Abstract
Zebrafish have the capacity to regenerate lost tissues and organs. Amputation of the caudal fin results in a rapid, transient increase in H2O2 levels emanating from the wound margin, which is essential for regeneration, because quenching of reactive oxygen species blocks regeneration. Protein-tyrosine phosphatases (PTPs) have a central role in cell signalling and are susceptible to oxidation, which results in transient inactivation of their catalytic activity. We hypothesized that PTPs may become oxidized in response to amputation of the caudal fin. Using the oxidized PTP-specific (ox-PTP) antibody and liquid chromatography-mass spectrometry, we identified 33 PTPs in adult zebrafish fin clips of the total of 44 PTPs that can theoretically be detected based on sequence conservation. Of these 33 PTPs, 8 were significantly more oxidized 40 min after caudal fin amputation. Surprisingly, Shp2, one of the PTPs that were oxidized in response to caudal fin amputation, was required for caudal fin regeneration. In contrast, Rptpα, which was not oxidized upon amputation, was dispensable for caudal fin regeneration. Our results demonstrate that PTPs are differentially oxidized in response to caudal fin amputation and that there is a differential requirement for PTPs in regeneration.
Highlights
Epimorphic regeneration is the perfect replacement of lost tissues, organs, or limbs
Oxidized Protein-tyrosine phosphatases (PTPs) can be detected by first lysing in the presence of N-ethylmaleimide (NEM), which alkylates all reduced cysteines, followed by reduction of the oxidized catalytic cysteines using DTT, and hyperoxidation using pervanadate (PV)
As the reduced catalytic cysteines are protected from detection by NEM, only the catalytic cysteines that were oxidized at the time of lysis will be detected using the oxidized PTP-specific (ox-PTP)-specific antibody
Summary
Epimorphic regeneration is the perfect replacement of lost tissues, organs, or limbs. Many genes have been implicated and multiple signalling pathways have been validated to be essential for regeneration to proceed, including fibroblast growth factor (FGF), sonic hedgehog, bone morphogenetic protein, Wnt, and Notch[3, 4]. It remains unclear how this complex process is initiated. The active-site cysteine of enzymes has a low pKa, which confers catalytic activity Reversible oxidation of these cysteine residues may temporarily activate or inactivate these enzymes[9]. Oxidation-mediated inhibition of PTPs results in the selective amplification or attenuation of specific signalling pathways, such as FGF, PI3K/AKT, and MAPK, regulating fundamental cellular processes, including proliferation, differentiation, and cell-cell adhesion[21]
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