Proteomics of Osmoregulatory Responses in Threespine Stickleback Gills.

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 Liquid chromatography/Tandem mass spectrometry 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.

Development of a Gill Assay Library for Ecological Proteomics of Threespine Sticklebacks (Gasterosteus aculeatus).

A data-independent acquisition (DIA) assay library for quantitative analyses of proteome dynamics has been developed for gills of threespine sticklebacks (Gasterosteus aculeatus). A raw spectral library was generated by data-dependent acquisition (DDA) and annotation of tryptic peptides to MSMS spectra and protein database identifiers. The assay library was constructed from the raw spectral library by removal of low-quality, ambiguous, and low-signal peptides. Only unique proteins represented by at least two peptides are included in the assay library, which consists of 1506 proteins, 5074 peptides, 5104 precursors, and 25,322 transitions. This assay library was used with DIA data to identify biochemical differences in gill proteomes of four populations representing different eco- and morpho-types of threespine sticklebacks. The assay library revealed unique and reproducible proteome signatures. Warm-adapted, low-plated, brackish-water fish from Laguna de la Bocana del Rosario (Mexico) show elevated HSP47, extracellular matrix, and innate immunity proteins whereas several immunoglobulins, interferon-induced proteins, ubiquitins, proteolytic enzymes, and nucleic acid remodeling proteins are reduced. Fully-plated, brackish-water fish from Westchester Lagoon (Alaska) display elevated ion regulation, GTPase signaling, and contractile cytoskeleton proteins, altered abundances of many ribosomal, calcium signaling and immunity proteins, and depleted transcriptional regulators and metabolic enzymes. Low-plated freshwater fish from Lake Solano (California) have elevated inflammasomes and proteolytic proteins whereas several iron containing and ion regulatory proteins are reduced. Gills of fully-plated, marine fish from Bodega Harbor (California) have elevated oxidative metabolism enzymes and reduced transglutaminase 2, collagens, and clathrin heavy chains. These distinct proteome signatures represent targets for testing ecological and evolutionary influences on molecular mechanisms of gill function in threespine sticklebacks. Furthermore, the gill assay library represents a model for other tissues and paves the way for accurate and reproducible network analyses of environmental context-dependent proteome dynamics in complex organisms.

Population-specific renal proteomes of marine and freshwater three-spined sticklebacks.

Quantitative proteomics was used to reveal biochemical differences in kidneys of marine and freshwater three-spined sticklebacks. More than 1500 unambiguous proteins were identified, 106 of which are robustly co-translationally modified. Amino-terminal acetylation sites for 94 and proline hydroxylation sites for 12 proteins, including 4 protein disulfide isomerases having the consensus motif APWCGHCK, were determined. More than 1500 proteins were quantified by LC-MS/MS yielding 120 proteins with consistent population-specific abundance differences. Twenty-five of these were selected for validation by data-independent acquisition (DIA) and spectral library based MS2 quantitation. A dense biochemical network was revealed, which promotes the synthesis of the organic osmolytes betaine, sorbitol, trimethylamine oxid (TMAO), and urea. It contains 33 of 49 proteins that are elevated in marine compared to freshwater sticklebacks, including the most highly elevated proteins (dimethylaniline monooxygenase, alanine-glyoxylate aminotransferase, glycine N-methyltransferase). Freshwater stickleback kidneys contain elevated levels of proteolytic, cytoskeletal, extracellular matrix, and calcium signaling proteins. Proteins that are most elevated in freshwater sticklebacks are ES1 protein homolog, apoptosis-associated speck-like protein containing a CARD and caspase 1. Protein-abundance network analysis demonstrates significantly higher levels of synchronized abundance control in marine sticklebacks. The significance of these findings for biochemical diversification of renal function in marine and FW sticklebacks is discussed.

Population-specific plasma proteomes of marine and freshwater three-spined sticklebacks (Gasterosteus aculeatus).

Molecular phenotypes that distinguish resident marine (Bodega Harbor) from landlocked freshwater (FW, Lake Solano) three-spined sticklebacks were revealed by label-free quantitative proteomics. Secreted plasma proteins involved in lipid transport, blood coagulation, proteolysis, plasminogen-activating cascades, extracellular stimulus responses, and immunity are most abundant in this species. Globulins and albumins are much less abundant than in mammalian plasma. Unbiased quantitative proteome profiling identified 45 highly population-specific plasma proteins. Population-specific abundance differences were validated by targeted proteomics based on data-independent acquisition. Gene ontology enrichment analyses and known functions of population-specific plasma proteins indicate enrichment of processes controlling cell adhesion, tissue remodeling, proteolytic processing, and defense signaling in marine sticklebacks. Moreover, fetuin B and leukocyte cell derived chemotaxin 2 are much more abundant in marine fish. These proteins promote bone morphogenesis and likely contribute to population-specific body armor differences. Plasma proteins enriched in FW fish promote translation, heme biosynthesis, and lipid transport, suggesting a greater presence of plasma microparticles. Many prominent population-specific plasma proteins (e.g. apoptosis-associated speck-like protein containing a CARD) lack any homolog of known function or adequate functional characterization. Their functional characterization and the identification of population-specific environmental contexts and selective pressures that cause plasma proteome diversification are future directions emerging from this study.

Alterations in the proteome of the respiratory tract in response to single and multiple exposures to naphthalene.

Protein adduction is considered to be critical to the loss of cellular homeostasis associated with environmental chemicals undergoing metabolic activation. Despite considerable effort, our understanding of the key proteins mediating the pathologic consequences from protein modification by electrophiles is incomplete. This work focused on naphthalene (NA) induced acute injury of respiratory epithelial cells and tolerance which arises after multiple toxicant doses to define the initial cellular proteomic response and later protective actions related to tolerance. Airways and nasal olfactory epithelium from mice exposed to 15 ppm NA either for 4 h (acute) or for 4 h/day × 7 days (tolerant) were used for label-free protein quantitation by LC/MS/MS. Cytochrome P450 2F2 and secretoglobin 1A1 are decreased dramatically in airways of mice exposed for 4 h, a finding consistent with the fact that CYPs are localized primarily in Clara cells. A number of heat shock proteins and protein disulfide isomerases, which had previously been identified as adduct targets for reactive metabolites from several lung toxicants, were upregulated in airways but not olfactory epithelium of tolerant mice. Protein targets that are upregulated in tolerance may be key players in the pathophysiology associated with reactive metabolite protein adduction. All MS data have been deposited in the ProteomeXchange with identifier PXD000846 (http://proteomecentral.proteomexchange.org/dataset/PXD000846).

Salinity-induced regulation of the myo-inositol biosynthesis pathway in tilapia gill epithelium.

The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol that protects cells from osmotic stress. Using proteomics, the enzymes that constitute the MIB pathway, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1), are identified in tilapia (Oreochromis mossambicus) gill epithelium. Targeted, quantitative, label-free proteomics reveals that they are both upregulated during salinity stress. Upregulation is stronger when fish are exposed to severe (34 ppt acute and 90 ppt gradual) relative to moderate (70 ppt gradual) salinity stress. IMPA1 always responds more strongly than MIPS, suggesting that MIPS is more stable during salinity stress. MIPS is N-terminally acetylated and the corresponding peptide increases proportionally to MIPS protein, while non-acetylated N-terminal peptide is not detectable, indicating that MIPS acetylation is constitutive and may serve to stabilize the protein. Hyperosmotic induction of MIPS and IMPA1 is confirmed using western blot and real-time qPCR and is much higher at the mRNA than at the protein level. Two distinct MIPS mRNA variants are expressed in the gill, but one is more strongly regulated by salinity than the other. A single MIPS gene is encoded in the tilapia genome whereas the zebrafish genome lacks MIPS entirely. The genome of euryhaline tilapia contains four IMPA genes, two of which are expressed, but only one is salinity regulated in gill epithelium. The genome of stenohaline zebrafish contains a single IMPA gene. We conclude that the MIB pathway represents a major salinity stress coping mechanism that is regulated at multiple levels in euryhaline fish but absent in stenohaline zebrafish.

Quantitative molecular phenotyping of gill remodeling in a cichlid fish responding to salinity stress.

A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into proteome changes that underlie the remodeling of tilapia gill epithelium in response to environmental salinity change.