The gill proteome of threespine sticklebacks (Gasterosteus aculeatus) differs greatly in populations that inhabit diverse environments characterized by different temperature, salinity, food availability, parasites, and other parameters. To assess the contribution of a specific environmental parameter to such differences it is necessary to isolate its effects from those of other parameters. In this study the effect of environmental salinity on the gill proteome of G. aculeatus was isolated in controlled mesocosm experiments. Salinity-dependent changes in the gill proteome were analyzed by LC/MSMS data-independent acquisition (DIA) and Skyline. Relative abundances of 1691 proteins representing the molecular phenotype of stickleback gills were quantified using previously developed MSMS spectral and assay libraries in combination with DIA quantitative proteomics. Non-directional stress responses were distinguished from osmoregulatory protein abundance changes by their consistent occurrence during both hypo- and hyper-osmotic salinity stress in six separate mesocosm experiments. If the abundance of a protein was consistently regulated in opposite directions by hyper- versus hypo-osmotic salinity stress, then it was considered an osmoregulatory protein. In contrast, if protein abundance was consistently increased irrespective of whether salinity was increased or decreased, then it was considered a non-directional response protein. KEGG pathway analysis revealed that the salivary secretion, inositol phosphate metabolism, valine, leucine and isoleucine degradation, citrate cycle, oxidative phosphorylation, and corresponding endocrine and extracellular signaling pathways contain most of the osmoregulatory gill proteins whose abundance is directly proportional to environmental salinity. Most proteins that were inversely correlated with salinity map to KEGG pathways that represent proteostasis, immunity, and related intracellular signaling processes. Non-directional stress response proteins represent fatty and amino acid degradation, purine metabolism, focal adhesion, mRNA surveillance, phagosome, endocytosis, and associated intracellular signaling KEGG pathways. These results demonstrate that G. aculeatus responds to salinity changes by adjusting osmoregulatory mechanisms that are distinct from transient non-directional stress responses to control compatible osmolyte synthesis, transepithelial ion transport, and oxidative energy metabolism. Furthermore, this study establishes salinity as a key factor for causing the regulation of numerous proteins and KEGG pathways with established functions in proteostasis, immunity, and tissue remodeling. We conclude that the corresponding osmoregulatory gill proteins and KEGG pathways represent molecular phenotypes that promote transepithelial ion transport, cellular osmoregulation, and gill epithelial remodeling to adjust gill function to environmental salinity.

中文翻译：

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三棘刺鱼鳃渗透调节反应的蛋白质组学

三棘刺鱼 (Gasterosteus aculeatus) 的鳃蛋白质组在栖息于以不同温度、盐度、食物供应、寄生虫和其他参数为特征的不同环境的种群中差异很大。为了评估特定环境参数对这种差异的贡献，有必要将其影响与其他参数的影响隔离开来。在这项研究中，环境盐度对 G. aculeatus 鳃蛋白质组的影响是在受控的中胚层实验中分离出来的。通过 LC/MSMS 数据独立采集 (DIA) 和 Skyline 分析鳃蛋白质组中盐度依赖性变化。使用先前开发的 MSMS 光谱和分析库结合 DIA 定量蛋白质组学，对代表棘鱼鳃分子表型的 1691 种蛋白质的相对丰度进行量化。非定向应激反应与渗透压调节蛋白丰度变化的区别在于它们在六个单独的中胚层实验中在低渗和高渗盐度胁迫期间一致发生。如果高渗透盐胁迫和低渗透盐胁迫在相反方向上一致地调节蛋白质的丰度，那么它被认为是渗透调节蛋白。相比之下，如果无论盐度是增加还是减少，蛋白质丰度都持续增加，那么它被认为是非定向响应蛋白。KEGG 通路分析表明，唾液分泌、磷酸肌醇代谢、缬氨酸、亮氨酸和异亮氨酸降解、柠檬酸循环、氧化磷酸化以及相应的内分泌和细胞外信号通路包含大部分渗透调节鳃蛋白，其丰度与环境盐度成正比。大多数与盐度呈负相关的蛋白质映射到代表蛋白质稳态、免疫和相关细胞内信号传导过程的 KEGG 通路。非定向应激反应蛋白代表脂肪和氨基酸降解、嘌呤代谢、粘着斑、mRNA 监视、吞噬体、内吞作用和相关的细胞内信号传导 KEGG 途径。这些结果表明 G. aculeatus 通过调节与瞬时非定向应激反应不同的渗透调节机制来响应盐度变化，以控制相容的渗透物合成、跨上皮离子转运和氧化能量代谢。此外，这项研究将盐度确定为导致调节众多蛋白质和 KEGG 通路的关键因素，这些通路在蛋白质稳态、免疫和组织重塑中具有既定功能。我们得出结论，相应的渗透调节鳃蛋白和 KEGG 通路代表了促进跨上皮离子转运、细胞渗透调节和鳃上皮重塑以调节鳃功能以适应环境盐度的分子表型。这项研究将盐度确定为导致调节众多蛋白质和 KEGG 通路的关键因素，这些通路在蛋白质稳态、免疫和组织重塑中具有既定功能。我们得出结论，相应的渗透调节鳃蛋白和 KEGG 通路代表了促进跨上皮离子转运、细胞渗透调节和鳃上皮重塑以调节鳃功能以适应环境盐度的分子表型。这项研究将盐度确定为导致调节众多蛋白质和 KEGG 通路的关键因素，这些通路在蛋白质稳态、免疫和组织重塑中具有既定功能。我们得出结论，相应的渗透调节鳃蛋白和 KEGG 通路代表了促进跨上皮离子转运、细胞渗透调节和鳃上皮重塑以调节鳃功能以适应环境盐度的分子表型。