Khib protein detection and identification
Based on the criteria, Pvalue < 0.05 , we identified that 156 upregulated proteins(fold change>1.5),124 downregulated proteins(fold change<0.66), including 220 Khib sites that were upregulated and 187 Khib sites that were downregulated. The proteins with higher fold change and multiple upregulated modifications were hemoglobin subunit gamma-2 (P69892), 40S ribosomal protein S9 (P46781), and 60S ribosomal protein L6 (Q02878). The proteins with higher fold change and multiple downregulated modifications were GRIP1-associated protein 1 (Q4V328), heterogeneous nuclear ribonucleoprotein A/B (Q99729), and heterogeneous nuclear ribonucleoproteins A2/B1 (P22626), the differentially expressed protein summary in table2. The number of modificantion sites per proteion in theFigure 1a, The proteins with > 7 Khib modification sites were Spectrin alpha chain, nonerythrocytic 1 (Q13183), Talin-1 (Q9Y490), Spectrin beta chain, erythrocytic (P11277)Figure. The 897 2-hydroxyisobutylated proteins, most contain 1-2 modification sites, some have 3-7 modification sites, and a few contain 8 or more modification sites. Most peptides vary in length from 8 to 20 amino acids, consistent with trypsin digestion(Figure 1b). To investigate the presence of Khib in SLE, Western blotting with Khib antibody was carried out in the total protein, we can saw a large number of protein bands, then a wide protein mass range was observed, revealing that Khib is highly abundant with the protein (Figure 2a). To identify Khib sites in SLE,we randomly selected H3K79hib site and H3K14hib loci for Western blotting analysis, and detected the expression of H3K79hib site and H3K14hib antibodies in SLE with H3 as a reference(Figure 2b).
Subcellular localization information and GO classifications of proteins with Khib
According to the subcellular localization analysis, the largest percentage of proteins with Khib was in the cytoplasm, and proteins with higher fold change modifications were localized in the cytoplasm. For the upregulated proteins with Khib shown in Figure 3(a), 49% were located in the cytoplasm 49%, 19% in the nucleus, and 10% in the mitochondria. Among the downregulated proteins in Figure 3(b), 42% were located in the cytosol, 30% in the nucleus, and 14% in the extracellular fraction. The GO classification indicated that the identified proteins with Khib fell into three categorizations (the cellular component, molecular function, and biological process). Of the upregulated proteins in the cellular component category, as shown in Figure 4(a), 20% were in the cell, 19% were in the organelle, 15% were in the extracellular region, and 13% were in the membrane. Among the downregulated proteins shown in Figure 4(b), 20% were in the cell, 20% were in the organelles; 14% were in the membrane and 14% were in the membrane-enclosed lumen. Based on their molecular function, of the upregulated proteins shown Figure 4(c) and the downregulated proteins shown Figure 3(d), 51% and 55%, respectively, were in the largest category: binding. Based on their biological process, of the upregulated proteins shown in Figure 4(e), 13% were involved in cellular processes, 12% were involved in biological regulation, and 11% were involved in single-organism processes. The percentages of the downregulated proteins were involved in the following biological processes: cellular processes, 14%; biological regulation, 12%; metabolic processes, 11%; and single-organism processes, 11% Figure 4(f).
Functional enrichment of proteins with Khib
Functional enrichment analysis revealed that the proteins with Khib were broadly involved in biological processes. Figure 4 shows the functional enrichment description based on the GO analysis (cellular component, molecular function, biological process), the Fisher exact test p-value was determined after the logarithmic transformation. The longer the bar in the graph, the more significant the enrichment of differentially expressed proteins in this classification or function. Based on the upregulated proteins with the red bar of Figure 5(a), the proteins were enriched for the adherens junctions, anchoring junctions, and cell-substrate junctions in the cellular components category. The proteins were enriched for the domain-specific binding, structural constituent of ribosome, and structural molecule activity in the molecular function categories. As determined by the biological process categories, the proteins localized at the endoplasmic reticulum, for actin cytoskeleton reorganization, and for the regulation of the viral life cycle were enriched. Based on the downregulated proteins, signified by the blue bar in Figure 5(b), for the cellular component category, the proteins were enriched in the cytosolic ribosomes, ribosomal subunits, and the cytosol; for the molecular function category, these proteins were enriched in the structural constituents of ribosomes for structural molecule activity and DNA binding; and for the biological process category, these proteins were enriched for ribosomal large subunit biogenesis, viral gene expression, and protein localization to the endoplasmic reticulum.
KEGG organically combines genomic information and gene function information, and is a powerful tool for metabolic analysis and metabolic network research in vivo. By analyzing metabolic pathways and interaction networks of disease-related genes, it is helpful to understand the molecular pathological basis of diseases.Of the 9 different signaling pathways revealed by the KEGG pathway enrichment analysis, the hsa03010 Ribosome pathway was the most relevant, as shown in Figure 6. The multiple modifications with an fold change>2.0 for the proteins with the upregulated proteins of the has03010 Ribosome pathway analysis were 40S ribosomal protein S9(P46781), 60S ribosomal protein L6(Q02878), 60S ribosomal protein L26(P61254), 40S ribosomal protein SA(P08865), 40S ribosomal protein S16(P62249), 40S ribosomal protein S3a(P61247). The 40S ribosomal protein S9(P46781) Khib modification sits locus was predicted by STRING (Figure 7), the 40S ribosomal protein S9(P46781) had 6 Khib modification sits with K121,K139,K93,K52,K91and K180 .
The protein domain functional descriptions identified for Khib were annotated by InterProScan (Figure 8). Upregulated proteins with Khib were significantly enriched for the Calponin homology domain, the Nucleotide-binding alpha-beta plait domain and FERM, and the N-terminal. The downregulated proteins with Khib were enriched for Globin-like, Globin/Protoglobin, Globin domains and so on.