken to investigate the proteomic changes in the liver of murrel Channa striatus exposed to high temperature stress. Fishes were
exposed to 36°C for 4 days and liver proteome changes were analyzed using gel­ based proteomics, i.e. 2DE, MALDI­TOF/TOF­MS, and validation by transcript
analysis. The study showed, besides others, increased abundance of two sets of proteins, the antioxidative enzymes superoxide dismutase (SOD), ferritin, cellular
retinol binding protein (CRBP), glutathione­S­transferase (GST), and the chaperones HSP60 and protein disulfide isomerase; this was validated by transcript
analysis. The proteome data are available via ProteomeXchange with identifier PXD002608. Further, gene expression analysis was also carried out in the fishes
exposed to thermal stress for longer durations (30 days experimental exposure in laboratory and for 30 days beyond, taking Channa collected from a hot spring
runoff at 36–38°C); sod, gst, crbp, and hsp60 were found to continue to remain upregulated at eight­, 2.5­, 2.4­, and 2.45­fold, respectively, in the hot spring runoff
fish. Pathway analysis showed that the upregulations of the antioxidant enzymes as well as molecular chaperones are induced by the transcription factor Nrf2
(nuclear factor erythroid 2­related factor 2). Thus, while short­term heat stress tolerance involves the antioxidative enzymes SOD, ferritin, CRBP, GST, and
chaperones HSP60 and protein disulfide isomerase, adaptation under chronic heat stress is associated with SOD, CRBP, GST, and HSP60.

KEYWORDS: Antioxidative enzymes; Channa striatus; High temperature stress; Liver proteome; Stress response

PMID:27058960; DOI:10.1002/elps.201500393

Supplement:
Temperature stress is one of the most important abiotic factors that hampers the normal physiology, productivity and is one of the leading causes of mortality and
morbidity. Heat stress has become a cause of concern because of the increase in average climatic temperature in the recent years. The causalities occurring due to
heat strokes have increased drastically in recent years. With steady increase in climatic temperatures, the scenario is expected to worsen; the Intergovernmental
Panel on Climate Change (IPCC) has predicted that around 20% of the species assessed so far are at the risk of extinction if the average climatic temperature
increases by 2­3 °C (IPCC 2014).

Therefore, to safeguard life forms from death upon sudden heat stroke, it is necessary to develop mitigation strategies and understanding the molecular mechanism
of heat stress tolerance is one of the primary prerequisites for it. One of the cellular mechanisms that influence the thermal tolerance of an organism is the heat
shock response (HSR) which is characterized by the synthesis of a group of proteins, the heat shock proteins (Hsps). The Hsps act as molecular chaperones,
stabilizing and refolding the denaturing proteins and also leading the already­denatured proteins to the proteolytic machinery (Feder and Hofmann 1999;
Mahanty et al. 2016b). To find out which Hsps are involved in acute and long term heat stress tolerance, we had earlier studied the expression of a battery of hsp
genes in liver of a lower vertebrate Channa striatus heat stressed for different time periods (36°C/4, 15, 30 days) and compared it with those maintained at ambient
temperature (25­27 °C). Further, fish collected from a hot spring runoff (Atri hot spring in India; 20°09′N 85°18′E) area were also studied to see the expression of
the hsp genes when the fish is exposed to chronic heat stress (beyond 30 days). It was found that while, Hsp90 and Hsp110, besides Hsp70, are required for
immediate survival of fish at high temperature whereas Hsp60, 70 and 78 are needed for long term survival at high temperature (Fig. 1) (Purohit et al. 2014). This
study suggested that hsp70, hsp78 and hsp60 could be used as biomarkers for monitoring heat stress in lower vertebrates.

Apart from the HSPs, many other proteins are likely to be involved in the temperature stress response. The present study was carried out to identify the proteins that
are altered in abundance and could possibly be in cross­talk with the HSPs in the liver of Channa striatus exposed to high temperature (36 °C/4 days) using 2­D
gel based proteomics platform followed by protein identification by MALDI­TOF­MS. The alterations in abundance of the identified proteins were validated by
semi quantitative reverse transcription PCR. Additionally, expressions of the transcripts of the identified proteins in liver samples from Channa exposed to longer
periods (30 days and much beyond 30 days) were also analyzed by q­PCR

It was found that the cellular response to short­term (36 °C/4 days) temperature stress led to increase in abundance of primarily two groups of proteins; the
antioxidative proteins superoxide dismutase (SOD), glutathione­S­transferase (GST), cellular retinol binding protein (CRBP), ferritin, and the chaperones, HSP60
and proteins disulfide isomerase (PDI) (Figure 1). The transcript levels of these proteins were also found to be up­regulated validating the results of the proteomics
study.

Further, pathway analysis using Ingenuity Pathway Analysis (IPA) software was carried out to infer the pathways influenced by the protein which increased in
abundance and their upstream regulators. Pathway analysis showed that major biochemical and cellular pathways that were impacted by heat stress include Nrf2
(nuclear factor erythroid 2­related factor 2)­mediated oxidative stress response, gluconeogenesis, glycolysis, and tryptophan degradation. Up­stream regulator
analysis showed that the transcription factor Nrf2 is the common inducer of both the antioxidative enzymes and molecular chaperones (Figure 3).

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Significance of the study: This study showed that Nrf­2 mediated increased synthesis of antioxidative proteins and chaperone helps the fish to withstand heat
stress. Based on these results, supplementation of potential Nrf­2 inducers could be tested as a strategy as therapeutic interventions in fish which could further be
extrapolated to humans and other animals.

Fig. 1. Trends in hsp gene expression in liver tissues of Channa striatus in response to heat stress. Based on the pattern of expression, the hsps has
been clustered into two groups. Hsp70, Hsp78, and Hsp60 were found to be at an elevated level of expression when the fish was heat stressed for
longer duration of time indicating these are needed for long term survival at high temperature whereas expression of Hsp90 and Hsp110 were found
elevated during short term heat stress which declined with increasing time period indicating that these are required for immediate survival of fish at
high temperature (Data source Purohit et al. 2014).

Fig. 2. Heat stress induced alteration in abundance of antioxidative proteins and chaperones. Liver proteomics in Channa striatus showed that in
response to heat stress, primarily the abundance of two sets of proteins, the antioxidative enzymes SOD, ferritin, CRBP, GST, and the chaperones
HSP60 and PDI increases which provides protection to fish against heat stress (Data source Mahanty et al. 2016).

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Fig. 3. Nrf­2 mediated induction of antioxidative proteins and molecular chaperones synthesis in response to heat stress. Pathway analysis (Ingenuity
Pathway Analysis, IPA) showed that the increase in abundance of both the antioxidative enzymes and chaperones is mediated by a common
transcription factor, Nrf­2. Heat stress induced production of reactive oxygen species (ROS) causes the dissociation of Nrf­2 from the Nrf­2­Keap­1
(Kelch­like ECH­associated protein 1) complex present in the cytoplasm which then translocate into the nucleus and binds with the antioxidative
response element (ARE) of DNA and induces synthesis of antioxidative enzymes and Hsps (Data source Mahanty et al. 2016).

References

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of Experimental Biology 213:912­920.
2. Mahanty A, Purohit GK, Yadav RP, Mohanty S, Mohanty BP 2016 hsp90 and hsp47 appear to play an important role in minnow Puntius sophore for
surviving in the hot spring runoff aquatic ecosystem. Fish Physiology and Biochemistry 43 (1), 89­102.
3. IPCC. 2014. Fifth Assessment Report – Climate Change 2014: Synthesis Report. IPCC, Geneva, Switzerland.
4. Purohit GK, Mahanty A, Suar M, Sharma AP, Mohanty BP, Mohanty S 2014 Investigating hsp gene expression in liver of Channa striatus under heat
stress for understanding the upper thermal acclimation. BioMed Research International 2014, 1­ 10.
5. Mahanty A, Purohit GK, Banerjee S, Karunakarn D, Mohanty S, Mohanty BP 2016 Proteomic changes in the liver of Channa striatus in response to
high temperature stress. Electrophoresis. 37(12):1704­1717.
6. Feder ME, Hofmann GE 1999 Heat­shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annual
Reviews in Physiology 61:243–282.

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