Current Proteomics - Volume 19, Issue 3, 2022

Background: The exact mechanism of acute pancreatitis (AP), which is an inflammation of the pancreas, still remains unclear. Objective: In this study, we examined the protein phosphorylation changes during the early stage of AP in mice using proteomic analysis. Methods: AP model in mice was constructed using an intraperitoneal injection of cerulein. Blood samples and pancreas were collected at 1, 3, 6, 9h after the final injection (n=3 at each time point). Samples collected 3h after the final injection were separately mixed and named S (saline group) and C1 (cerulein group); samples collected 6h after the final injection from the cerulein group were mixed and named C2. Proteins from S, C1, and C2 were extracted, digested by trypsin, and subjected to LC-MS/MS analysis, bioinformatics analysis, and Western blotting. Results: A total of 549 sites (426 proteins) were upregulated, and 501 sites (367 proteins) were downregulated in C1 compared to S; while 491 phosphorylation sites (377 proteins) were upregulated and 367 sites (274 proteins) were downregulated in C2 compared to S. Motif analysis showed that proline-directed kinase and basophilic kinase had a key role during early AP. During an early AP stage, the cellular distributions of proteins slightly changed. The types of domains changed with the development of AP. Phosphorylation proteins associated with calcium signaling, especially IP3R mediated calcium release, lysosome and autophagosome pathway, pancreatic digestive activation, and secretion, were found to be involved in the development of early AP independent of NF-kB activation. Moreover, the MAPK family was found to have a greater impact at the early stage of AP. We also found differentially expressed phosphorylations of amylase and trypsinogen and increased phosphorylation of MAPK6 S189 in early AP. Conclusion: IP3R mediated calcium release and activation of MAPK family are key events promoting the development of early AP.

Background: Amylase used in the market is mostly medium-temperature enzyme or high-temperature enzyme and has poor enzyme activity under low-temperature environment. Acid α-amylase can be used to develop digestion additives in the pharmaceutical and healthcare industries. The amino acid sequence and structural differences among α-amylases obtained from various organisms are high enough to confer interesting biochemical diversity to the enzymes. However, low- temperature (0-50°C) amylase, with an optimum temperature and heat sensitivity, has a greater potential value than medium (50-80°C) and high (80-110°C) temperature amylases. Methodology: The gene amy48 from encoding extracellular α-amylase in Bacillus subtilis YX48 was successfully cloned into the pET30a (+) vector and expressed in Escherichia coli BL21 (DE3) for biochemical characterization. Results and Conclusion: The molecular weight of α-amylase was 75 kDa. The activity of α-amylase was not affected by Ca²⁺, and Amy48 had the best activity at pH 5.0 and 37°C. AMY48 has high stability over a narrow pH and temperature range (5.0-8.0 and 30-45°C). Amylase activity was strongly inhibited by Zn²⁺, Mn²⁺, Cu²⁺, and Fe²⁺ ions, but Na+, K+, and Co²⁺ ions stimulate its activity slightly. The purified enzyme showed gradually reduced activity in the presence of detergents. However, it was remarkably stable against EDTA and urea.

Background: Identification of a peripheral biological marker might aid in identifying patients at high risk of attempting suicide and might help in effective early intervention. Objective: In the present study, we extend the findings of our previous multidimensional proteomics study by examining the levels of plasma Apolipoprotein-AIV in patients diagnosed with major depression with and without suicidal ideation compared to age and gender-matched controls. Methods: Using the mass spectrometry platform, we quantified the levels of plasma Apolipoprotein- AIV in patients with major depressive disorder with and without suicidal ideation compared to matched controls with isotope-labelled peptides-based quantitative proteomics approach. Results: The targeted quantitative proteomics approach with isotope-labelled peptides showed that plasma Apolipoprotein-AIV was significantly downregulated in depressed patients having suicidal ideation 1.45 (CI:1.11-1.90) compared to those without suicidal ideation 0.88 (CI:0.77-1.003). Conclusion: These findings extend our earlier observation of downregulation of plasma Apolipoprotein- AIV in patients with suicidal attempts to depressed patients with suicidal ideation. The consistent downregulation of plasma Apolipoprotein-AIV observed in both the proteomics studies suggests Apolipoprotein-AIV might be a plasma-based biomarker for suicidal behaviour.

Background: H7N9 influenza virus poses a high risk to human beings, and proteomic evaluations of this virus may help better understand its pathogenic mechanisms in human systems. Objective: This study aimed at determining membrane proteins related to H7N9 infection. Methods: In this study, we infected primary human alveolar adenocarcinoma epithelial cells (A549) with H7N9 (including wild and mutant strains) and then produced enriched cellular membrane isolations, which were then evaluated by western blot. The proteins in these cell membrane fractions were analyzed using the isobaric Tags for Relative and Absolute Quantitation (iTRAQ) proteome technologies. Results: Differentially expressed proteins (n = 32) were identified following liquid chromatography- tandem mass spectrometry, including 20 down-regulated proteins, such as CD44 antigen and CD151 antigen, and 12 up-regulated proteins, such as tight junction protein ZO-1 and prostaglandin reductase 1. Gene Ontology database searching revealed that 20 out of the 32 differentially expressed proteins were localized to the plasma membrane. These proteins were primarily associated with the cellular component organization (n = 20) and enriched in the reactome pathway of extracellular matrix organization (n = 4). Conclusion: These findings indicate that H7N9 may dysregulate cellular organization via specific alterations to the protein profile of the plasma membrane.

Background: Spiroplasma eriocheiris is a novel pathogen of freshwater crustaceans and is closely related to S. mirum. They have no cell wall and a helical morphology. They have the ability to infect mammals with an unclear mechanism. Objective: In this study, our aim was to investigate the profile of protein expression in 3T6 cells infected with S. eriocheiris. Methods: The proteome of 3T6 cells infected by S. eriocheiris was systematically investigated by iTRAQ. Results: We identified and quantified 4915 proteins, 67 differentially proteins were found, including 30 up-regulated proteins and 37 down-regulated proteins. GO term analysis shows that dysregulation of adhesion protein , interferon and cytoskeletal regulation are associated with apoptosis. Adhesion protein Vcam1 and Interferon-induced protein GBP2, Ifit1, TAPBP, CD63 ,Arhgef2 were up-regulated. A key cytoskeletal regulatory protein, ARHGEF17 was down-regulated. KEGG pathway analysis showed the NF-kappa B signaling pathway, the MAPK signaling pathway , the Jak-STAT signaling pathway and NOD-like receptor signaling are closely related to apoptosis in vivo. Conclusion: Analysis of the signaling pathways involved in invasion may provide new insights for understanding the infection mechanisms of S. eriocheiris.

Objective: The tissue remodeling process and cellular migration relate to the activities of Matrix Metalloproteinases (MMPs). The aim of this study was to investigate the effects of a predicted motif from TIMPs on the MMP-2 and MMP-9 activities secreted from the differentiated macrophages. Materials and Methods: The monocytes were isolated from the healthy individuals by RosetteSep kit and were differentiated into macrophages using M-CSF. A 4-amino acid motif (TCAP) was predicted using bioinformatics tools. Zymography technique was applied for the measurement of MMP activities. The docking studies were also investigated between MMPs, tetrapeptide, and Batimastat. Results: The TCAP inhibited significantly the differentiated macrophage MMP-2 and MMP-9 activities (p=0.0001and p=0.01, respectively). The docking results suggested that some MMP amino acids are involved with both tetrapeptide (TCAP) and Batimastat, Conclusion: The data showed that the small motif (TCAP) of TIMPs inhibits effectively the MMP- 2 activity.

Aim: Proteomic profile analysis of pulmonary artery in a rat model under hypoxic pulmonary hypertension. Background: Hypoxic pulmonary hypertension (HPH) is a pathological condition exemplified by a constant rise in pulmonary artery pressure in high-altitudes. Objective: To investigated the proteome profile and response mechanisms of SD rats under hypoxia over a period of four-weeks. Methods: Proteomic profile analysis of pulmonary artery in a rat model under hypoxic pulmonary hypertension. Results: With 3, 204 proteins identified, 49 were up-regulated while 46 were down-regulated. Upregulated genes included Prolargin, Protein S100-A6 and Transgelin-2, whereas Nascent polypeptide- associated complex and Elongator complex protein 1 were down-regulated. KEGG enriched pathways had purine metabolism, cancer and lipolysis regulation as significantly enriched in the hypoxic group. Conclusion: In conclusion, our findings submit a basis for downstream studies on tissue hypoxia mechanisms alongside the associated physiological conditions. Hypoxic pulmonary hypertension (HPH) is a pathological condition exemplified by a constant rise in pulmonary artery pressure in high altitudes. Herein, we investigated the proteome profile and response mechanisms of Sprague-Dawley (SD) rats under hypoxia over a period of four weeks. Unbiased iTRAQ-based quantitative proteomics was utilized in proteome profile analysis of a rat model exposed to HPH. With 3, 204 proteins identified, 49 were upregulated while 46 were downregulated. Upregulated genes included Prolargin, Protein, S100-A6 and Transgelin-2, whereas Nascent polypeptide-associated complex and Elongator complex protein 1 were downregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched pathways had purine metabolism, cancer, and lipolysis regulation as significantly enriched in the hypoxic group. In conclusion, the findings from this study submit a basis for downstream studies on tissue hypoxia mechanisms alongside the associated physiological conditions.

Background: The absence of absolute clinical indicators and suitable biomarkers hinders the timely diagnosis of women at risk of preterm birth. It influences roughly 12% of births. At delivery and clinical presentation, preterm births are generally inspected based on the gestational period. Different disturbed pathways are associated with the signs of at-risk pregnancies. Objective: The main purpose of this study is to analyze and explore the serum proteome of early deliveries and help health care professionals to improve the understanding of the progression of preterm birth. Methods: In the present study, 200 pregnant females of 20-30 years of age were selected. We collected samples of second and third-trimester pregnant females, out of which 40 females delivered preterm. We further divided them into three groups, i.e., extremely preterm group, very preterm, and controls. Overall comparison of serum profiles of all the three groups expressing fourteen proteins ranging between 200-10kDa was made. Serum proteins were isolated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photographed by totalLab quant software. Groups were evaluated using the ANOVA Tukey’s Post Hoc analysis. Results: Proteins of 69kDa and 15kDa expressed a significant decrease when compared with control subjects. In contrast, the proteins of 23kDa expressed a significant increase, while the proteins of 77kDa, 45kDa, and 25kDa demonstrated no considerable variation. Conclusion: The serum proteins showing significant difference as compared to the control group will serve as predictive biomarkers for at-risk pregnancies. The present study is expected to considerably improve the understanding of the disease pathogenesis along with improved diagnostic and therapeutic approaches leading to better management of pregnancy and reducing the risk of preterm birth.

Background: Preeclampsia (PE) is a common pregnancy-specific disease with potential adverse maternal and neonatal outcomes. Objective: We aimed to estimate proteomic profiles of serum-derived exosomes obtained from PE offspring with bioinformatics methods. Methods: Serum samples were collected from 12 h, 24 h, and 72 h newborns delivered by preeclamptic and normal pregnant women. Exosomes were extracted, and the concentration and size distribution were determined. The exosome surface markers CD9, CD63, CD81, and TSG101, were assayed by Western blot. The exosome proteins were screened by quantitative proteomics with tandem mass tag (TMT). All the identified proteins were subjected to the Weighted Gene Co- Expression Network Analysis (WGCNA), GO function, and KEGG pathway analysis. A proteinprotein interaction network (PPI) was used to extract hub proteins through the Cytohubba plugin of Cytoscape. Results: The extracted exosomes were round or oval vesicular structures at a 100-200 nm concentration, and the size distribution was standard and uniform. Exosome surface markers CD9, CD63, and CD81 were detected, and TSG101 was not detected. A total of 450 expressed proteins were selected, and 444 proteins were mapped with gene names. A blue module with 66 proteins highly correlated with phenotype at 12 h. Functional analyses revealed that module proteins were mainly enriched in the extracellular matrix. The top 10 selected hub proteins were identified as hub proteins, including COL6A2, HSPG2, COL4A1, COL3A1, etc. Conclusion: Our study provides important information for exploring molecular mechanisms of preeclampsia and potential biomarkers for future diagnosis and treatment in the clinic.