Proteome and phosphoproteome profiling reveals the regulation mechanism of hibernation in a freshwater leech (Whitmania pigra)
Graphical abstract
The leech Whitmania pigra as an important medicinal resource in Asia is an excellent model freshwater invertebrate for studies of environmentally-induced hibernation. The present study provides the first quantitative proteomics and phosphoproteomic analysis of leech hibernation using isobaric tag based TMT labeling and high-resolution mass spectrometry. These data significantly improve our understanding of the regulatory mechanisms when ectotherm animals face environmental stress and provides substantial candidate phosphorylated proteins that could be important for functionally adapt in freshwater animals.
Introduction
Leech is used in large quantities in Asia, North America and Europe for various medical applications. Whitmania pigra is a common medicinal leech species in China and has been used as a traditional Chinese medicine in treatment of cardiovascular diseases due its potent anticoagulant [1,2], antiatherosclerosis [[3], [4], [5]] and antiplatelet aggregation effects [6]. Whitmania pigra is widely farmed in aquaculture in Asia. However, hibernation is commonly observed when Whitmania pigra exposed to low temperature. Environmentally induced hibernation of Whitmania pigra usually begins in November and lasts until next April, and renews when the environmental temperature rises to 20 °C, which attracts attention from researchers because the hibernation seriously affects the cultivation of leeches.
Hibernation is used as an effective strategy by many animals, both terrestrial and aquatic, to cope with cold environments and/or other environmental stresses. Key features of long-term hibernation involve substantial decreases in metabolic rate depression and descent into a hypometabolic state characterized by reductions in energy expensive processes with both biochemical (transcription, translation, cell cycle, etc.) and physiological processes (e.g. body temperature and cardiac rhythm, etc.) are suppressed [[7], [8], [9]].
We found that hypometabolism in hibernating leech was evidenced by strong decreases in the rates of oxygen consumption. And the intestine degenerated into a very thin filament. Once hibernation is completed, the intestine regenerated in a very short time. The activity of enzymes participating in energy metabolism decreased significantly in deep hibernation and rapidly elevated during arousal. In addition, antioxidant defenses of intestine over cycles of hibernation-arousal were also examined, which decreased during deep hibernation, but increased during entrance into hibernation and arousal from hibernation. These results indicate that the Whitmania pigra has an adaptive antioxidant defenses mechanism that protects it from the injurious effects of free radicals either during periods of entrance into hibernation and arousal [10].
The molecular mechanisms underlying this remarkable physiological phenotype during hibernation have been examined in several vertebrates such as ground squirrels [11], bats [12] and bear [13]. The liver proteome between summer-active and winter-hibernating 13-lined ground squirrels demonstrated that a dramatic metabolic fuel shift away from oxidation of dietary carbohydrates and toward oxidation of stored fat for energy in winter. A large number of metabolic enzymes involved in the TCA cycle decreased. On the contrary, proteins involved in fatty acid metabolism and transport elevated [11]. It is revealed that protein expressions associated with amino acid metabolism, proteostasis, energy metabolism, cytoskeletal structure, and stress response were significantly altered in hibernating bats' brain by a proteomic approach [12]. In addition, hibernating animals have developed well antioxidant defenses during the hypometabolic period or periods of arousal back to euthermia [[14], [15], [16], [17]]. Several cellular signaling pathways are known to mediate hypometabolism during hibernation,such as AMP-activated protein kinase which regulated glycogen and lipid synthesis [18,19], mTOR signaling which regulated protein synthesis required for growth [20].
Multiple regulatory controls could be involved in metabolic arrest during hibernation. It is well known that a primary regulatory mechanism is reversible protein phosphorylation (RPP) [[21], [22], [23]]. RPP is known to regulate the activities and kinetic properties of huge numbers of metabolic enzymes, ion motive ATPases, and protein synthesis (ribosomal initiation and elongation factors). Reversible phosphorylation also controls many steps in signal transduction cascades (e.g., many protein kinases are themselves subject to on/off control by other kinases) as well as the activation of many transcription factors, so the mechanism undoubtedly participates in all of the cell signaling and gene expression events that occur over hibernation-arousal cycles [24]. The Comparative phosphoproteomic analysis of intestins from active versus aestivating sea cucumbers indicated that six major functional classes of proteins including protein synthesis transcriptional regulators, kinases, signaling, transporter and DNA binding, exhibited changes in their phosphorylation status during sea cucumbers aestivation [25]. Although previous studies in other species have revealed important universal regulation of metabolism by reversible protein phosphorylation, only selected proteins with potential roles were examined [[26], [27], [28]].
Despite the discovery of some important molecular mechanisms of adaptation to hibernation, limited research been directed toward the hibernation phenomenon in aquatic environments. In the present study, we used quantitative proteomic and phosphoproteomic analyses to understand the mechanisms underpinning the regulation during the hibernation-arousal cycles in leech Whitmania pigra, an excellent model freshwater invertebrate for studies of environmentally-induced hibernation. And we identified a group of differentially expressed proteins and phosphorylated proteins that could be important for functionally adapt in freshwater animals facing environmental stress.
Section snippets
Animals
Adult leech (one year old, body weight 12–15 g) were collected from culture ponds in Nanjing Agricultural University. Animals deep hibernation (DH) were collected after 50%–60% of the duration of the hibernation bout (120–150 days). Animals sampled early after spontaneously arousing from hibernation (CDM) were collected 0.5 h after water temperature had increased above 25 °C during rewarming. Animals sampled in October (water temperature about 25 °C) were not in the hibernation period, these
Impaired energy metabolism and abnormal intestine histology
In intestine, ATP, ADP and AMP content decreased significantly during deep hibernation (DH), and increased in early arousal (EA) compared with DH. Meanwhile, energy charge, defined as (ATP + 1/2 ADP) / (ATP + ADP + AMP), was fell slightly in deep hibernation, and they were not significantly different between active controls (AC) and EA (Fig. 1a). These results showed that hibernation involved strong metabolic rate depression. The histological comparison of intestine over the hibernation-arousal
Discussion
Through analysis of gene expression, detection of protein and protein phosphorylation and detection of enzyme activity, we found that reversible phosphorylation can regulate energy metabolism enzymes, ion-dynamic ATPase, protein synthesis (ribosomal initiation and elongation factors), signal transduction Cascades (eg, many protein kinases themselves are subject to on/off control of other kinases).We detailed a list of cellular stressors, their effects on leech that are not stress tolerant, and
Conclusion
In summary, this study indicates a global involvement of protein expression and phosphorylation in the control of many aspects of hypometabolism during hibernation in leech intestine. Proteome and phosphoproteome analysis demonstrated that hibernating leech invoked their intrinsic “cold-against” proteomic mechanisms for long-term viability in a hypometabolic state. These mechanisms include regulated energy metabolism, elevated autophagy and depressed apoptosis, and suppressed protein synthesis
Author contributions
Hongzhuan Shi and Jia Wang conducted the experiments and prepared the manuscript; Fei Liu and Xiangjing Hu analyzed the data; Yiming Lu, Shimeng Yan, Daoxin Dai, Manjun Wu, Xibin Yang and Zaibiao Zhu collected the samples; Hongzhuan Shi and Qiaosheng Guo designed and coordinated the study. All authors read and approved the final manuscript.
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgments
This study has been supported the National Natural Science Foundation of China (No. 81673538, No. 81872960 and No. 81473306).
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These authors have contributed equally to this work.