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. 2026 Mar 2;27:354. doi: 10.1186/s12864-026-12663-5

Parkin-mediated ubiquitination of hif-α modulates hypoxia signaling in Megalobrama amblycephala

Zhi Li 1,#, Xiaoqian Leng 2,#, Runkun Yan 1, Jing Wang 1, Juan Du 3,✉
PMCID: PMC13059482  PMID: 41772419

Abstract

Background

Megalobrama amblycephala is an economically important fish species in Chinese aquaculture, but its high sensitivity to hypoxia poses a major challenge for farming. This study aims to investigate the role of the E3 ubiquitin ligase parkin in regulating hypoxia signaling in fish.

Results

Under hypoxic stress, parkin expression was significantly upregulated in oxygen-sensitive tissues (brain and gills) and embryos of M. amblycephala, consistent with the expression of hypoxia-responsive genes. Ma-parkin was found to exhibit high conservation in its key functional domains. Mechanistically, it directly interacts with Ma-hif-α, promoting their ubiquitination and proteasomal degradation via E3 ligase activity, thereby suppressing hif-mediated transcriptional activation. Critical residues T74 and C333 were identified as essential for this activity; mutations at these sites impaired Ma-hif-α degradation and ubiquitination, as well as HRE transactivation. Furthermore, we found that Ma-hif-α can regulate the transcription of parkin, forming a negative feedback loop to fine-tune the hypoxic response.

Conclusions

Our study demonstrates that parkin serves as a key negative regulator of the hypoxia signaling pathway in M. amblycephala, operating through a finely tuned feedback mechanism. The E3 ubiquitin ligase activity of parkin, dependent on critical residues T74 and C333, directly mediates hif-α ubiquitination and degradation. The reciprocal regulation between hif-α and parkin forms a regulatory circuit that modulates the hypoxic response. These findings not only reveal a conserved adaptive mechanism to hypoxia in fish but also identify specific molecular targets for genetic improvement of hypoxia tolerance in aquaculture species.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12663-5.

Keywords: Hypoxia, Parkin, Hif-α, Ubiquitination, Megalobrama amblycephala, Aquaculture

Introduction

Megalobrama amblycephala (hereafter referred to as M. amblycephala, commonly known as Wuchang bream), represents a vital herbivorous cyprinid species within Chinese aquaculture, contributing significantly to annual yields exceeding 7 × 10 ^ 5 tons [1]. Nevertheless, the commercial production of M. amblycephala encounters considerable obstacles due to its limited tolerance to hypoxia when compared to other economically significant cyprinids, such as Cyprinus carpio and Ctenopharyngodon Idella [2]. Notably, exposure to dissolved oxygen (DO) levels below 0.5 mg/L for durations of two hours or more results in lethal hypoxic stress in M. amblycephala [3].

Oxygen serves as a crucial regulator of aerobic metabolism and is essential for survival in aquatic ecosystems [4, 5]. The increasing prevalence of aquatic hypoxia (DO < 2 mg/L), driven by factors such as climate change, eutrophication, and intensive aquaculture practices [6], is anticipated to intensify due to global warming-induced stratification, eutrophication-driven oxygen depletion, and environmental changes associated with aquaculture [7–9]. For M. amblycephala, a species highly sensitive to hypoxia, these worsening hypoxic conditions present a significant threat. Therefore, understanding the molecular mechanisms underlying hypoxic adaptation in M. amblycephala is imperative for mitigating this risk.

Fish have developed advanced molecular mechanisms to detect and adapt to variations in oxygen availability. Recent research has elucidated the pivotal role of the hypoxia response pathway, mediated by hypoxia-inducible factor (HIF), in regulating this adaptive process [10, 11]. HIF, a transcription factor that is highly sensitive to oxygen levels, is tightly regulated by intracellular oxygen partial pressure. This functional transcription factor complex is a heterodimer composed of two subunits: an oxygen-sensitive HIF-α subunit and a constitutively and stably expressed HIF-β subunit. Only when the two subunits assemble into a complete dimer can HIF translocate into the nucleus, recognize and bind to the hypoxia response element (HRE) on target genes, thereby initiating the transcription of downstream genes. In this mechanism, HIF-α serves as the key functional subunit that determines the transcriptional activity of HIF [12]. Under normoxic conditions, the proline residues of hif-α (hif-1αa, hif-1αb, hif-2αa, hif-2αb in M. amblycephala) [11] undergo hydroxylation by prolyl hydroxylase (phd). Among them, hif-1αa/b orchestrates metabolic reprogramming in acute hypoxia, whereas HIF-2αa/b is dedicated to regulating physiological homeostasis during chronic hypoxia [13]. The von Hippel-Lindau protein (pVHL) identifies hydroxylated hif-α for ubiquitination and proteasomal degradation, keeping their expression low and ensuring cellular homeostasis [14]. In hypoxic conditions, the activity of phd is inhibited, resulting in the stabilization and accumulation of hif-α. These isoforms translocate to the nucleus, where they form a heterodimeric complex with hif-1β, initiating the transcription of hypoxia-responsive genes that enhance the organism’s tolerance to low oxygen environments [11, 15–17]. In zebrafish and M. amblycephala, the hif signaling pathway is notably associated with hypoxia tolerance, serving as a crucial regulatory axis for elucidating mechanisms of hypoxia adaptation [5, 11, 18–20].

PARKIN, a RING-between-RING (RBR) E3 ubiquitin ligase, has mutations that contribute to familial Parkinson’s disease [21]. Within cellular physiology, PARKIN facilitates the ubiquitination-mediated degradation of various proteins, thereby playing a critical role in essential cellular processes. Recent advancements in proteomics have enabled comprehensive quantitative analyses of the PARKIN-dependent ubiquitinome, identifying HIF-1α as a potential substrate for PARKIN-mediated ubiquitination [22]. Subsequent studies have confirmed PARKIN’s involvement in modulating HIF-1α expression and function. For instance, in glioblastoma cells, increased PARKIN levels significantly diminish HIF-1α expression [23] ; furthermore, PARKIN inhibits tumor progression in breast cancer by promoting the ubiquitination and degradation of HIF-1α [24]. Collectively, these findings underscore PARKIN’s regulatory influence on HIF-1α.

While PARKIN is known to regulate HIF-1α in human tumors, the role of Parkin in the hypoxia adaptation of fish has not been previously characterized. This study provides the first elucidation of the regulatory mechanism of the parkin-hif signaling axis in M. amblycephala. The research reveals that parkin expression is significantly upregulated under hypoxic conditions in this species. Ma-parkin directly interacts with multiple Ma-hif-α, promoting their ubiquitination and subsequent proteasomal degradation through its E3 ligase activity, thereby attenuating the hypoxia signaling pathway. These findings identify potential targets of parkin within the hypoxia signaling pathway of M. amblycephala and may offer avenues for enhancing resilience in aquaculture.

Materials and methods

Sequence analysis of parkin in M. amblycephala

We obtained various species’ parkin protein amino acid sequences from the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/) and analyzed them using MEGA 7.0 software. To investigate the functional mechanism structurally, we predicted the three-dimensional structures of parkin using SWISS-MODEL (https://swissmodel.expasy.org/).

Cultivation and hypoxia exposure of M. amblycephala

Adult M. amblycephala were maintained in tanks (120 cm× 100 cm× 45 cm) equipped with continuous oxygenation systems. A 12-hour light/12-hour dark photoperiod regimen was maintained (lights on from 08:00 to 20:00 daily). The water temperature was maintained at a constant 25.0 ± 1.0 °C. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Hydrobiology, Chinese Academy of Sciences (Approval No. IHB-2022-039) and were conducted in strict accordance with its guidelines.

Hypoxic conditions were established using a Ruskinn INVIVO2 400 workstation with 10% O₂ or 12% O₂ and 5% CO₂. Dissolved oxygen was monitored using an LD0101 probe (HQ 30d, HACH). Fish and embryos were exposed to hypoxia for experiments.

Cell culture and hypoxia exposure

HEK293T cells were maintained in DMEM medium (HyClone) with 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂, while Epithelioma Papulosum Cyprini (EPC) cells were cultured in M199 medium (VivaCell Biosciences) at 28 °C with 5% CO₂. Both cell lines were incubated under humidified conditions.

To investigate the effects of hypoxia on EPC cells, the Ruskinn INVIVO2 400 workstation was used as the hypoxic-treatment platform. Prior to treatment, the incubator within the workstation was set to a precisely controlled 1% oxygen environment. EPC cells were then placed within this hypoxic chamber for an 18-hour exposure period.

Plasmid construction

The parkin gene sequence was amplified using the cDNA template of M. amblycephala (accession number: XP_048061823.1 in the NCBI database). The primers information for the cloning is presented in Supplemental Table 1. Following ligation of the amplified parkin gene fragment into the pCMV-Flag and pCMV-Myc vector (Clontech), the Flag-Ma-parkin and Myc-Ma-parkin fusion protein expression systems were constructed.

Quantitative Real-Time PCR (qRT-PCR) analysis

Phenol/chloroform-based RNA extraction from biological samples used TransZol reagent (TransGen Biotech). Reverse transcription with Thermo Fisher Scientific’s cDNA Synthesis Kit generated cDNA templates. Target genes were quantified by qRT-PCR on Bio-Rad’s CFX Connect platform with SYBR Green Mix (High Rox, Monad Biotech) kit. All primer sequences, including that of the internal reference gene β-actin, are provided in detail in Supplemental Table 1.

Western blotting, immunoprecipitation and ubiquitination assays

This section describes methodologies based on previous studies [25, 26]. In brief, cellular proteins extracts underwent separation via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer onto polyvinylidene difluoride (PVDF) membranes. Protein detection utilized western blot analysis with a Fujifilm LAS4000 imaging system.

Following transfection with target plasmids, cellular lysates for immunoprecipitation were incubated with either Anti-Flag affinity gel (Bimake, B23101, USA) or Anti-Myc affinity gel (Beyotime, P2285, China). Subsequently, protein expression was evaluated through Western blot analysis.

To assess the polyubiquitination level of Ma-hif-α, we conducted a co-transfection of HEK293T cells with plasmids encoding Myc-Ma-hif-α, His-ubiquitin (His-Ub), and Flag- Ma-parkin. Following transfection, the cells were treated with the proteasome inhibitor MG132. Subsequently, ubiquitinated proteins were affinity-purified using Ni²⁺-NTA resin (Novagen) via the His-ubiquitin tag. Polyubiquitinated Myc-hif-α in the purified samples was detected using an anti-Myc antibody in immunoblotting.

Luciferase reporter assay

EPC cells were transfected with the hypoxia response elements (HRE) - Luc reporter plasmid (100 ng/well), the CMV - Renilla internal control plasmid (10 ng/well), and experiment-specific expression plasmids. The HRE-luc reporter was constructed using a synthetic DNA fragment containing multiple tandem repeats of the “human” HRE core sequence “ACGTG”, which was cloned into a basic luciferase reporter vector [27]. Although this study focuses on M. amblycephala, the HRE core sequence is highly conserved across species [28]. The use of a human HRE-based reporter gene system has been widely applied in studies of hypoxia response mechanisms in fish [29–31] and does not compromise the core conclusions of this study regarding hypoxia signaling pathway activation. Luciferase activity was assessed 24 h post-transfection using the Promega Dual-Luciferase Reporter Assay System.

Statistical analysis

In accordance with established laboratory protocols, each experiment was conducted in triplicate (n = 3), with each iteration comprising three independently produced parallel samples. The data were subjected to statistical analysis, utilizing the mean ± standard deviation derived from the repeated experiments. GraphPad Prism 8.3.0 facilitated statistical analysis and figure generation. Significance thresholds: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Results

Ma-parkin exhibits high evolutionary conservation in sequence and structure

The parkin gene (GenBank: XP_048061823.1) in M. amblycephala spans 1101 bp, encoding a 366-amino acid protein. This protein contains an N-terminal ubiquitin-like domain and a C-terminal ubiquitin ligase domain.

Comparative sequence analysis across multiple species, including Homo sapiens (humans), Mus musculus (mice), Danio rerio (zebrafish), and M. amblycephala, revealed significant conservation in the amino acid sequence of parkin protein (Fig. 1A). To investigate the functional implications of this sequence conservation, we generated predictive three-dimensional structural models of parkin for these species (Fig. 1B–E). Structural analysis further revealed conserved features at functional interfaces, most notably the C-terminal RING2 domain (also referred to as the catalytic RING domain). This domain, which comprises an α-helix and a β-sheet across all four species (Fig. 1B–E), directly catalyzes ubiquitin transfer and serves as the core region responsible for E3 ubiquitin ligase activity [32]. Collectively, these findings indicate that Ma-parkin exhibits high conservation across both amino acid sequence and structural dimensions.

Fig. 1.

Fig. 1

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Alignment and structural prediction of Parkin protein sequences. A Multiple sequence alignment of Parkin proteins across selected species. The red box highlights the amino acid residues of the C-terminal domain. B-E Predicted structural models of Parkin proteins from selected species. The blue section highlights the C-terminal domain. Source of the sequence: Homo sapiens PARKIN: NCBl accession number XM_011535863.2; Mus musculus Parkin: NCBl accession number XM_017317572.3; Danio rerio Parkin: NCBl accession number NM_001423707.3; Megalobrama amblycephala parkin: NCBl accession number XM_048205866.1

Effect of hypoxia treatment on parkin expression in M. amblycephala

To investigate the potential role of parkin in the hypoxic response, we first asked whether its expression is regulated by oxygen availability. We hypothesized that if parkin is involved in hypoxia adaptation, its mRNA levels would be upregulated under hypoxic stress. We therefore measured parkin mRNA levels in various adult tissues of M. amblycephala exposed to hypoxia. Consistent with our hypothesis, parkin expression was most significantly elevated in oxygen-demanding organs such as the heart, liver, and muscle [11] (Fig. 2A). Considering that the brain exhibits the highest rate of oxygen consumption and that the gills are the primary interface for oxygen sensing [11], we reasoned that these tissues might represent key sites for hypoxic regulation and thus focused our subsequent analysis on them. To determine whether parkin is integrated into the canonical hypoxic signaling network, we tested the hypothesis that parkin expression would be co-regulated with established hypoxia-responsive genes. Our results confirmed that hypoxia significantly induced the expression of phd3, vegfa, glut1, and parkin in both the brain and gills (Fig. 2B and C). This coordinated upregulation strongly suggests that parkin is an integral component of the hypoxic transcriptional response.

Fig. 2.

Fig. 2

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Impact of hypoxic conditions on parkin mRNA Expression in M. amblycephala. A Levels of parkin mRNA expression in various tissues of adult fish subjected to hypoxia. B, C Expression levels of vegfa, glut1, and parkin mRNA in the brain (B) and gill tissues (C) of adult fish under hypoxic conditions. D Expression levels of parkin, glut1 and vegfa mRNA in larvae fish exposed to hypoxia for 3 h. E Temporal expression levels of phd3 and parkin mRNA in larvae fish at different intervals (0 h, 4 h, 8 h, 12 h) during hypoxic treatment

Next, we sought to determine whether the role of parkin in the hypoxic response is conserved across developmental stages. Hypoxic treatment significantly elevated the mRNA levels of parkin, as well as the control genes phd3, glut1, and vegfa, in the embryos (Fig. 2D). This indicates that parkin’s involvement in hypoxia signaling is not limited to adult tissues but is also functional during early development. Finally, to gain insights into the kinetics of parkin induction and to infer its potential function as an immediate-early or sustained responder, we analyzed its temporal expression profile during hypoxic exposure. We found that parkin expression changed dynamically, peaking at 8 h, a pattern that closely paralleled the induction of the well-characterized immediate-early hypoxia-responsive gene phd3 (Fig. 2E). This similar kinetic profile supports the notion that parkin is an early respondent within the hypoxic signaling cascade. Taken together, these findings establish parkin as a hypoxia-inducible modulator that is integrated into the canonical hypoxic response pathway in M. amblycephala.

Interaction between Ma-parkin and Ma-hif-α

To elucidate the molecular mechanism underlying parkin’s involvement in the hypoxia response in M. amblycephala, we conducted an investigation into the interaction between parkin and hif-α. Utilizing the co-immunoprecipitation technique, we observed that Ma-parkin demonstrated a strong binding affinity for several hif-α, specifically Ma-hif-1αa, Ma-hif-1αb, Ma-hif-2αa, and Ma-hif-2αb (Fig. 3). These results imply that Ma-parkin may regulate hypoxia response in M. amblycephala via binding to Ma-hif-α.

Fig. 3.

Fig. 3

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Specific interaction between Ma-parkin and Ma-hif-α. A-D HEK293T cells were co-transfected with Flag-parkin and Myc-hif-1αa (A), Myc-hif-1αb (B), Myc-hif-2αa (C), or Myc-hif-2αb (D). Total cell lysates were immunoprecipitated (IP) with anti-Flag agarose beads 24 h post-transfection. The presence of each Myc-hif-α isoform in the immunoprecipitated complexes was detected using an anti-Myc antibody. E-H HEK293T cells were co-transfected with Flag-parkin and Myc-hif-1αa (E), Myc-hif-1αb (F), Myc-hif-2αa (G), or Myc-hif-2αb (H). Total cell lysates were immunoprecipitated (IP) with anti-Myc agarose beads 24 h post-transfection. The presence of Flag-parkin in the immunoprecipitated complexes was detected using an anti-Flag antibody

Ma-parkin decreases Ma-hif-α protein abundance and transcriptional activation activity

Based on the elucidation of the interaction between Ma-parkin and Ma-hif-α, we undertook a series of experimental studies to further investigate how Ma-parkin modulates the response of M. amblycephala to hypoxic conditions through its interaction with Ma-hif-α.

Initially, we established a gradient of Ma-parkin overexpression in HEK293T and EPC cells. This intervention markedly reduced protein levels of Ma-hif-α (Ma-hif-1αa, Ma-hif-1αb, Ma-hif-2αa, and Ma-hif-2αb) (Fig. 4), suggesting that Ma-parkin can suppress the stability of Ma-hif-α protein.

Fig. 4.

Fig. 4

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Ma-parkin overexpression reduces the protein abundance of Ma-hif-α in HEK293T and EPC cells. HEK293T and EPC cells were transfected with increasing amounts of a Ma-parkin expression plasmid (or an empty vector as a control). Whole-cell lysates were then subjected to immunoblot analysis using antibodies against Ma-hif-α and Ma-parkin

HIF-α is known to activate the transcription of various downstream anti-hypoxia genes under hypoxic conditions, thereby aiding the organism’s adaptation to low oxygen environments. Building on this foundation, we further examined the impact of Ma-parkin on Ma-hif-α transcriptional activation. A dual-luciferase reporter assay system was used to evaluate the expression of HRE, as hif-1α binds to HRE in its target genes to modulate their expression [12, 22]. Ma-parkin suppressed HRE-luciferase reporter activity both in cells overexpressing Ma-hif-α (Ma-hif-1αa, Ma-hif-1αb, Ma-hif-2αa, Ma-hif-2αb) (Fig. 5A-D) and under hypoxic conditions (Fig. 5E). Simultaneously, the overexpression of Ma-parkin in a gradient manner also substantially decreased the mRNA expression levels of glut1 under hypoxic conditions (Fig. 5F). In conclusion, Ma-parkin was found to attenuate both the expression of Ma-hif-α protein and its transcriptional activation potential.

Fig. 5.

Fig. 5

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Regulation of transcriptional activation by Ma-parkin on Ma-hif-α. A-D The impact of gradient overexpression of Ma-parkin on the activity of the HRE-luciferase reporter (HRE-luc) activated by Myc-hif-1αa (A), Myc-hif-1αb (B), Myc-hif-2αa (C), or Myc-hif-2αb (D) in EPC cells. E The influence of gradient overexpression of Ma-parkin on hypoxia-induced HRE-luciferase reporter (HRE-luc) activity in EPC cells. F The effect of gradient overexpression of Ma-parkin on hypoxia-induced glut1 mRNA expression levels in EPC cells

Ma-parkin promotes the ubiquitin-proteasome-dependent degradation of Ma-hif-α protein

Previous studies have shown that Ma-parkin suppresses Ma-hif-α protein levels. To investigate whether this regulatory process is mediated by the ubiquitin-proteasome pathway, we performed a sequence of experiments. Treatment with the proteasome inhibitor MG-132 reversed the down-regulation of Ma-hif-α protein by Ma-parkin (Fig. 6A-D). Further ubiquitination immunoprecipitation experiments revealed that Ma-parkin overexpression significantly enhanced Ma-hif-α protein ubiquitination levels (Fig. 6E-H). These collective findings confirm that Ma-parkin promotes Ma-hif-α degradation through the ubiquitin-proteasome system.

Fig. 6.

Fig. 6

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Ma-parkin downregulates Ma-hif-α protein expression via the ubiquitin-proteasome pathway. A-D Effect of gradient overexpression of Ma-parkin combined with proteasome inhibitor MG132 treatment on the protein expression levels of Myc-hif-1αa (A), Myc-hif-1αb (B), Myc-hif-2αa (C), or Myc-hif-2αb (D) in HEK293T cells. E-H Effect of Ma-parkin overexpression on the polyubiquitination levels of Myc-hif-1αa (E), Myc-hif-1αb (F), Myc-hif-2αa (G), or Myc-hif-2αb (H) proteins in HEK293T cells

Ma-parkin down-regulates Ma-hif-α protein expression dependent on its key sites

To determine whether the inhibitory effect of Ma-parkin on the hypoxic signaling pathway depends on its E3 ubiquitin ligase activity, we performed site-directed mutagenesis at key functional residues. According to the alignment results (Fig. 7A), we constructed the following Ma-parkin mutants: T74A (corresponding to human T173A), P195S (corresponding to the human P294S), and C333A (corresponding to human C431A), in order to systematically evaluate the functional conservation of these residues in fish. In contrast to the overexpression of wild-type Ma-parkin and Ma-parkin-P195S, the mutants Ma-parkin-T74A or Ma-parkin-C333A did not significantly reduce Ma-hif-α protein levels (Fig. 7B-E), enhance the ubiquitination of Ma-hif-α (Fig. 8A-D), or decrease the activity of the HRE reporter and glut1 gene induced by Ma-hif-α/hypoxia (Fig. 9A-E). This indicates that Ma-parkin’s ability to inhibit the hypoxia pathway relies on its E3 ubiquitin ligase function, with the T74 and C333 residues being essential for this function.

Fig. 7.

Fig. 7

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Active site-dependence of Ma-parkin-mediated downregulation of hif-α protein expression. A Multiple sequence alignment of ubiquitin ligase active sites in Parkin proteins from representative species. B-E Effect of overexpressing wild-type Ma-parkin or its active site mutants (T74A, P195S, C333A) on the protein expression levels of Myc-hif-1αa (B), Myc-hif-1αb (C), Myc-hif-2αa (D), and Myc-hif-2αb (E)

Fig. 8.

Fig. 8

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Active site-dependence of Ma-parkin-mediated ubiquitination of hif-α proteins. A-D Effect of overexpressing wild-type Ma-parkin or its active site mutants (T74A, P195S, C333A) on the ubiquitination levels of Myc-hif-1αa (A), Myc-hif-1αb (B), Myc-hif-2αa (C), and Myc-hif-2αb (D), respectively

Fig. 9.

Fig. 9

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Active site-dependence of Ma-parkin regulation on hif-α transcriptional activation activity. A-D Effect of gradient overexpression of Ma-parkin or its active site mutants (T74A, P195S, C333A) on HRE-luciferase reporter (HRE-luc) activity activated by Myc-hif-1αa (A), Myc-hif-1αb (B), Myc-hif-2αa (C), or Myc-hif-2αb (D) in EPC cells. E-F Effect of gradient overexpression of Ma-parkin or its active site mutants (T74A, P195S, C333A) on hypoxia-stimulated HRE-luciferase reporter (HRE-luc) activity (E) and glut1 gene mRNA expression (F) in EPC cells

Ma-parkin and Ma-hif-α form a negative feedback regulatory loop

To further elucidate the regulatory role of parkin in the hypoxic response, this study investigated the transcriptional regulation mechanism of the parkin gene itself. The HRE is a DNA sequence bound by transcription factors such as HIF, which typically activates the transcription of target genes under hypoxic conditions [27]. Notably, the promoter region of the parkin gene contains the typical HRE sequence “ACGTG” (Supplemental Fig. 1), suggesting that it may be directly regulated by the HIF pathway. To test this hypothesis, we performed dual-luciferase reporter assays and confirmed that hypoxia and Ma-hif-α can directly activate the transcription of parkin (Fig. 10). This finding suggests that Ma-hif-α may directly regulate parkin transcription. Combined with the previously described degradation of Ma-hif-α protein by Ma-parkin, we propose that the two may form a negative feedback regulatory loop to precisely control the intensity and duration of the hypoxic signal.

Fig. 10.

Fig. 10

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Ma-hif-α directly transcriptionally upregulates parkin. A Luciferase activity of the parkin promoter-driven reporter under normoxic and hypoxic conditions. B Luciferase activity of the parkin promoter-driven reporter following overexpression of Ma-hif-α

Discussion

The M. amblycephala species demonstrates limited tolerance to hypoxic conditions, a challenge that is further intensified by the increasing frequency of low oxygen stress in aquaculture environments [33], thereby presenting significant barriers to its large-scale cultivation. To facilitate the development of hypoxia-tolerant strains through genetic breeding, it is crucial to elucidate the molecular mechanisms underlying hypoxic tolerance, with a particular focus on the key oxygen-sensing pathways.

The HIF signaling pathway is a central component of the hypoxic response [34–36], and its regulation is dependent on the oxygen-mediated degradation of HIF by E3 ubiquitin ligases, such as pVHL [36, 37]. Theoretically, editing the vhl gene could enhance hypoxia tolerance in fish; however, studies in mice and zebrafish have shown that vhl knockout leads to embryonic lethality, indicating that a similar outcome may occur in M. amblycephala [11]. Consequently, it is essential to investigate the regulatory roles of other E3 ubiquitin ligases. Notably, the mammalian E3 ubiquitin ligase parkin inhibits tumor progression, a process intricately linked to hypoxia, by promoting hif-1α degradation [24, 38–40]. In the present study, we have identified that the conserved amino acid sequence and structural domains of Ma-parkin, and speculated that its potential role in hypoxia signaling pathways similar to that in mammals.

Our comprehensive multi-dimensional experiments have confirmed the essential role of parkin in hypoxia adaptation in M. amblycephala. Initially, we observed a significant upregulation of parkin expression in the brain and gill under hypoxic conditions, which showed synergistic changes with the expression profiles of classical hypoxia-responsive genes such as phd3, vegfa, and glut1, indicating its integration into the hypoxia signaling network. Notably, parkin exhibited an exceptionally rapid response to hypoxia during embryonic development, reaching its peak within 8 h.

The second aspect concerns the regulatory mechanism. Ma-parkin exerts a negative regulatory influence on the hypoxia signaling pathway by directly binding to Ma-hif-α and promoting its ubiquitination and subsequent degradation. This mechanism mirrors the parkin-mediated degradation of hif-1α observed in mammals. Importantly, in mammals, mutations in Parkin, specifically T173A, P294S, and C431A, have been demonstrated to impair its ubiquitin ligase activity [24]. In this study, site-directed mutagenesis of Ma-parkin was performed based on amino acid sequence comparisons, identifying the residues T74 and C333 as essential for maintaining E3 ubiquitin ligase activity in M. amblycephala. The mutation at the C333 site (corresponding to C431 in human PARKIN [22, 41] ) abolished its effect on Ma-hif-α protein stability, ubiquitination and HRE activity, indicating that this site is critical for substrate recognition or catalytic efficiency. The precise mechanism by which the parkin mutants (T74A, C333A) lose their ability to promote hif-α degradation remains to be fully elucidated and warrants further investigation. Future studies will focus on exploring whether these mutations impair the E3 ubiquitin ligase activity of Parkin or disrupt its proper conformational change, which is essential for substrate recognition and ubiquitin transfer. While these findings identify the key residues in parkin responsible for degrading hif-α, the corresponding ubiquitination sites on hif-α itself remain to be identified. Therefore, we plan to systematically screen for critical ubiquitination sites by constructing a series of Ma-hif-α point mutants in future studies, aiming to fully delineate this regulatory pathway. Our study reveals that overexpression of Ma-hif-α activates the parkin promoter, providing a new perspective for understanding the fine-tuning of hypoxic signaling. Based on this, we propose a working model: during the initial phase of hypoxic stress, accumulated Ma-hif-α initiates the transcription of various target genes, including parkin. Subsequently, the newly synthesized Ma-parkin protein, through its E3 ubiquitin ligase activity, tags Ma-hif-α and promotes its degradation via the proteasomal pathway. This potential negative feedback mechanism helps prevent overactivation of the hypoxic response and is crucial for maintaining cellular homeostasis. However, to unequivocally confirm this as a direct transcriptional circuit, future studies employing chromatin immunoprecipitation (ChIP) assays are needed to demonstrate whether endogenous Ma-hif-α directly binds to the promoter region of the parkin gene under physiological hypoxic conditions, thereby ultimately validating this direct transcriptional regulatory circuit.

Evolutionarily, this regulatory mechanism parallels the parkin-hif negative regulation observed in mammals, a process crucial for maintaining energetic homeostasis in fish residing in environments with fluctuating dissolved oxygen levels. From a physiological adaptation standpoint, the highly efficient interaction between Ma-parkin and Ma-hif-α highlights the extensive adaptation of fish to hypoxic aquatic environments. Nevertheless, the present study has not fully elucidated the comprehensive network through which Ma-parkin regulates the hypoxic response in M. amblycephala. Subsequent research could utilize genome editing to knockout parkin in M. amblycephala, allowing for direct assessment of endogenous hif protein levels and overall hypoxia tolerance under low-oxygen conditions to confirm its function in vivo.

Conclusion

This study elucidates the role of parkin as a pivotal negative regulator of hypoxia signaling in the economically significant fish species M. amblycephala. Our findings indicate that hypoxic stress significantly upregulates parkin expression in oxygen-sensitive tissues and embryos. Evolutionary analyses further reveal that the functional domains of Ma-parkin are conserved across vertebrates. Mechanistically, Ma-parkin interacts with Ma-hif-α, facilitating its ubiquitin-mediated degradation through E3 ligase activity, thereby attenuating hif-driven transcriptional activation of HREs. Site-directed mutagenesis has elucidated the critical role of the T74 and C333 residues in Ma-parkin’s regulatory function. Mutations at these sites disrupt the abrogation of Ma-hif-α degradation and ubiquitination, consequently inhibiting HRE repression. Furthermore, we identified that Ma-hif-α transcriptionally activates parkin expression, establishing a negative feedback loop that precisely controls hypoxia signaling. In summary, our study establishes parkin as a key negative regulator in the hypoxic signaling pathway of M. amblycephala. The T74 and C333 residues were identified as critical for maintaining its E3 ligase activity and substrate interaction. These findings reveal a novel mechanism through which parkin activity regulates hif-α stability, providing both a theoretical foundation and candidate targets for developing potential strategies to breed hypoxia-tolerant aquaculture species.

Supplementary Information

Supplementary Material 1. (16.6KB, docx)
Supplementary Material 2. (1.3MB, tif)
Supplementary Material 3. (56.4KB, docx)
Supplementary Material 4. (1.4MB, pdf)

Acknowledgements

Not applicable.

Authors’ contributions

Zhi Li, Xiaoqian Leng and Runkun Yan prepared figures, Jing Wang and Juan Du wrote the main manuscript text. All authors reviewed the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 32202940), Natural Science Foundation of Wuhan (No. 2024020801020403). Wuhan Talents, Excellent Young Talents Project (2021) (JD).

Data availability

All materials and protocols used during the current study are available from the corresponding author on reasonable request. The datasets generated and/or analyzed during the current study, including gene sequences, are available in the National Center for Biotechnology Information (NCBI) repository (https://www.ncbi.nlm.nih.gov/). The accession numbers are provided in the main text and Figure 1 legend: Ma-parkin (XP_048061823.1, XM_048205866.1), Homo sapiens PARKIN (XM_011535863.2), Mus musculus Parkin (XM_017317572.3), Danio rerio Parkin (NM_001423707.3). Structural predictions were performed using the SWISS-MODEL repository (https://swissmodel.expasy.org/).

Declarations

Ethics approval and consent to participate

All animal care protocols complied with the guidelines of the Institute of Hydrobiology, Chinese Academy of Sciences. Every effort was made to minimize animal suffering. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Hydrobiology, Chinese Academy of Sciences (Approval No. IHB-2022-039) and were conducted in strict accordance with its guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zhi Li and Xiaoqian Leng shared first authorship.

References

  • 1.Sun S, et al. Dynamic mRNA and MiRNA expression analysis in response to hypoxia and reoxygenation in the blunt snout Bream (Megalobrama amblycephala). Sci Rep. 2017;7(1):12846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Shen RJ, et al. HIF-1alpha and – 2alpha genes in a hypoxia-sensitive teleost species megalobrama amblycephala: cDNA cloning, expression and different responses to hypoxia. Comp Biochem Physiol B Biochem Mol Biol. 2010;157(3):273–80. [DOI] [PubMed] [Google Scholar]
  • 3.Wang C, et al. Hypoxia-inducible factor 1alpha from a high-altitude fish enhances cytoprotection and elevates nitric oxide production in hypoxic environment. Fish Physiol Biochem. 2020;46(1):39–49. [DOI] [PubMed] [Google Scholar]
  • 4.Rytkonen KT, et al. Comparison of hypoxia-inducible factor-1 alpha in hypoxia-sensitive and hypoxia-tolerant fish species. Comp Biochem Physiol Part D Genomics Proteom. 2007;2(2):177–86. [DOI] [PubMed] [Google Scholar]
  • 5.Liao Q, et al. Disruption of Sirtuin 7 in zebrafish facilitates hypoxia tolerance. J Biol Chem. 2023;299(8):105074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Huang KJ, et al. Hypoxia leads to gill Endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): mitigation effect of thiamine. J Hazard Mater. 2024;469:134005. [DOI] [PubMed] [Google Scholar]
  • 7.Lai KP, et al. Hypoxia causes transgenerational impairment of ovarian development and hatching success in fish. Environ Sci Technol. 2019;53(7):3917–28. [DOI] [PubMed] [Google Scholar]
  • 8.Wang Y, et al. Hypoxia aggravates the burden of yellowstripe goby (Mugilogobius chulae) under Atorvastatin exposure. Aquat Toxicol. 2023;255:106381. [DOI] [PubMed] [Google Scholar]
  • 9.Yu XX, et al. Effects of hypoxia on the gill morphological structure, apoptosis and hypoxia-related gene expression in blunt snout Bream (Megalobrama amblycephala). Fish Physiol Biochem. 2023;49(5):939–49. [DOI] [PubMed] [Google Scholar]
  • 10.Xiao W. The hypoxia signaling pathway and hypoxic adaptation in fishes. Sci China Life Sci. 2015;58(2):148–55. [DOI] [PubMed] [Google Scholar]
  • 11.Li J, et al. Fih attenuates hypoxic tolerance by regulating hypoxia-inducible factors (hif-α) in megalobrama amblycephala. Aquaculture Rep. 2024;37:102235. [Google Scholar]
  • 12.Slawski J, et al. Regulation of the HIF switch in human endothelial and cancer cells. Eur J Cell Biol. 2024;103(2):151386. [DOI] [PubMed] [Google Scholar]
  • 13.Mu W, et al. HIFs genes expression and hematology indices responses to different oxygen treatments in an ovoviviparous teleost species Sebastes schlegelii. Mar Environ Res. 2015;110:142–51. [DOI] [PubMed] [Google Scholar]
  • 14.Ivan M, Kaelin WJ. The EGLN-HIF O(2)-Sensing system: multiple inputs and feedbacks. Mol Cell. 2017;66(6):772–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Shi X, et al. Role of HIF in fish inflammation. Fish Shellfish Immunol. 2023;143:109222. [DOI] [PubMed] [Google Scholar]
  • 16.Zhang B, et al. Molecular response and association analysis of megalobrama amblycephala fih-1 with hypoxia. Mol Genet Genomics. 2016;291(4):1615–24. [DOI] [PubMed] [Google Scholar]
  • 17.Li J et al. Zebrafish usp3 loss promotes hypoxic tolerance by disrupting deubiquitination of K63-polyubiquitinated hif-1αa. Water Biology Secur. 2024;3(2):100245.
  • 18.Wang Z, et al. Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity. J Biol Chem. 2022;298(12):102633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Liao Q, et al. Deletion of Prolyl hydroxylase domain-containing enzyme 3 (phd3) in zebrafish facilitates hypoxia tolerance. J Biol Chem. 2023;299(12):105420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Cai X, et al. Deletion of the Fih gene encoding an inhibitor of hypoxia-inducible factors increases hypoxia tolerance in zebrafish. J Biol Chem. 2018;293(40):15370–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Roverato ND, et al. Parkin is an E3 ligase for the ubiquitin-like modifier FAT10, which inhibits parkin activation and mitophagy. Cell Rep. 2021;34(11):108857. [DOI] [PubMed] [Google Scholar]
  • 22.Sarraf SA, et al. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature. 2013;496(7445):372–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Maugeri G, et al. Parkin modulates expression of HIF-1alpha and HIF-3alpha during hypoxia in gliobastoma-derived cell lines in vitro. Cell Tissue Res. 2016;364(3):465–74. [DOI] [PubMed] [Google Scholar]
  • 24.Liu J, et al. Parkin targets HIF-1alpha for ubiquitination and degradation to inhibit breast tumor progression. Nat Commun. 2017;8(1):1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Du J, et al. march2 negatively regulates antiviral immune response by targeting tbk1 in grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol. 2023;140:108965. [DOI] [PubMed] [Google Scholar]
  • 26.Li Z, et al. Zebrafish F-box protein fbxo3 negatively regulates antiviral response through promoting K27-Linked polyubiquitination of the transcription factors irf3 and irf7. J Immunol. 2020;205(7):1897–908. [DOI] [PubMed] [Google Scholar]
  • 27.Janji B, Chouaib S. The promise of targeting hypoxia to improve cancer immunotherapy: Mirage or reality? Front Immunol. 2022;13:880810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Rees BB, Bowman JA, Schulte PM. Structure and sequence conservation of a putative hypoxia response element in the lactate dehydrogenase-B gene of fundulus. Biol Bull. 2001;200(3):247–51. [DOI] [PubMed] [Google Scholar]
  • 29.Wang Z, et al. Spring viremia of carp virus infection induces hypoxia response in zebrafish by stabilizing hif1alpha. J Virol. 2025;99(1):e0149124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Cai X et al. Zebrafish Hif3alpha modulates erythropoiesis via regulation of gata1 to facilitate hypoxia tolerance. Development. 2020;147(22):185116. [DOI] [PubMed]
  • 31.Li J, et al. MYLIP attenuates hypoxia tolerance by inducing K27-linked polyubiquitination and subsequent proteasomal degradation of HIF-alpha. Commun Biol. 2025;8(1):774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ye X et al. Cooperative substructure and energetics of allosteric regulation of the catalytic core of the E3 ubiquitin ligase parkin by phosphorylated ubiquitin. Biomolecules. 2024;14(10):1338. [DOI] [PMC free article] [PubMed]
  • 33.Zhao SS, et al. The transcriptomic responses of blunt snout Bream (Megalobrama amblycephala) to acute hypoxia stress alone, and in combination with bortezomib. BMC Genomics. 2022;23(1):162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Tirpe AA et al. Hypoxia: overview on hypoxia-mediated mechanisms with a focus on the role of HIF genes. Int J Mol Sci. 2019;20(24):6140. [DOI] [PMC free article] [PubMed]
  • 35.Dai W, et al. Hypoxia and the endometrium: an indispensable role for HIF-1alpha as therapeutic strategies. Redox Biol. 2024;73:103205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Maxwell PH, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999;399(6733):271–5. [DOI] [PubMed] [Google Scholar]
  • 37.Semenza GL. The genomics and genetics of oxygen homeostasis. Annu Rev Genomics Hum Genet. 2020;21:183–204. [DOI] [PubMed] [Google Scholar]
  • 38.Koyano F, et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature. 2014;510(7503):162–6. [DOI] [PubMed] [Google Scholar]
  • 39.Jing X, et al. Role of hypoxia in cancer therapy by regulating the tumor microenvironment. Mol Cancer. 2019;18(1):157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Wicks EE, Semenza GL. Hypoxia-inducible factors: cancer progression and clinical translation. J Clin Invest. 2022;132(11):758-70. [DOI] [PMC free article] [PubMed]
  • 41.Riley BE, et al. Structure and function of parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases. Nat Commun. 2013;4:p1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (16.6KB, docx)
Supplementary Material 2. (1.3MB, tif)
Supplementary Material 3. (56.4KB, docx)
Supplementary Material 4. (1.4MB, pdf)

Data Availability Statement

All materials and protocols used during the current study are available from the corresponding author on reasonable request. The datasets generated and/or analyzed during the current study, including gene sequences, are available in the National Center for Biotechnology Information (NCBI) repository (https://www.ncbi.nlm.nih.gov/). The accession numbers are provided in the main text and Figure 1 legend: Ma-parkin (XP_048061823.1, XM_048205866.1), Homo sapiens PARKIN (XM_011535863.2), Mus musculus Parkin (XM_017317572.3), Danio rerio Parkin (NM_001423707.3). Structural predictions were performed using the SWISS-MODEL repository (https://swissmodel.expasy.org/).

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