When extracts of newly fertilized eggs from N = 4 F3 homozygous vtg1-KO females and from N = 4 Wt females were compared in terms of their proteomic profiles via label-free LC-MS/MS, a total of N = 301 proteins were identified. Of these, N = 126 proteins were detected in at least four samples and showed a ≥ 1.5-fold difference in normalized spectral counts (N-SC) between groups (vtg1-KO versus Wt), or were unique to a certain group, and, on this basis, were considered to be ‘differentially regulated’ (Table S1). The frequency distribution of differentially regulated proteins among 10 categories of physiological function chosen to represent most (≥ 90%) of the proteins significantly differed (χ2, p < 0.05) between vtg1-KO and Wt eggs (Fig. 1a). Frequencies of proteins related to protein degradation and synthesis inhibition were significantly higher in vtg1-KO eggs, whereas frequencies of proteins related to energy metabolism and vitellogenins were higher in Wt eggs. Individual proteins that were up-regulated in vtg1-KO eggs (N = 94) (Fig. 1a) were mainly related to protein degradation and synthesis inhibition (23%), cell cycle, division, growth and fate (19%), lectins (19%), and protein synthesis (10%), with the remaining categorized proteins being related to energy metabolism (8%), redox-detox activities (8%), vitellogenins (6%) and immune functions (5%). Two percent of proteins which were up-regulated in vtg1-KO eggs were placed in the category of ‘others’. Individual proteins that were higher in abundance in Wt eggs (N = 32), and so down-regulated in vtg1-KO eggs, were mainly related to energy metabolism (28%), Vtgs (19%), lectins (16%), and redox-detox activities (13%) with the remaining categorized proteins being related to cell cycle, division, growth and fate (9%), immune functions (9%), and protein degradation and synthesis inhibition (6%).
Figure 1. Distribution of proteins up-regulated in vtg-KO and Wild type (Wt) zebrafish eggs among functional categories. Panel a. vtg1-KO Experiment. Panel b. vtg3-KO Experiment. Only proteins that were identified in ≥ 4 biological samples and that exhibited a ≥ 1.5-fold difference in N-SC between groups (vtg-KO versus Wt), or proteins unique to a certain group, were includeed in this analysis. In both experiments, the overall distribution of up-regulated proteins among the functional categories significantly differed between KO and Wt eggs (χ2, p < 0.05). Asterisks indicate significant differences between different groups in the proportion of up-regulated proteins within a functional category (χ2, p < 0.05). The corresponding Ensembl Protein IDs and associated gene, transcript and protein names, functional categories (shown above), regulation (unique or up-regulated), and fold-difference in N-SC between KO and Wt eggs for proteins included in this analysis for the vtg1-KO and vtg3-KO experiments are given in Tables S1 and S2, respectively.
When protein extracts of eggs from N = 4 F3 homozyous vtg3-KO females, and from N = 4 Wt females, were compared in terms of their protein profiles, a total of N = 238 proteins were identified. Based on the criteria mentioned above, N = 74 of these proteins were identified as ‘differentially regulated’ (Table S2). The frequency distribution of differentially regulated proteins among the 10 categories of physiological function chosen to represent most (≥ 90%) of the proteins significantly differed (χ2, p < 0.05) between vtg3-KO and Wt eggs (Fig. 1b). The frequency of up-regulated proteins related to protein degradation and synthesis inhibition was significantly higher in vtg3-KO eggs, whereas the frequencies of up-regulated proteins related to lipid metabolism, and to redox-detox activities were significantly higher in Wt eggs (χ2, p < 0.05). Individual proteins that were up-regulated in vtg3-KO eggs (N = 53) were mainly related to protein degradation and synthesis inhibition (36%), cell cycle, division, growth and fate (23%), and energy metabolism (15%) with the remaining categorized proteins being related to protein synthesis (7%), Vtgs (7%), lectins (6%), and immune functions (4%). Two percent of these proteins were placed in the ‘others’ category. Individual proteins that were higher in abundance in Wt eggs (N = 21) and so were down-regulated in vtg3-KO eggs were mainly related to lipid metabolism (19%), energy metabolism (19%), protein synthesis (14%), redox-detox activities (14%) and vitellogenins (14%), with the remaining proteins being related to immune function (5%) and protein degradation and synthesis inhibition (5%).
Figure 2. PANTHER GO Biological Processes found to be overrepresented by proteins up-regulated in Wt and vtg-KO zebrafish eggs. Panel a. vtg1-KO Experiment. Panel b. vtg3-KO Experiment. Only proteins that were identified in ≥ 4 biological samples and that exhibited a ≥ 1.5-fold difference in N-SC between groups (vtg1-KO versus Wt), or proteins unique to a certain group, were includeed in this analysis. Horizontal bars indicate the number of proteins attributed to each GO term for which statistically significant results (Fisher’s Exact test, p ≤ 0.05, followed by Bonferroni correction for multiple testing (p < 0.05)) were observed (Results shown for Wt egg proteins in Fig. 2b. Top Panel based on FDR only, no Bonferronni correction was applied). Numbers next to the bars indicate the fold-enrichment with proteins attributed to each term and the number of asterisks indicates the significance level of the enrichment, as follows p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). Where possible, horizontal bars are colored to indicate corresponding protein functional categories shown in Fig. 1; cell cycle, division, growth and fate (lavender), protein synthesis (light blue), protein degradation and synthesis inhibition (dark blue), energy metabolism (magenta).
An automated Protein ANalysis THrough Evolutionary Relationships (PANTHER) overrepresentation test of the 126 proteins regulated differentially between vtg1-KO and Wt eggs revealed three Gene Ontology (GO) Biological Process terms, Cellular response to chemical stimulus, Response to organic cyclic compound and Response to estradiol, to be significantly overrepresented by proteins with higher abundance in Wt eggs (Fig. 2a. Top Panel), The GO Molecular Function term, Lipid transporter activity, was also significantly overrepresented by proteins with higher abundance in Wt eggs (Fig. 3a. Top Panel). In contrast, many Biological Process terms were significantly overrepresented by proteins increased in abundance in vtg1-KO eggs. These included mainly terms related to cell cycle, division, growth and fate (Anatomical structure morphogenesis, Cell division, Cytokinesis), to protein synthesis (Chaperone-mediated protein folding), and to protein degradation and synthesis inhibition (Response to unfolded protein, Response to topologically incorrect protein, Cellular response to unfolded protein, Response to heat, Cellular response to heat) (Fig. 2a. Bottom Panel). The remaining terms related to vesicle-mediated transport (Vesicle-mediated transport, Exocytosis). Similarly, the several GO Molecular Function terms that were significantly overrepresented by proteins up-regulated in vtg1-KO eggs were related to cell cycle, division, growth and fate (Structural molecule activity, Nucleotide binding, Purine ribonucleotide binding, ATP binding), or to protein degradation and synthesis inhibition (Unfolded protein binding, Ubiquitin protein ligase binding, Heat shock protein binding) (Fig. 3a. Bottom Panel). The broad agreement between Biological Process and Molecular Function terms is consistent with a proteome tailored to cytoskeletal activities and to protein degradation and synthesis inhibition shown in Fig. 1a. Accordingly, a PANTHER analyses conducted to identify GO Cellular Component and Protein Class terms overrepresented by proteins up-regulated in vtg1-KO eggs returned mainly proteins related to cytoskeletal regulation and protein degradation and synthesis inhibition (Table S3).
Figure 3. PANTHER GO Molecular Functions found to be overrepresented by proteins up-regulated in Wt and vtg-KO zebrafish eggs. Panel a. vtg1-KO Experiment. Panel b. vtg3-KO Experiment. Only proteins which were identified in ≥ 4 biological samples and that exhibited a ≥ 1.5-fold difference in N-SC between groups (vtg3-KO versus Wt), or proteins unique to a certain group, were includeed in this analysis. Horizontal bars indicate the number of proteins attributed to each GO term for which statistically significant results (Fisher’s Exact test, p ≤ 0.05, followed by Bonferroni correction for multiple testing (p < 0.05)) were observed. Numbers next to the bars indicate the fold-enrichment with proteins attributed to each term and the number of asterisks indicates the significance level of the enrichment, as follows p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). Where possible, horizontal bars are colored to indicate corresponding protein functional categories shown in Fig. 1; lipid metabolism (yellow), cell cycle, division, growth and fate (lavender), protein degradation and synthesis inhibition (dark blue).
A PANTHER overrepresentation test of the 74 proteins differentially regulated between vtg3-KO and Wt eggs revealed the GO Biological Process terms, Response to estradiol, Response to organic cyclic compound, Monosaccharide metabolic process and Carbohydrate metabolic processes to be overrepresented by proteins with higher abundance in Wt eggs (Fig. 2b. Top Panel). The Molecular Function terms, Phosphatidylinositol phosphate binding and Phosphatidylinositol-3-phosphate binding were significantly over represented by proteins with higher abundance in Wt eggs (Fig. 3b. Top Panel), which is consistent with the proteomic emphasis on lipid metabolism activities revealed by the frequency distribution analysis (Fig. 1b), and also with the overrepresentation of the Molecular Function term, Lipid transporter activity, by proteins with higher abundance in Wt eggs in the vtg1-KO experiment (Fig. 3a. Top Panel). Similar to the results of the vtg1-KO experiment, many GO Biological Process terms were significantly overrepresented by proteins up-regulated in vtg3-KO eggs, and these included mainly terms related to Cell cycle, division, growth and fate (Developmental process, Anatomical structure development, Anatomical structure morphogenesis, Modification-dependent protein catabolic process, Cell division, cytokinesis), to protein synthesis (Protein folding, Chaperone-mediated protein folding), and to Protein degradation and synthesis inhibition (Response to unfolded protein, Response to topologically incorrect protein, Cellular response to unfolded protein, Response to heat, Cellular response to heat), as well as some terms related to vesicle trafficking (Endocytosis, Transport, Vesicle-mediated transport, Exocytosis) (Fig. 2b. Bottom Panel). As was the case with proteins up-regulated in vtg1-KO eggs, all of the many Molecular Function terms that were significantly overrepresented by proteins up-regulated in vtg3-KO eggs were related to cell cycle, division, growth and fate (Structural molecule activity, ATPase activity, ATPase activity-coupled, Small molecule binding, Nucleotide binding, Ribonucleotide binding, Purine ribonucleotide binding, ATP binding), or to Protein degradation and synthesis inhibition (Unfolded protein binding, Ubiquitin protein ligase binding, Heat shock protein binding) (Fig. 3b. Bottom Panel). A PANTHER analysis conducted to identify GO Cellular Component and Protein Class terms overrepresented by proteins with higher abundance in Wt eggs returned only the membrane components Autophagosome and Lysosomal membrane, whereas the same analysis for proteins up-regulated in vtg3-KO eggs returned Actin cytoskeleton component, many cytoskeletal protein classes and class Ribosomal protein (Table S4).
Figure 4. PANTHER GO Biological Pathways which are significantly over-represented by differentially abundant proteins in vtg-KO eggs Panel a. vtg1-KO Experiment Panel b. vtg3-KO Experiment. Only proteins which were identified in ≥ 4 biological samples and those with a ≥ 1.5-fold difference in N-SC, or proteins unique to any group, were included in this analysis. Horizontal bars indicate the number of proteins attributed to each pathway for which statistically significant results (Fisher’s Exact test, p ≤ 0.05, followed by Bonferroni correction for multiple testing (p < 0.05)) were observed. Numbers next to the bars indicate the fold-enrichment with proteins attributed to each pathway and the number of asterisks indicates the significance level of the enrichment, as follows p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).
A PANTHER Pathways overrepresentation analysis revealed the Wnt signaling, Inflammation mediated by chemokine and cytokine signaling, Huntington disease, Alzheimer disease-presenilin, Cadherin signaling, Nicotinic acetylcholine receptor signaling, and Cytoskeletal regulation by Rho GTPase pathways to be significantly overrepresented by proteins up-regulated in vtg1-KO eggs (Fig. 4a). All of these pathways were also significantly overrepresented by proteins up-regulated in vtg3-KO eggs (Fig. 4b). The Apoptosis signaling pathway, and the Parkinson disease pathway, were also overrepresented by proteins up-regulated in vtg3-KO eggs. The PANTHER Pathways analyses revealed no pathways to be significantly overrepresented by proteins with higher abundance in Wt eggs.
Figure 5. STRING Network Analysis of the differentially regulated proteins in vtg1-KO and vtg3-KO experiments. A total of 32 proteins which were up-regulated in wild type eggs and 94 proteins which were up-regulated in vtg1-KO eggs in vtg1-KO experiment, and a total of 21 proteins which were up-regulated in wild type eggs and 53 proteins which were up-regulated in vtg3-KO eggs in vtg3-KO experiment, were over-represented in specific biological pathways in the PANTHER Pathways enrichment analyses (Tables 1 and 2). Each network node (sphere) represents all proteins produced by a single, protein-coding gene locus (splice isoforms or post-translational modifications collapsed). Only nodes representing query proteins are shown. Nodes are named for the transcript(s) to which spectra were mapped; for full protein names, see Tables S1 and S2. Edges (colored lines) represent protein-protein associations meant to be specific and meaningful, e.g. proteins jointly contribute to a shared function but do not necessarily physically interact. Model statistics are presented at the top left and at the top right of each panel for proteins increased in wild type eggs and for proteins increased in KO eggs, respectively. Explanation of edge colors is given below panels. For each experiment, the subnetwork formed by proteins up-regulated in Wt eggs is shown to the upper left above the diagonal dashed line, and the subnetwork formed by proteins up-regulated in vtg-KO eggs is shown to the lower right below the diagonal dashed line. Where possible, solid lines encircle clusters of transcripts encoding interacting proteins involved in physiological processes distinct from other such clusters (see text for details). Dashed lines identify subclusters of two or more transcripts encoding proteins of a common type.
When the 126 differentially regulated proteins with significant differences in abundance between vtg1-KO and Wt eggs were submitted separately (Wt; N = 32, vtg1-KO; N = 94) to a protein-protein interactions network analysis using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and the zebrafish protein database, they resolved into networks with significantly and substantially greater numbers of known and predicted interactions between proteins than would be expected of the same size lists of proteins randomly chosen from the zebrafish database (Fig. 5a). The subnetwork formed by proteins with higher abundance in Wt eggs (N = 32) is made up of, a cluster of three type-I Vtgs (Vtg1, Vtg4 and Vtg5) loosely interacting with a second cluster having at its center two strongly interacting peroxidases, catalase (Cat) and a peroxiredoxin (Prdx2), responsible for protecting the cell from oxidative damage by reactive oxygen species (ROS). Catalase interacts with all remaining members of the cluster, including two C-reactive proteins (Crp2), pattern recognition receptors that activate the complement system, a broad spectrum antiprotease, alpha-2-macroglobulin-like (A2ML), two forms of creatinine kinase (Ckba, Ckbb), an enzyme essential for cellular energy homeostasis, and the common heat shock protein (Hsp90), a key regulator of proteostasis including protein folding, stabilization and degradation when damaged. Aside from Cat, the Prdx2 reacted only with Hsp90 and the two creatinine kinases (Fig. 5a. Left Panel, PPI network enrichment value P = 2.75 × 10− 5).
The subnetwork formed by proteins up-regulated in vtg1-KO eggs (N = 94) is made up of four interacting protein clusters. The first cluster includes two vitellogenins (Vtg7 and Vtg3), an aspartic protease, nothepsin (Nots), secreted by the liver of vitellogenic females and stored in the egg yolk, and two serpins (Serpina1 and Serpina 1I), broad spectrum serine protease inhibitors produced by the liver of vitellogenic females, that bind to Vtgs for transport into the yolk where they associate with the surface of Vtg-drived yolk platelets. The second cluster includes several proteins related to energy metabolism, including glyceraldehyde-3-phosphate dehydrogenase (Gapdh), lactate dehydrogenase (Ldhba), malate dehydrogenase (Mdh2), and the mitochondrial enzyme, ATP synthase F1 subunit beta (ATP5B). The third cluster seems to be mainly related to purine metabolism and redox-detox activities and contains several quinoid dihydropteridine reductases (Qdprb2), an A2ML, and a nucleoside diphosphate kinase (Nme2b.1). The remaining large cluster contains most proteins present in the subnetwork. These are mainly concerned with protein homeostasis and can be placed into subclusters by type. The subclusters include eight forms of the cytoskeletal protein, actin (ACTG2, Acta2, two forms of Actc1a, Acta1a, Actc1, Actc1b and Acta1b), which are involved in cell structure and motility and intracellular vesicle trafficking, three forms of eukaryotic translation elongation factor alpha subunit (Eef1a1a, Eef1a1b, Eef1a2), four Hsps (Hsp70, Hsp70.1, Hsp8, Hspa8b), three forms of ubiquitin (UBC, Uba52, Rps27a), proteins that modulate Hsps and coordinate elimination of damaged/unfolded proteins and protein aggregates via the 26S proteasome, and two protein disulfide isomerase family members (Pdia3 and P4hb). Remaining proteins not assigned to subclusters are cerulolplasmin (Cp), which is engaged in iron transport, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (Atic), which is engaged in purine biosynthesis, and S-adenosylhomocysteine hydrolase (Ahcy), which is engaged in hydrolysis of methionine (Fig. 5a. Right Panel, PPI network enrichment value P = 5.95 × 10− 9).
When the 74 differentially regulated proteins with significant differences in abundance between vtg3-KO and Wt eggs were submitted separately (Wt; N = 21, vtg1-KO; N = 53) to the STRING protein-protein interactions network analysis by mapping to the public zebrafish protein database, they resolved networks with significantly and substantially greater numbers of known and predicted interactions between proteins than would be expected from the same size lists of proteins randomly chosen from the zebrafish database (Fig. 5b). The subnetwork revealed by proteins with higher abundance in Wt eggs (N = 21) is made up of a Vtg (vtg3) weakly interacting with a cluster of five interacting proteins. Proteins in this cluster include three strongly interacting proteins, an enolase involved in glucose metabolism (Eno3), a heat shock protein (Hspa5), and a eukaryotic translation elongation factor alpha subunit (Eef1a1|), two of which (Hsp5 and Eef1a1|1) interact with a catalase (Cat) that, in turn reacts with Crp2 (Fig. 5a. Left Panel; PPI network enrichment value P = 6.99 × 10− 6).
The subnetwork revealed by proteins up-regulated in vtg3-KO eggs (N = 53) is made up of two type-I Vtgs (Vtg5 and Vtg7) that interact with three protein clusters. Two of these clusters are made up of proteins concerned with energy metabolism, the first including two forms of lactate dehydrogenase (Ldhba and Ldha) plus malate dehydrogenase (Mdh2), and the second containing three forms of creatinine kinase (Ckma, Ckmb and Ckba). The third, largest, cluster contains proteins concerned with protein homeostasis that can be arranged as subclusters of proteins by type. These subclusters include seven forms of actin (Acta2, ACTC1, Acta1a, Acta1b, Actc1a, Actc1b and ZGC:86709, a form of Actc2), three Hsps (HSPA8, Hspa8, and Hsp70), three forms of ubiquitin (Uba52, rps27a, and UBC), and three protein disulfide isomerases (Pdia3, Pdia4 and P4hb) (Fig. 5b. Right Panel; PPI network enrichment value P = 0.0517).
Table 1
KEGG and Reactome Pathways revealed by Network enrichment analyses for differentially regulated proteins which were resolved in a STRING subnetwork in the vtg1-KO Experiment. A) proteins up-regulated in wild type eggs. B) proteins up-regulated in vtg1-KO eggs. Only statistically significant results are reported (χ2, p < 0.05). See Table S5 for full analysis report.
A) vtg1-KO Experiment: Network stats for proteins up-regulated in Wt eggs (N = 32) |
number of nodes: 17 |
number of edges: 18 |
average node degree: 2.12 |
clustering coefficient: 0.64 |
expected number of edges: 6 |
PPI enrichment p-value: 2.75E-05 |
confidence level: 0.15 |
KEGG Pathways |
pathway | description | protein count | FDR |
dre00330 | Arginine and proline metabolism | 2 of 58 | 0.0074 |
Reactome Pathways |
pathway | description | protein count | FDR |
DRE-71288 | Creatine metabolism | 2 of 10 | 0.0001 |
B) vtg1-KO Experiment: Network stats for proteins up-regulated in vtg1-KO eggs (N = 94) |
number of nodes: 63 |
number of edges: 107 |
average node degree: 3.4 |
clustering coefficient: 0.387 |
expected number of edges: 58 |
PPI enrichment p-value: 5.95E-09 |
confidence level: 0.40 |
KEGG Pathways |
pathway | description | protein count | FDR |
dre00270 | Cysteine and methionine metabolism | 3 of 47 | 0.0072 |
dre04260 | Cardiac muscle contraction | 3 of 89 | 0.0135 |
dre04141 | Protein processing in endoplasmic reticulum | 4 of 176 | 0.0135 |
dre01100 | Metabolic pathways | 10 of 1278 | 0.0135 |
dre00790 | Folate biosynthesis | 2 of 26 | 0.0135 |
dre00620 | Pyruvate metabolism | 2 of 41 | 0.0244 |
dre03013 | RNA transport | 3 of 151 | 0.0263 |
dre04261 | Adrenergic signaling in cardiomyocytes | 3 of 180 | 0.0368 |
dre00983 | Drug metabolism - other enzymes | 2 of 61 | 0.0368 |
dre00010 | Glycolysis / Gluconeogenesis | 2 of 74 | 0.0439 |
Reactome Pathways |
pathway | description | protein count | FDR |
DRE-71182 | Phenylalanine and tyrosine catabolism | 3 of 14 | 0.003 |
DRE-450408 | AUF1 (hnRNP D0) binds and destabilizes mRNA | 4 of 54 | 0.003 |
DRE-3371568 | Attenuation phase | 3 of 19 | 0.003 |
DRE-3371497 | HSP90 chaperone cycle for steroid hormone receptors | 3 of 21 | 0.003 |
DRE-6798695 | Neutrophil degranulation | 7 of 490 | 0.0088 |
DRE-168256 | Immune System | 11 of 1379 | 0.0154 |
DRE-3371453 | Regulation of HSF1-mediated heat shock response | 3 of 69 | 0.0238 |
DRE-1430728 | Metabolism | 12 of 1751 | 0.0258 |
DRE-2262752 | Cellular responses to stress | 5 of 321 | 0.0313 |
DRE-168249 | Innate Immune System | 8 of 875 | 0.0313 |
DRE-140837 | Intrinsic Pathway of Fibrin Clot Formation | 2 of 19 | 0.0313 |
DRE-140875 | Common Pathway of Fibrin Clot Formation | 2 of 23 | 0.0365 |
DRE-901042 | Calnexin/calreticulin cycle | 2 of 25 | 0.0387 |
Results of a STRING enrichment analysis of differentially abundant proteins also forming the resolved protein-protein interaction networks in Wt eggs (N = 32) and in vtg1-KO (N = 94) eggs for Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways and Rectome Pathways are given in Table 1. Results for other terms such as the UniProt Keywords, Protein Families Database (PFAM) Protein Domains, and Interpro Protein Families Database (INTERPRO) Protein Domains and Features and Simple Modular Architecture Research Tool (SMART) Protein domains are additionally reported in Table S5. A single KEGG pathway (Arginine and proline metabolism) and a single Reactome pathway (Creatine metabolism) were found to be enriched in proteins with higher abundance in Wt eggs (p ≤ 0.05). KEGG pathways that were enriched in the subnetwork resolved by proteins up-regulated in vtg1-KO eggs were; Cysteine and methionine metabolism, Cardiac muscle contraction, Protein processing in endoplasmic reticulum, Metabolic, Folate biosynthesis, Pyruvate metabolism, RNA transport, Andrenergic signaling of cardiomyocytes, Drug metabolism, and Glycolysis/Gluconeogenesis (p ≤ 0.05). The corresponding Reactome pathways were Phenylalanine and tyrosine catabolism, AUFI (hnRNP D0) binds and destabilizes mRNA, Attenuation phase, HSP90 chaperone cycle for steroid hormone receptors, Neutrophil degranulation, Immune system, Regulation of HSFI-mediated heat shock response, Metabolism, Cellular response to stress, Innate immune system, Intrinsic pathway of fibrin clot formation, Common pathway of fibrin clot formation, and Calnexin/calreticulin cycle (p ≤ 0.01).
Table 2
KEGG and Reactome Pathways revealed by Network enrichment analyses for differentially regulated proteins which were resolved in a STRING subnetwork in the vtg3-KO Experiment. A) proteins up-regulated in wild type eggs. B) proteins up-regulated in vtg3-KO eggs. Only statistically significant results are reported (χ2, p < 0.05). See Table S6 for full analysis report.
A) vtg3-KO Experiment: Network stats for proteins up-regulated in Wt eggs (N = 21) |
number of nodes: 11 |
number of edges: 7 |
average node degree: 1.27 |
clustering coefficient: 0.394 |
expected number of edges: 1 |
PPI enrichment p-value: 6.99E-06 |
confidence level: 0.4 |
KEGG Pathways |
pathway | Description | Protein counts | FDR |
dre01200 | Carbon metabolism | 2 of 125 | 0.0147 |
B) vtg3-KO Experiment: Network stats for proteins up-regulated in vtg3-KO eggs (N = 53) |
number of nodes: 36 |
number of edges: 46 |
average node degree: 2.56 |
clustering coefficient: 0.417 |
expected number of edges: 36 |
PPI enrichment p-value: 0.0517 |
confidence level: 0.4 |
KEGG Pathways |
pathway | description | protein count | FDR |
dre04141 | Protein processing in endoplasmic reticulum | 6 of 176 | 3.04E-06 |
dre00270 | Cysteine and methionine metabolism | 4 of 47 | 6.54E-06 |
dre00620 | Pyruvate metabolism | 3 of 41 | 0.00019 |
dre00330 | Arginine and proline metabolism | 3 of 58 | 0.00037 |
dre04260 | Cardiac muscle contraction | 3 of 89 | 0.001 |
dre01100 | Metabolic pathways | 8 of 1278 | 0.001 |
dre03040 | Spliceosome | 3 of 131 | 0.0022 |
dre00640 | Propanoate metabolism | 2 of 31 | 0.0022 |
dre04261 | Adrenergic signaling in cardiomyocytes | 3 of 180 | 0.0041 |
dre00010 | Glycolysis / Gluconeogenesis | 2 of 74 | 0.0091 |
dre04144 | Endocytosis | 3 of 293 | 0.013 |
dre04010 | MAPK signaling pathway | 3 of 359 | 0.0206 |
dre03010 | Ribosome | 2 of 126 | 0.0206 |
Reactome Pathways |
pathway | description | protein count | FDR |
DRE-450408 | AUF1 (hnRNP D0) binds and destabilizes mRNA | 5 of 54 | 5.35E-06 |
DRE-3371568 | Attenuation phase | 4 of 19 | 5.35E-06 |
DRE-3371497 | HSP90 chaperone cycle for steroid hormone receptors (SHR) | 4 of 21 | 5.35E-06 |
DRE-71288 | Creatine metabolism | 3 of 10 | 3.53E-05 |
DRE-3371453 | Regulation of HSF1-mediated heat shock response | 4 of 69 | 0.00014 |
DRE-2262752 | Cellular responses to stress | 6 of 321 | 0.0002 |
DRE-71406 | Pyruvate metabolism and Citric Acid (TCA) cycle | 3 of 55 | 0.002 |
DRE-189085 | Digestion of dietary carbohydrate | 2 of 11 | 0.0037 |
DRE-71291 | Metabolism of amino acids and derivatives | 4 of 249 | 0.0076 |
DRE-901042 | Calnexin/calreticulin cycle | 2 of 25 | 0.0111 |
DRE-70268 | Pyruvate metabolism | 2 of 31 | 0.0157 |
DRE-168256 | Immune System | 7 of 1379 | 0.0343 |
DRE-390522 | Striated Muscle Contraction | 2 of 54 | 0.0372 |
DRE-5358346 | Hedgehog ligand biogenesis | 2 of 58 | 0.0408 |
STRING enrichment analysis of differentially up-regulated proteins also forming the resolved protein-protein interaction networks in Wt eggs (N = 21) and in vtg1-KO eggs (N = 53) for KEGG Pathways and Rectome Pathways are given in Table 2. Results for other terms such as UniProt Keywords, PFAM Protein Domains, INTERPRO Protein Domains and Features and SMART Protein domains are additionally reported in Table S6. A single KEGG pathway, Carbon metabolism, was found to be enriched in the network revealed by proteins with higher abundance in Wt eggs (N = 21) (p ≤ 0.05). KEGG pathways that were enriched in the subnetwork resolved by proteins up-regulated in vtg3-KO eggs were Protein processing in endoplasmic reticulum, Cysteine and methionine metabolism, Pyruvate metabolism, Arginine and proline metabolism, Cardiac muscle contraction, Metabolic, Spliceosome, Propanoate metabolism, Andrenergic signaling of cardiomyocytes, Glycolysis/Gluconeogenesis, Endocytosis, MAPK signaling and Ribosome (p ≤ 0.05). The corresponding Reactome pathways were AUFI (hnRNP D0) binds and destabilizes mRNA, Attenuation phase, HSP90 chaperone cycle for steroid hormone receptors, Creatine metabolism, Regulation of HSFI-mediated heat shock response, Cellular response to stress, Pyruvate metabolism and citric acid (TCA) cycle, Digestion of dietary carbohydrate, Metabolism of amino acids and derivatives, Calnexin/calreticulin cycle, Pyruvate metabolism, Immune system, Striated muscle contraction, and Hedgehog ligand biosynthesis (p ≤ 0.01) (Table 2).
Figure 6. Proteins with significant differences in abundance between vtg1-KO eggs and Wt eggs in the vtg1-KO Experiment. Panel a. HeatMap representation of differences in abundance based on normalized spectral counts. Panel b. Vitellogenins with significant differences in abundance between vtg1-KO eggs and Wt eggs. Panel c. Other proteins with significant differences in abundance between vtg1-KO eggs and Wt eggs. All proteins are named for the transcript(s) to which spectra were mapped; for full protein names, see Table S1. Only proteins that were identified in ≥ 4 samples were included in this analysis (t-test, p ≤ 0.05, followed by Benjamini Hochberg correction for multiple tests, p ≤ 0.05). Vertical bars indicate mean N-SC values (n = 4 per group) and vertical brackets indicate SEM. Protein (transcript) labels are color-coded to indicate functional categories to which the proteins were attributed (Fig. 1).
The list of total identified proteins in the vtg1-KO experiment (N = 301) and in the vtg3-KO experiment (N = 238) were subjected to filtering by Perseus 1.5.5.3 (available online at https://maxquant.org/perseus/) for candidates to further analyze for statistically significant differences in abundance. No fold difference was taken into account at this step of analysis, but only proteins which were detected in ≥ 4 samples (N = 132 for vtg1-KO and N = 102 for vtg3-KO) were carried further for statistical analyses of abundances based on N-SC (t-test followed by Benjamini-Hochberg correction for multiple testing, p < 0.05). A total of 45 proteins displayed statistically significant differences in abundance between vtg1-KO eggs and Wt eggs (Fig. 6a). Two variants of Vtg1 and two variants of Vtg4 were found to be uniquely identified in Wt eggs and variants of Vtg7 and Vtg3 were 3x and 1.8x more abundant, respectively, in vtg1-KO eggs (p < 0.05) (Fig. 6b). Three forms of a brain type creatine kinase (Ckbb) and two variants of Cat were significantly more abundant in Wt eggs (p < 0.05). Among other differentially regulated proteins with significant differences in abundance between vtg1-KO eggs and Wt eggs were a nucleoside diphosphate kinase (Nme2b), a microfibril-associated protein 4 (MFAP4), several variants of quinoid dihydropteridine reducatase (Qdprb), two variants of glyceraldehyde-3-phosphate dehydrogenase (Gapdh), a malate dehydrogenase 2 (Mdh2), a protein disulfide-isomerase (Pdia3), several eukaryotic translation elongation factor alpha subunits (Eef1a), two variants of si:dkey-46g23.5 (predicted to have endopeptidase inhibitor activity), two variants of DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (Ddx41), two variants of serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1, like (Serpina1), and many variants of a protein predicted to contain a C-type lectin domain (Fig. 6c).
Figure 7. Proteins with significant differences in abundance between vtg3-KO eggs and Wt eggs in the vtg3-KO Experiment. Panel a. HeatMap representation of differences in abundance based on normalized spectral counts. Panel b. Vitellogenins with significant differences in abundance between vtg3-KO eggs and Wt eggs. Panel c. Other proteins with significant differences in abundance between vtg3-KO eggs and Wt eggs. All proteins are named for the transcript(s) to which spectra were mapped; for full protein names, see Table S2. Only proteins that were identified in ≥ 4 samples were included in this analysis (t-test, p ≤ 0.05, followed by Benjamini Hochberg correction for multiple tests, p ≤ 0.05). Vertical bars indicate mean N-SC values (n = 4 per group) and vertical brackets indicate SEM. Protein (transcript) labels are color-coded to indicate functional categories to which the proteins were attributed (Fig. 1).
The exact same series of statistical analysis revealed 18 proteins with significant differences in abundance (N-SC) between vtg3-KO eggs and Wt eggs (Fig. 7a). Three variants of Vtg3, two isoforms of si:ch211-251f6.7 and two isoforms of zgc:136254, proteins predicted to be autophagosome/lysosome components with phosphatidylinositol-3-phosphate binding activity in zebrafish (UniprotKB-F1Q1D9_DANRE and UniProtKB Q1RMB1_DANRE), and two variants of solute carrier family 45 member 4 protein (Slc45a4) were found to be uniquely present in Wt eggs (Fig. 7b and 7c), while Hspa5, a Eukaryotic translation elongation factor 1 alpha 1 like 1 (Eef1a1l1), and two variants of Cat were significantly higher in abundance in Wt eggs versus vtg3-KO eggs (Fig. 7c). Two Vtg7 variants, a malate dehydrogenase 2 (Mdh2), a heat shock 70 kDa protein 8 (HSPA8), and a variant of protein disulfide-isomerase (Pdia4) were found to be expressed in significantly higher abundance in vtg3-KO eggs than in Wt eggs (p < 0.05) (Fig. 7b and Fig. 7c). Of these, Vtg7 was more abundant (1.5-fold) in vtg3-KO eggs, just as it was in vtg1-KO eggs, potentially compensating for the missing Vtgs in both experiments. Catalases showed a similar pattern of up-regulation in both the vtg1-KO and vtg3-KO experiments, increasing in abundance 7.2 x and 4.5 x, respectively (p < 0.05). Mdh2 was also more abundant in Wt eggs in both the vtg1-KO experiment (3.6 x) and the vtg3-KO experiment (3.4 x) (p < 0.05). Finally, two protein disulfide-isomerases (Pdia3 and Pdia4) seem to be upregulated in vtg1-KO eggs and in vtg3-KO eggs in comparison to Wt eggs with 4.5-fold and 4.3-fold differences, respectively (p < 0.05) (Fig. 6 and Fig. 7).