Current Proteomics - Volume 17, Issue 5, 2020

Infectious diseases have caused historical pandemics in the world. Three strategies, including sanitation programs, antimicrobial drugs, and vaccines are considered for the prevention and treatment of infectious diseases. Today, some infectious diseases cause millions of mortalities universally. Due to the emergence of antibiotic-resistant pathogens, as well as some limitations of traditional vaccines, focusing on novel strategies is essential. Multi-Epitope Vaccines (MEVs), as a novel strategy, have been designed based on immunoinformatics methods; epitope prediction by authentic servers, attachment of epitopes using proper linkers, physicochemical, immunological and structural evaluation by bioinformatics tools that are basic stages in MEVs designing. Advantages such as cost-effective, high safety, less time consumption in designing, the application of natural adjuvants, and satisfactory preclinical evaluation outstand MEVs than other types of vaccines. Therefore, MEVs are promising vaccines against resistant diseases such as lower respiratory infection and diarrhea.

Background: Licorice is an herbal medicine applied extensively worldwide, and most of the licorice for clinical consumption is provided by Glycyrrhiza uralensis Fisch. Evidence suggests that there is a significant difference in the metabolite composition of licorice from different ecotypes. Objective: To better understand the proteomic changes and molecular mechanisms of metabolite formation in wild and cultivated Glycyrrhiza uralensis Fisch. Methods: Firstly, we established a proteome database by annotating protein sequences according to the genomic and transcriptomic data of G. uralensis. Then, iTRAQ and LC-MS/MS were applied to detect significant protein changes between cultivated and wild G. uralensis. A total of 2751 validated proteins were obtained with high confidence, and 333 were differentially expressed. Differentially expressed proteins were identified and analysed by GO, KEGG, and STRING for network and pathway enrichment. Ultimately, we combined the iTRAQ results with our previous investigation on metabolites to understand the molecular mechanisms underlying metabolite accumulation. Results: The results showed that differentially expressed proteins were mainly involved in the anabolism of carbohydrates and important amino acids that participate in primary metabolism and secondary metabolite synthesis. Another important pathway is the synthesis of flavonoids, which are generally accepted as important bioactive constituents of G. uralensis, and the accumulation of flavonoids in different synthesis stages in two ecotypes of G. uralensis was diverse. Therefore, the differentially abundant proteins in wild and cultivated G. uralensis possibly resulted in differences in medicinal compounds. Conclusion: Our study will provide novel clues for revealing the molecular mechanism of secondary metabolite synthesis as well as quality formation in wild and cultivated G. uralensis.

Background: The tolerogenic homeostasis in Breast Cancer (BC) can be surpassed by rationally designed immune-encouraging constructs against tumor-specific antigens through immunoinformatics approach. Objective: Availability of high throughput data providing the underlying concept of diseases and awarded computational simulations, lead to screening the potential medications and strategies in less time and cost. Despite the extensive effects of Placenta Specific 1 (PLAC1) in BC progression, immune tolerance, invasion, cell cycle regulation, and being a tumor-specific antigen the fundamental mechanisms and regulatory factors were not fully explored. It is also worth to design an immune response inducing construct to surpass the hurdles of traditional anti-cancer treatments. Methods and Result: The study was initiated by predicting and modelling the PLAC1 secondary and tertiary structures and then engineering the fusion pattern of PLAC1 derived immunodominant predicted CD8+ and B-cell epitopes to form a multi-epitope immunogenic construct. The construct was analyzed considering the physiochemical characterization, safety, antigenicity, post-translational modification, solubility, and intrinsically disordered regions. After modelling its tertiary structure, proteinprotein docking simulation was carried out to ensure the attachment of construct with Toll-Like Receptor 4 (TLR4) as an immune receptor. To guarantee the highest expression of the designed construct in E. coli k12 as an expressional host, the codon optimization and in-silico cloning were performed. The PLAC1 related miRNAs in BC were excavated and validated through TCGA BC miRNA-sequencing and databases; the common pathways then were introduced as other probable mechanisms of PLAC1 activity. Conclusion: Regarding the obtained in-silico results, the designed anti-PLAC1 multi-epitope construct can probably trigger humoral and cellular immune responses and inflammatory cascades, therefore may have the potential of halting BC progression and invasion engaging predicted pathways.

Background: Post-Translational Modifications (PTMs), such as phosphorylation, are an essential regulatory mechanism of protein function and are associated with a range of biological processes beyond the genome and transcriptome. Spiroplasma eriocheiris, a wall-less helical bacterium, is one of the smallest self-replicating bacteria, and is a novel pathogen of freshwater crustaceans. Methods: Protein phosphorylation in S. eriocheiris was systematically investigated by iTRAQ analyzed by LC-MS/MS to study the physiological characteristics and regulatory mechanisms of this bacteria. Data are available via ProteomeXchange with identifier PXD015055. Results: We identified 465 phosphorylation sites in 246 proteins involved in a broad spectrum of fundamental biological processes ranging from the regulation of metabolic pathways to protein synthesis. Notably, most proteins involved in glycolysis and all proteins in the arginine deiminase system were phosphorylated. Cytoskeleton proteins (Fibril, Mrebs and EF-Tu) were also phosphorylated suggesting that phosphorylation may play a crucial role in the formation of the cell skeleton. The analysis identified a number of highly conserved proteins and phosphorylation sites that predominantly participate in glucose metabolism and protein synthesis. Crosstalk analysis with protein-protein interaction networks in relation to phosphorylated proteins and acetylated proteins found that the two PTMs are involved in a number of crucial physiological processes in S. eriocheiris. Comparison of the relative positions of acetylation versus phosphorylation revealed that the two modifications are often located in close proximity to the same protein. Conclusion: The results indicate a previously unreported role of phosphorylation in defining the functional state of Spiroplasma.

Background: Silver nanoparticles pose high antibacterial properties against multi drugresistant and non-resistant bacteria. However, bacteria acquire resistance against chemically synthesized silver nanoparticles after repeated exposure. Therefore, there is an inevitable need to understand the mechanistic behavior of silver nanoparticles. Objective: In this study, we have performed a complete proteomic analysis of Escherichia coli after the treatment with silver nanoparticles to find out the mechanism of bactericidal action of silver nanoparticles (AgNPs). Methods: Silver nanoparticles were synthesized using Artemisia annua leaf extract and incubated with Escherichia coli to elucidate the antibacterial assay by determining MIC and the effect on the growth pattern. Further total genome proteins were isolated from control and silver nanoparticles treated bacteria, which were identified by LC MS and Label free quantification analysis technique. Results: Total identified proteins were 293, out of which 11 proteins were exclusively present in treated bacteria; these are the proteins mainly expressed in stress conditions. Fold change analysis shows that 65 proteins were upregulated where stress proteins are overexpressed while membrane proteins were downregulated. Conclusion: This study reveals that silver nanoparticles inhibit the expression of cellular proteins and cause cell death. Such a study may be helpful in designing drugs against resistant microbes.

Background: Invasion of HIV in human occurs through DC-SIGN’s interaction via the mucosal lining during sexual transmission. Bovine Lactoferrin (bLF) has been known to hinder this invasion via its interaction with DC-SIGN. Hitherto, protein assays have taken place but molecular-level studies remain unexplored. Methodology: The 3D structures of the three proteins were studied. After protein docking (bLF_DCSIGN and gp120_DC-SIGN), the complexes underwent simulation. Stability parameters and binding patterns with residues were explored. Results and Conclusion: ΔG values, net area for solvent accessibilities and conformational fluctuations in DC-SIGN affirm the binding of bLF with DC-SIGN to be more spontaneous and steadier contrary to that with gp120. Residue participation inferred more interactions to occur from bLF complex with a greater percentage of arginine (which strengthens the interaction) while electrostatic interaction between Lys45 (bLF) and Glu26 (DC-SIGN) strengthened the complex. Arg37 played an active role from DC-SIGN to form the stabilizing charged-neutral H-bond, while Lys63 from bLF formed two more such stabilizing charged-neutral H-bond with DC-SIGN. The prime binding sites in DC-SIGN; Arg37 and Gln34 occupy helices. The binding pockets in DC-SIGN may be blocked by bLF spontaneously, to hinder their interaction with gp120. No ionic-ionic interaction was observed from gp120_DCSIGN complex. 88^th residue, which was a predominant residue in the binding pocket was found to experience a conformational shift from coils to sheets after interaction of DC-SIGN with bLF. This would instigate the pharmaceutical research as non-toxic LF would be economic as a remarkable peptide inhibitor opposing HIV.

Background: Breast cancer is the most occurring cancer in women with high incidence rates both in developed and developing countries. Among different types of breast cancers, Triple Negative Breast Cancer (TNBC) is the most aggressive type as it lacks receptors of Estrogen, Progesterone and Human Epidermal Growth Factor Receptor 2, common diagnostic biomarkers for the disease. Since early detection of TNBC can save thousands of lives, there is a dire need to discover and develop effective and affordable methods for early detection. Different Post Translational Modifications (PTMs) have been proposed as potential biomarker for various clinical conditions. Ubiquitination is a type of PTM involved in the stability and regulation of cellular proteins. Objective: It is hypothesized that reticence of ubiquitination may lead to cell death. Current study focuses on the inhibition of Ubiquitin Specific Protease (USP), USP2 using its inhibitor, ML364 in HTB- 132 triple negative breast cancer cell line to induce cell death. The aim of the current study was to evaluate anticancer property of ML364 that might be a promising novel therapeutic agent for TNBC. Furthermore, current investigations focus on USP2 and their focal stabilizing substrates i.e. Fatty acid Synthase (FAS) and Cyclin D1 could be potential prognostic markers for the disease. Methods: Quantitative PCR of CyclinD1, USP2, MDM2, and Fatty Acid Synthase (FAS) was performed to identify the deubiquitination effect of ML364 in breast cancer cells, which complemented our results with studies on normal and breast cancerous tissue samples. Results: Expression of USP2 and its substrates Cyclin D1and FAS was found to be down regulated in ML364 treated breast cancer cell line whereas higher expression was observed in breast cancer tissue, indicating therapeutic potential of USP2 inhibitor. Conclusion: Our findings suggest that USP2, Cyclin D1 and FAS could be used as prognostic marker and therapeutic target for TNBC.

Background: Aspergillus terreus is a major etiological agent among fungal pathogens and emerged as an opportunistic pathogen in immunocompromised patients. Objective: To explore mycelial proteins to shed light on chemical and molecular details that contribute to pathogenesis including intrinsic resistant to amphotericin B (AmB), and biomolecules associated with biofilm formation. Methods: The mycelial proteins of A. terreus cultured for 48h at 37°C have been analysed by using nLC-ESI-MS/MS. Protein data analysis performed by Proteome Discoverer (V/2.2) against the UniProt database. Also, Scanning Electron Microscopy (SEM) analysis of biofilm formation under optimum conditions was carried out. qRT-PCR was carried out for genes encoding for functionally important proteins. Results: A total of 389 proteins were detected at FDR 0.01. Gene ontology showed the abundance of proteins from energy metabolism, cellular homeostasis, ribosome biogenesis, cell wall, and structural components. We observed catalase, superoxide dismutase, thioredoxin, glutathione S-transferase involved in redox homeostasis and heat shock proteins (Hsp90 and Hsp70). These proteins may provide insight into the resistance mechanism of A. terreus against AmB. Additionally, SEM analysis of A. terreus showed the formation of dense hyphal network covered with porous extracellular matrix (ECM). Using SECRETOOL, 8 proteins were predicted as secretory. Three proteins, such as glucanase Crf1/allergen (Asp F9), 1, 3-β-glucanosyltransferase and β-hexosaminidase were reported in biofilm establishment. Conclusion: Our proteome data provided experimental evidence to the annotated set of proteins in A. terreus that comprehend biochemical and cellular events involved in the establishment of mycelial network and contributing to the pathogenesis. This work also provides resource for further molecular studies in A. terreus.