Current Proteomics - Volume 17, Issue 3, 2020

Background: Arecoline is known as a carcinogenic toxicant. The refreshment effect of arecoline is mainly due to the increase in vasodilation and blood flow. This is essential to understand whether arecoline can induce the production of Nitric Oxide (NO•) and regulate the subsequent protein S-nitrosylation in Endothelial Cells (ECs). Objective: The present study is focused on the promotion effect of arecoline in NO• production and the subsequent regulation of S-nitrosoproteome. Methods: The phosphorylation of endothelial nitric oxide synthase serine 1177 residue (peNOS^Ser1177) was investigated by western blot. By using a specific FA-OMe fluorescent probe, the NO• molecules could be observed by fluorescent microscopy or flow cytometry. S-nitrosylated proteins were purified by biotin switch and then subjected to the Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-labeled shotgun proteomic analysis. Results: Our study reveals that a lower concentration of arecoline can increase the phosphorylation of peNOS^Ser1177. Pretreatment of N^G-nitro-L-arginine methyl ester (L-NAME) indicated that arecolineinduced NO• production was mediated by e-NOS. We identified 224 proteins with up-regulated S-nitrosylation and 159 proteins with down-regulated S-nitrosylation. The NO• binding sites of seven representative S-nitrosoproteins were illustrated. The effect of arecoline on the S-nitrosylation of HSP60 chaperonin and calnexin was verified. Conclusion: Our experimental results proved that a lower concentration of arecoline could modulate the production of NO• and the subsequent protein S-nitrosylation. Therefore, it is worthy for further investigation and discussion if these S-nitrosoproteomes are important in maintaining endothelium homeostasis.

Background: Gestational Diabetes Mellitus (GDM) is one of the most common medical complications of pregnancy. The untreated GDM affects both the baby and mother during gestation as well as presents the risk of subsequent type 2 diabetes in mothers and babies. Objective: To determine the effect of GDM on plasma proteomics as variable plasma proteins can be secreted by the cells at the pathological sites and can serve as a biomarker of the disease. Methods: Blood samples were taken from 400 pregnant women at early second trimester and they were followed till early third trimester or until the development of GDM. All the pregnant females were sampled again in 3^rd trimester. Overall 42 pregnant females developed GDM. These females were grouped as GDM I and GDM II, before and after the development of gestational diabetes, respectively. An equivalent number of pregnant women who did not develop GDM served as control I and control II in early second and third trimester, respectively. Blood samples from all the groups were subjected to 2D gel electrophoresis. Results: Nineteen protein spots were differently expressed between GDM and control groups, two spots were further confirmed by LC-MS/MS as Retinol binding protein A4 and Transthyretin. These two proteins were found to be up regulated in GDM group in early second trimester as well as early 3^rd trimester. Conclusion: Transthyretin and Retinol binding protein 4 can be used as predictive plasma biomarkers of GDM that might help in the identification of at-risk pregnancies, hence, providing the best opportunity for early treatment in order to prevent the onset or progression of the disease.

Objective: This study aimed to detect hepatic oxidative damage caused by aflatoxin B1 (AFB1), as well as to examine how propolis protects against hepatotoxic effects of AFB1. Methods: Rats were split into four groups as control group, AFB1 group, propolis group, AFB1+ propolis group. Results: There was significant increase in malondialdehyde (MDA) level and tumor suppressor protein (TP53) gene expression, Glutathione (GSH) level, Catalase (CAT) activity, CAT gene expression decreased in AFB1 group in blood. MDA level and Glutathione-S-Transferase (GST) activity, GST and TP53 gene expressions increased in AFB1 group, whereas GSH level and CAT activity alongside CAT gene expression decreased in liver. AFB1+propolis group showed significant decrease in MDA level, GST activity, TP53 and GST gene expressions, GSH level and CAT activity and CAT gene expression increased in liver compared to AFB1 group. Conclusion: These results suggest that propolis may potentially be natural agent that prevents AFB1- induced oxidative stress and hepatotoxicity.

Background: Collagenase is a type of proteolytic enzyme that specifically hydrolyzes native collagen with a three-dimensional helical structure. The structure and properties of collagenase vary with different sources and types. In addition to the well-characterized Clostridium collagenase, other collagenases are largely unknown. Various gene and protein databases have been widely used to mine novel functional genes in the genome. Gene mining and sequence analysis are effective methods for studying these enzymes. Objective: The present study aimed to understand the molecular, structural, and functional characteristics of collagenase from Bacillus cereus MH19 using a bioinformatics approach. Methods: Based on the three-generation sequencing PacBio technique, Sequencing the Bacillus cereus MH19 genome. Function annotation is completed by blasting genes with different databases. Collagenases were investigated based on the physiochemical properties, phylogenetic relation, and domain architecture. The 3D structure model of the selected collagenase has been constructed and verified by SAVES. Results: There were 5 collagenases in Bacillus cereus MH19 with a molecular weight distribution ranging from 36-110 kDa. The analysis of evolutionary relationship between different collagenases indicating that the BCC000504 and BCC003388 collagenase gene sequences are closer to the typical collagenase genes ColG, ColA, and ColH, followed by BCC003615. The domain and function analysis showed that the collagenases BCC000504 and BCC003388 were similar to the collagenases ColG, ColA and ColH. BCC004271 was similar to BCC004272, and BCC003615 might be quite different from other collagenases. The secondary structure of collagenase was analyzed. The SAVES evaluation indicates that 3D structural modeling of the selected collagenase is acceptable. Conclusion: This study provides an overview of the molecular, functional, and structural characteristics of collagenase from Bacillus cereus MH19, which helps to understand the bacterial collagenase. The characterization of the collagenase will certainly expand the application range of collagenase.

Background: Wilson’s disease is a rare autosomal recessive genetic disorder of copper metabolism, which is characterized by hepatic and neurological disease. ATP7B encodes a transmembrane protein ATPase (ATP7B), which functions as a copper-dependent P-type ATPase. The mutations in the gene ATP7B (on chromosome 13) lead to Wilson’s disease and is highly expressed in the liver, kidney, and placenta. Consequently, this enzyme was considered a special topic in clinical and biotechnological research. For in silico analysis, the 3D molecular modeling of this enzyme was conducted in the I-TASSER web server. Methods: For a better evaluation, the important characteristics of this enzyme such as the rare codons of the ATP7B gene were evaluated by online software, including a rare codon calculator (RCC), ATGme, LaTcOm, and Sherlocc program. Additionally, the multiple sequence alignment of this enzyme was studied. Finally, for evaluation of the effects of rare codons, the 3D structure of ATP7B was modeled in the Swiss Model and I-TASSER web server. Results: The results showed that the ATP7B gene has 35 single rare codons for Arg. Additionally, RCC detected two rare codons for Leu, 13 single rare codons for Ile and 28 rare codons for the Pro. ATP7B gene analysis in minmax and sliding_window algorithm resulted in the identification of 16 and 17 rare codon clusters, respectively, indicating the different features of these algorithms in the detection of RCCs. Analyzing the 3D model of ATP7B protein showed that Arg816 residue constitutes hydrogen bonds with Glu810 and Glu816. Mutation of this residue to Ser816 cause these hydrogen bonds not to be formed and may interfere in the proper folding of ATP7B protein. Furthermore, the side chain of Arg1228 does not form any bond with other residues. By mutation of Arg1228 to Thr1228, a new hydrogen bond is formed with the side chain of Arg1228. The addition and deletion of hydrogen bonds alter the proper folding of ATP7B protein and interfere with the proper function of the ATP7B position. On the other hand, His1069 forms the hydrogen bonds with the His880 and this hydrogen bond adhere two regions of the protein together, which is critical in the final structural folding of ATP7B protein. Conclusion: Previous studies show that synonymous and silent mutations have been linked to numerous diseases. Given the importance of synonymous and silent mutations in diseases, the aim of this study was to investigate the rare codons (synonymous codons) in the structure of ATP7B enzyme. By these analyses, a new understanding was developed and our findings can further be used in some fields of the clinical and industrial biotechnology.

Background: Tea is one of the most popular calming drinks. Drought is a major environmental factor that limited the growth and development of plants. Objective: Therefore, the identification of proteins under the drought stress conditions in tea can have an essential role in the breeding programs and tea production. Methods: For this purpose, 14 tea clones were studied under normal and drought stress conditions in two separate experiments, and the leaves of the clones were stored at −80°C. After the identification of two clones (100 and 278) as tolerant and sensitive clones, respectively, the proteomics approach was used to compare the leaf protein profile changes under both conditions. Results: The results of proteomics showed about 500 detectable protein spots, of which 250 spots were repeatable. Among the 250 reproducible spots, 16 spots responded to the drought stress, which showed the highest amount of variation among the treatments. Thioredoxin, peroxidase, superoxide dismutase, ribosomal protein, and hsp70 were mentioned among the identified proteins. These proteins were involved in various cellular functions. Conclusion: Identified proteins also had a crucial role in regulating carbohydrate and nitrogen metabolism and the scavenging of the Reactive Oxygen Species (ROS). Upregulation of proteins involved in protein processing (ribosomal protein), oxygen species scavenging, and defense (Superoxide dismutase, Peroxidase, and thioredoxin) may increase plant adaptation to drought stress. This study was the first report that showed ribosomal protein L32 was significantly changed in tea against drought stress response. Therefore, these proteins can protect the plant against drought stress. This study partially identified the drought stress proteins in the tea plant.

Background: Deinagkistrodon acutus (D. acutus) and Bungarus multicinctus (B. multicinctus) as traditional medicines have been used for hundreds of years in China. The venoms of these two species have strong toxicity on the victims. Objective: The objective of this study is to reveal the profile of venom proteins and peptides of D. acutus and B. multicinctus. Methods: Ultrafiltration, SDS-PAGE coupled with in-gel tryptic digestion and Liquid Chromatography- Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS) were used to characterize proteins and peptides of venoms of D. acutus and B. multicinctus. Results: In the D. acutus venom, 67 proteins (16 protein families) were identified, and snake venom metalloproteinases (SVMPs, 38.0%) and snake venom C-type lectins (snaclecs, 36.7%) were dominated proteins. In the B. multicinctus venom, 47 proteins (15 protein families) were identified, and three-finger toxins (3FTxs, 36.3%) and Kunitz-type Serine Protease Inhibitors (KSPIs, 32.8%) were major components. In addition, both venoms contained small amounts of other proteins, such as Snake Venom Serine Proteinases (SVSPs), Phospholipases A2 (PLA2s), Cysteine-Rich Secreted Proteins (CRISPs), 5'nucleotidases (5'NUCs), Phospholipases B (PLBs), Phosphodiesterases (PDEs), Phospholipase A2 Inhibitors (PLIs), Dipeptidyl Peptidases IV (DPP IVs), L-amino Acid Oxidases (LAAOs) and Angiotensin-Converting Enzymes (ACEs). Each venom also had its unique proteins, Nerve Growth Factors (NGFs) and Hyaluronidases (HYs) in D. acutus, and Cobra Venom Factors (CVFs) in B. multicinctus. In the peptidomics, 1543 and 250 peptides were identified in the venoms of D. acutus and B. multicinctus, respectively. Some peptides showed high similarity with neuropeptides, ACE inhibitory peptides, Bradykinin- Potentiating Peptides (BPPs), LAAOs and movement related peptides. Conclusion: Characterization of venom proteins and peptides of D. acutus and B. multicinctus will be helpful for the treatment of envenomation and drug discovery.