Dynamic changes in the heart proteome and phosphoproteome of Pelophylax nigromaculatus during salt stress and recovery.
TL;DR
A multi-layered regulatory framework encompassing kinase-driven signal transduction, energy metabolic reprogramming, and RNA homeostasis maintenance is revealed, providing novel molecular insights into the salt tolerance and reversible repair capacity of the amphibian heart.
Abstract
Salinization of freshwater ecosystems poses an escalating threat to amphibian populations worldwide, yet the molecular mechanisms underlying cardiac responses to salt stress remain poorly understood. In this study, we employed DIA-based quantitative proteomics and phosphoproteomics to systematically investigate the dynamic protein expression and phosphorylation profiles in the heart of Pelophylax nigromaculatus across low-salt, high-salt, and recovery stages. A total of 3747 proteins and 623 phosphopeptides were identified. Kinase prediction analysis revealed that ERK2, along with AGC, TKL, TK, and STE kinase families, may serve as key upstream regulators of salt-stress signaling. Cluster analysis classified the proteins shared among all treatment groups into six expression patterns, among which cluster 3 (metabolism-related) and cluster 5 (stress-responsive) emerged as two core responsive protein populations. Functional enrichment and protein-protein interaction analyses demonstrated that high-salt stress suppressed mitochondrial aerobic respiration via down-regulation of TCA cycle enzymes, while compensatorily activating glycolysis to sustain basal energy supply-a metabolic reprogramming strategy that was largely reversed upon stress relief. Concurrently, RNA-binding proteins and stress granule assembly factors formed tight interaction networks, suggesting that RNA homeostasis maintenance serves as an auxiliary protective mechanism against salt-induced cellular damage. These findings reveal a multi-layered regulatory framework encompassing kinase-driven signal transduction, energy metabolic reprogramming, and RNA homeostasis maintenance, providing novel molecular insights into the salt tolerance and reversible repair capacity of the amphibian heart. This study not only advances our understanding of osmotic adaptation in amphibians but also establishes a foundational proteomic resource for assessing the ecological impacts of freshwater salinization on amphibian biodiversity.