Current Proteomics - Volume 18, Issue 2, 2021

The current global pandemic outbreak of a novel type of coronavirus termed by the World Health Organization as COVID-19 became a grave concern and worry to human health and the world economy. Intense research efforts are now underway worldwide to combat and prevent the spread of this deadly disease. This zoonotic virus, a native to bat population, is most likely transmitted to a human via a host reservoir. Due to its close similarity to previously known SARS CoV (Severe Acute Respiratory Syndrome Corona Virus) of 2002 and related MERS CoV (Middle East Respiratory Syndrome Corona Virus) of 2012, it is also known as SARS CoV2. But unlike them, it is far too infectious, virulent and lethal. Among its various proteins, the surface spike glycoprotein “S” has drawn significant attention because of its implication in viral recognition and host-virus fusion process. A detailed comparative analysis of “S” proteins of SARS CoV (now called SARS CoV1), SARS CoV2 (COVID-19) and MERS CoV based on structure, sequence alignment, host cleavage sites, receptor binding domains, potential glycosylation and Cys-disulphide bridge locations has been performed. It revealed some key features and variations that may elucidate the high infection and virulence character of COVID-19. Moreover, this crucial information may become useful in our quest for COVID-19 therapeutics and vaccines.

Background: In our previous study, six cultivars of Mungo (Vigna radiata) were exposed to 100-250 mM NaCl and studied for changes in growth and biomass. Among them, AEM-96 cultivar of the Mung bean [Vigna radiata (L.) Wilczek cv.] was found to tolerate NaCl stress at 250 mM. Objective: The soluble proteome of salt-tolerant mungo cultivar (AEM-96) was compared to the proteome of control mungo to investigate the possible mechanism of salinity tolerance. Methods: Gel-based two-dimensional gel electrophoresis was employed for comparative proteomics. PDQuest-based image analysis of 2D SDS-PAGE was performed. Scatter plots were prepared and total spots were analyzed for 2-fold changes in abundance of protein spot intensities in control and treated gels. Results: In total 517 protein spots were detected; 36 with high significance. Among these 36 spots, 2-fold expression change was analyzed in 27 protein spots. Seven protein spots were upregulated, eight spots were down-regulated, 3 spots were newly induced and 9 spots were silenced, while 9 protein spots did not change their 2-fold abundance under salinity. Protein spots (9 in total) which were 2-fold upregulated and newly induced were excised from the respective gels. The spots were tryptically digested and run on LC-MS/MS for generating peptides and performing a comparative fingerprinting of the proteins. The peptide signal data was loaded on the Mascot (Swissprot) database to retrieve protein IDs. Proteins with the best score were selected, namely isomers of oxygen- evolving enhancer protein 1 (S1-S3), RuBisCO (S4), oxygen-evolving enhancer protein 2 (S5), Heat shock protein 70 isomers (S6-S7), RuBisCO activase (S8), rubber elongation factor (S9) and pathogen-related protein 10 (S10). Conclusion: The identified proteins were found to play important roles in photosynthesis, stress response and plant growth.

Background: Leptospirosis is a prevalent zoonotic disease caused by Leptospira interrogans bacterium. Despite the importance of this disease, traditional strategies including attenuated and inactivated vaccines have not been able to prevent leptospirosis. Objective: Hence, this study was designed to develop a novel poly-epitope fusion protein vaccine against Leptospirosis. Methods: To do so, the best epitopes of OmpA, LipL45, OmpL1, LipL41 and LipL21 proteins were predicted. Then, the best-predicted epitopes were applied to assemble IFN-γ, MHC I binding, B cell and MHC II binding fragments, and heparin-binding hemagglutinin adhesion was used as a molecular adjuvant. After designing the vaccine, the most important features of it, including physicochemical parameters, protein structures and protein-protein interaction, were evaluated. Finally, the nucleotide sequence of the designed vaccine was used for codon adaptation. Results: The results showed that the designed vaccine was a stable protein with antigenicity of 0.913, which could dock to its receptor. The results also suggested that the nucleotide sequence of the designed vaccine could be expressed in the prokaryotic system. Conclusion: Based on the results of this study, it can be concluded that the vaccine can be a promising candidate to control Leptospirosis.

Background: Excluding humans, the peroxisomal uricase is responsible for the catabolism of uric acid into allantoin in many species like microorganisms, plants, and invertebrates. Particularly in humans, the synthesis and excretion of uric acid are naturally balanced. When the uric acid concentration crosses 7 mg/dl, it results in conditions such as hyperuricemia and gout. Uricase is one of the potential sources for the reduction of uric acid in humans. Uricase is also widely used as a commercial diagnostic reagent in medical and clinical biochemistry to estimate the uric acid concentration in blood and other biological fluids. Computational approaches can be used for screening and investigation of uricase enzyme with desirable characteristics that can be employed in diverse industrial applications. Objectives: The present study deals with computational-based structural, functional, and phylogenetic analyses of uricase enzymes from various Bacillus species. Methods: Seventy uricase protein sequences from Bacillus species were selected for multiple sequence alignment, phylogenetic analysis, motif assessment, domain architecture examination, understanding of basic physicochemical properties and in silico identification of the composition of amino acids in uricase. Further, structural (secondary and tertiary structure prediction), and functional (CYS_REC, MOTIF scan, CD-search, STRING, SOSUI, and PeptideCutter) analyses of uricase were performed. Results: Bacillus simplex (WP_063232385.1) was chosen as the representative species of the Bacillus genera. The three-dimensional (3D) structure of B. simplex uricase was predicted and validated using QMEAN, RAMPAGE, ERRAT, Verify 3D and PROQ servers. The analysis revealed that the tertiary structure of the selected uricase has good quality and acceptability. Conclusion: Computational analysis of uricase from various Bacillus sources revealed that all the selected Bacillus uricases are active within acidic to a neutral environment, and thermally stable with a molecular weight ranging from 35.59-59.85kDa. The secondary structure analysis showed that all uricases are rich in alpha-helices and sheets. The CDD tool identified two conserved domains, one of which belongs to OHCU decarboxylase and another belongs to Uricase superfamily. The quality estimation of 3D modeled protein gave a high overall quality factor score of 94.64. Also, all Bacillus species of uricase enzyme and their corresponding genes showed a strong correlation from the phylogenetic comparison of the selected taxa. The present detailed computational investigation on the uricase protein could help in screening a suitable uricase producing microbe with desirable characteristics for industrial application.

Background: Diabetic Retinopathy (DR), one of the major microvascular complications commonly occurring in diabetic patients, can be classified into Proliferative Diabetic Retinopathy (PDR) and Non-Proliferative Diabetic Retinopathy (NPDR). Currently, available therapies are only targeted for later stages of the disease in which some pathologic changes may be irreversible. Thus, there is a need to develop new treatment options for earlier stages of DR through revealing pathological mechanisms of PDR and NPDR. Objective: The purpose of this study was to characterize the proteomes of diabetes through quantitative analysis of PDR and NPDR. Methods: Vitreous body was collected from three groups: control (non-diabetes mellitus), NPDR, and PDR. Vitreous proteins were digested to peptide mixtures and analyzed using LC-MS/MS. MaxQuant was used to search against the database and statistical analyses were performed using Perseus. Gene ontology analysis, related-disease identification, and protein-protein interaction were performed using the differential expressed proteins. Results: Twenty proteins were identified as critical in PDR and NPDR. The NPDR group showed different expressions of kininogen-1, serotransferrin, ribonuclease pancreatic, osteopontin, keratin type II cytoskeletal 2 epidermal, and transthyretin. Also, prothrombin, signal transducer and activator of transcription 4, hemoglobin subunit alpha, beta, and delta were particularly up-regulated proteins for PDR group. The up-regulated proteins related to complement and coagulate cascades. Statherin was down-regulated in PDR and NPDR compared with the control group. Transthyretin was the unique protein that increased its abundance in NPDR compared with the PDR and control group. Conclusion: This study confirmed the different expressions of some proteins in PDR and NPDR. Additionally, we revealed uniquely expressed proteins of PDR and NPDR, which would be differential biomarkers: prothrombin, alpha-2-HS-glycoprotein, hemoglobin subunit alpha, beta, and transthyretin.

Background: The rate of Human Immunodeficiency Virus type 1 (HIV-1) infection in Iran has increased dramatically in the last few years. Objective: The aim of this study was to investigate the HIV subtype amongst all Iranian HIV sequences, using 8 websites. Methods: In this study, 637 sequences of polymerase, and gag genes of HIV-1 were obtained from NCBI. HIV-1 subtyping was done, using 8 reliable software. Results: The final results of the 8 online tools indicated that the majority of sequences were HIV-1 subtype CRF35 AD. However, it appeared that in some genes, a few programs could not determine specific subtypes and in some cases they described different subtypes. Conclusion: Considering the CRF35 AD diagram, it was clear that integrase was not an appropriate region to define this subtype. Also the full length of gag gene should be used for subtyping. For CRF1, AE envelop gene is a reliable region to define this subtype. Stanford software was used to determine the drug resistance prevalence and in 5.7% of the sequences, drug resistance mutations were found.

Aims: The importance of cathelicidin-1 as an indicator of the severity of mammary infection in ewes. Background: Mastitis is an important disease of sheep, affecting their health and welfare. Objective: The association of the presence of cathelicidin-1 in milk samples from ewes with mastitis with the severity of the infection. Methods: Ewes were intramammarily inoculated with Mannheimia haemolytica or Staphylococcus chromogenes. Conventional (clinical, bacteriological and cytological examinations; milk yield measurements) and proteomics evaluation (2-DE, MALDI-TOF MS) to record cathelicidin-1 spot optical densities in milk samples were recorded. Results: Ewes challenged with M. haemolytica developed clinical and ewes challenged with S. chromogenes subclinical mastitis (P=0.05). The challenged organism was isolated from milk samples from inoculated mammary glands; increased somatic cell counts were also recorded. Cathelicidin- 1 was detected in milk samples from the inoculated side of udders of all ewes. Mean spot density of cathelicidin-1 from samples from inoculated glands of ewes challenged with M. haemolytica was higher than from ewes challenged with S. chromogenes: 2896 ± 973 versus 1312 ± 361 (P =0.034). There were significant correlations between the presence of clinical mastitis / somatic cell counts with the spot density of cathelicidin-1 on 2-DE gels (P=0.043 and P=0.023, respectively). There was also a significant inverse correlation between the mean spot densities of cathelicidin-1 in milk samples and the milk yield of respective ewes on D10 (P =0.031). Conclusion: Potentially, cathelicidin-1 could be used as a marker to indicate the severity of damage to the mammary parenchyma.

Background: Lipid homeostasis and gut flora can be related to many metabolic diseases, especially autism. Lipid metabolism in the brain can control neuronal structure and function and can also take part in signal transduction pathways to control metabolism in peripheral tissues, especially in the liver. Impaired phospholipid metabolism promotes oxidative stress and neuroinflammation and is, therefore, directly related to autism. Objective: The effect of propionic acid (PPA) toxicity on lipid homeostasis in the gut-liver-brain axis was evaluated to understand their inter-connection. Cytosolic phospholipase A2 (cPLA2) concentration and activity was measured in autistic model and protective role of omega-3 (ω-3) and vitamin B12 was evaluated. Methods: Animals were divided into five groups: Group I (control group); Group II (autistic model treated with neurotoxic dose of PPA); Group III (treated with vitamin B12 (16.7 mg/kg/day) for 30 days post PPA treatment); Group IV (treated with ω-3 (200 mg/kg body weight/day) for 30 days post PPA treatment; Group V (combined dose of ω-3 and Vitamin B12, for 30 days post PPA treatment). Phospholipase A2 activity and protein expression level in the liver homogenate of all the groups was analyzed by western blotting and was compared to brain cPLA2. Results: PPA increased the levels of liver and brain cPLA2. However, independent or combined treatment with ω-3 and vitamin B12 was effective in neutralizing its effect. Moreover, PPA-induced dysbiosis, which was ameliorated with the above treatments. Conclusion : This study showed the role of cPLA2 as a lipid metabolism marker, related to PPA-induced inflammation through a highly interactive gut-liver-brain axis.

Background: Human Papillomavirus (HPV) is responsible for substantial morbidity and mortality worldwide. We predicted immunogenic promiscuous monovalent and polyvalent T-cell epitopes from the polyprotein of the Human Papillomavirus (HPV) using a range of bioinformatics tools and servers. Methods: We used immunoinformatics and reverse vaccinology-based approaches to design prophylactic peptides by antigenicity analysis, T-cell epitopes prediction, proteasomal and conservancy evaluation, host-pathogen protein interactions, and in silico binding affinity analysis. Results: We found two early proteins (E2 and E6) and two late proteins (L1 and L2) of HPV as potential vaccine candidates. Of these proteins (E2, E6, L1 & L2), 2-epitopes of each candidate protein for multiple alleles of MHC class I and II were found, bearing significant binding affinity (>-6.0 kcal/mole). These potential epitopes for CD4+ and CD8+ T-cells were also linked to design polyvalent construct using GPGPG linkers. Cholera toxin B and mycobacterial heparin-binding hemagglutinin adjuvant with a molecular weight of 12.5 and 18.5 kDa were used for epitopes of CD4+ and CD8+ T-cells, respectively. The molecular docking indicated the optimum binding affinity of HPV peptides with MHC molecules. This interaction showed that our predicted vaccine candidates are suitable to trigger the host immune system to prevent HPV infections. Conclusion: The predicted conserved T-cell epitopes would contribute to the imminent design of HPV vaccine candidates, which will be able to induce a broad range of immune-responses in a heterogeneous HLA population.

Background: Oxaliplatin(Oxa) is a major chemotherapy drug for colorectal cancer. However, drug resistance is a major cause of treatment failure for late-stage colorectal cancer. Therefore, it is necessary to explore the mechanism of resistance to oxaliplatin in HCT116 colorectal cancer cells. Objective: Therefore, this study explored the mechanisms of HCT116 cells resistance to oxaliplatin by combining the results of proteomic and phosphoproteomic analyses. Methods: In this study, firstly,we constructed oxaliplatin-resistant HCT116 cells called HCT116/ Oxa. Then, we conducted a quantitative study of phosphoproteomics in HCT116 and HCT116/ Oxa cells via TMT labeling, bio-material-based PTM enrichment, HPLC fractionation, and LC-MS/MS analyses. At the same time, we applied TMT/iTRAQ labeling, HPLC fractionation, and LC-MS/MS to conduct proteomic and phosphoproteomic analyses of the cell lines. Finally, we analyzed the results from Gene Ontology (GO), protein domain, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the 1.5 change rate as a meaningful change threshold. Results: Our analysis confirmed the previously described mechanisms of colon cancer resistance and revealed the important role of phosphorylation in drug resistance. Conclusion: Collectively, this study provides a new direction for the study of oxaliplatin resistance in colorectal cancer.

Background: Cancers result due to the dysregulation of gene expression. They can be identified on the basis of driver mutations and genetic signatures. Proteins are macromolecules that regulate the structure and function of body organs. Missense somatic mutations play a critical role in the development of cancer by altering the underlying properties of corresponding proteins. The extent to which the chemical properties and composition of amino acid are changed in cancer is still under investigation. Objective: The main objective of this study is to identify amino acid changes that might be responsible for causing liver cancer. It also aims to identify frequently mutated genes associated with liver cancer. Methods: The mutation data of Hepatocellular Carcinoma (HCC) in coding variants was retrieved from COSMIC (Catalogue of Somatic Mutations in Cancer) databases. Different bioinformatics tools were used to study genetic alterations at the protein level. The identified amino acid replacements were compared with Grantham’s distance to determine similarity/ dissimilarity between substituted amino acids. Results: The results show that TP53, CTNNB1, MUC16, PCLO, and TTN genes were frequently mutated in liver cancer. This study also reveals that the non-synonymous mutations, in analyzed dataset, cause loss of Alanine. Conclusion: The amino acid replacements, identified in this study, may act as signatures for early diagnosis of liver cancer. They may also be helpful in understanding the development of liver cancer.

Background: Although the food quality of soy protein is known to be as good as that of animal proteins, some soybean proteins are not susceptible to digestion and remain undigested in the intestine. We hypothesized that digestion-resistant soy proteins might interact with the intestinal membrane, microbes, and metabolites, and change the intestinal physiology or the profile of the gut microbiome. Objective: To identify the Protease-Resistant Soy Proteins (PRSPs) and their interaction with intestinal membrane proteins by MS, and to assess the functions of PRSPs in the small intestine. Methods: Soy proteins were sequentially digested with pepsin and pancreatin, and the PRSPs were identified by SDS-PAGE and MS. Intestinal cell membrane proteins interacting with PRSPs were isolated by affinity purification and photo-affinity crosslinking, and identified using MS/MS. Inhibition of cholesterol absorption to lipoprotein-depleted intestinal cells, CaCo-2, and hepatic cells, HepG2, was measured in the presence and absence of PRSPs. FITC-conjugated Gram-positive, Lactobacillus plantarum, and Gram-negative bacteria, Escherichia coli, were incubated with Ca- Co-2 cells in the presence of PRSPs to investigate the regulation of bacterial cell binding to intestinal epithelial cells by PRSPs. Results: MS/MS of PRSPs identified glycinin, β-conglycinin, trypsin inhibitors, lipoxygenase, and sucrose-binding protein. MS analysis also identified the intestinal membrane proteins bound to PRSPs. The functions of the identified interacting proteins included ion transportation, carbohydrate- binding, cytoskeleton formation, hydrolysis, cell-cell junction formation, and cholesterol/steroid- binding. In particular, apolipoprotein E, aminopeptidase N, and Niemann-Pick C1-like protein 1 are known to be involved in cholesterol absorption in the small intestine. The inhibition of cholesterol absorption by CaCo-2 and HepG2 cells by PRSPs confirmed the MS results. Binding of L. plantarum and E. coli to CaCo-2 cells was efficiently inhibited by PRSPs. Conclusion: PRSPs can interact with intestinal membrane proteins, and regulate cholesterol absorption by intestinal epithelial cell and interactions of the gut microbiome. Soy protein in the intestine acts as a nutrient, and triggers changes in intestinal functions by interacting with intestinal cells, microorganisms, and nutrients. These findings will provide valuable new functional information about the effects of soy proteins on human health.

Background: To investigate the interactions between RNA and proteins is essential to understand how these macromolecule complexes exert their functions. RNA pull-down is a classic technique to enrich RNA binding proteins, however, a large number of non-specific binding proteins may be enriched during sample preparation, interfering with the downstream mass spectrometric analyses and also causing false positives. Objective: In this study, we examined the background contaminates in RNA pull-down experiment using mass spectrometric analysis. Methods: Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses. Results: 191 protein groups were identified as non-specific binding proteins in RNA pull-down samples. In addition, a comparison between different sample preparation conditions showed that the level of background contaminates was mostly induced by the solid phase support rather than the studied RNA. In addition, using more stringent detergent and streptavidin magnetic beads with smaller size could reduce the amount of background interfering proteins. Conclusion: This study provides a reference to distinguish bona fide RNA interacting proteins from the background contaminants. The results also demonstrate that different sample preparation conditions have great impacts on the level of enriched background contaminates, shedding new light on the optimization of RNA pull-down experiments.

Background: Symptomatic intracranial atherosclerotic Disease (sICAD) is associated with the risk of recurrence of cerebral ischemic events in 4-19% of stroke patients annually. Previous studies indicate elevated high-sensitivity C-Reactive Protein (hs-CRP) and lipoprotein-associated phospholipase A2 (Lp-PLA2) to be associated with risk of recurrence. Objective: This prospective, observational study investigated serum levels of hs-CRP and the activity of Lp-PLA2 in patients with sICAD in predicting the risk of long-term stroke recurrence. Methods: We enrolled 48 patients with sICAD at 3 months from onset. The demographics, clinical, and imaging characteristics were recorded. Serum hs-CRP and Lp-PLA2 activity were assessed using automated high-sensitivity C-reactive protein assay and photometric technique, respectively. Patients were followed up at 6 months and 1 year and the presence of new vascular events was recorded. Results: The mean age of our study population was 59.5 ± 10.3 years and 91.7% were men. Four patients developed recurrent strokes during follow-up. The mean Hs-CRP was elevated in patients with events than in patients without events (5.9 ± 10.4 mg/L vs. 1.7 ± 2.4 mg/L, P=0.03). However, there was no significant association of mean Lp-PLA2 activity (118.3±42.9 nmol/min/ml vs. 111.9 ± 34.2 nmol/min/ml, P=0.73) with recurrence. Elevation of hs-CRP (3.02 ± 4.8 mg/L vs. 0.95 ± 0.57mg/L, P=0.02) and Lp-PLA2 activity (120.1±40.3 nmol/min/ml vs. 103.3 ± 23.9 nmol/min/ml, P=0.04) was correlated with high-grade stenosis in these patients. Conclusion: Our study suggests serum levels of hs-CRP may serve as a predictor of long-term stroke recurrence risk in sICAD and elevation of hs-CRP and Lp-PLA2 correlated with the severity of stenosis in symptomatic intracranial atherosclerotic disease.

Background: Helicobacter pylori infection and its treatment still remain a challenge for human health worldwide. A variety of antibiotics and combination therapies are currently used to treat H. pylori induced ulcers and carcinoma; however, no effective treatment is available to eliminate the pathogen from the body. Additionally, antibiotic resistance is also one of the main reasons for prolonged and persistent infection. Aims: Until new drugs are available for this infection, vaccinology seems the only alternative opportunity to exploit against H. pylori induced diseases. Methods: Multiple epitopes prioritized in our previous study have been tested for their possible antigenic combinations, resulting in 169-mer and 183-mer peptide vaccines containing the amino acid sequences of 3 and 4 epitopes respectively, along with adjuvant (Cholera Toxin Subunit B adjuvant at 5’ end) and linkers (GPGPG and EAAAK). Results: Poly-epitope proteins proposed as potential vaccine candidates against H. pylori include SabA-HP0289-Omp16-VacA (SHOV), VacA-Omp16-HP0289-FecA (VOHF), VacA-Omp16-HP0289- SabA (VOHS), VacA-Omp16-HP0289-BabA (VOHB), VacA-Omp16-HP0289-SabA-FecA (VOHSF), VacA-Omp16-HP0289-SabA-BabA (VOHSB) and VacA-Omp16-HP0289-BabA-SabA (VOHBS). Structures of these poly-epitope peptide vaccines have been modeled and checked for their affinity with HLA alleles and receptors. These proposed poly-epitope vaccine candidates bind efficiently with A2, A3, B7 and DR1 superfamilies of HLA alleles. They can also form stable and significant interactions with Toll-like receptor 2 and Toll-like receptor 4. Conclusion: Results suggest that these multi-epitopic vaccines can elicit a significant immune response against H. pylori and can be tested further for efficient vaccine development.

Aims: Exploring molecular mechanism of abiotic stress tolerance in plants is needed to overcome the deterioration of yield and quality of crop plants to meet the food security challenges of the growing population. Background: MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate target gene expression for modulating plant growth, development, and response to different stresses. Agave belonging to CAM plants’ has remarkable tolerance to extreme conditions of drought and heat; however, molecular mechanisms underlying this excellence are yet to explore. Objective: This study applies comparative genomics approach on available Transcriptome (RNA- Seq) data of Agave deserti to identify potential miRNAs, and miRNA targets. Methods: Transcriptome datasets consisting of 128,869 Agave contigs was processed to create local database, for nucleotide homology analysis with 6,028 non-redundant plant miRNAs as query sequences. Protein coding sequences were removed, and potential pre-miRNA sequences were tested for stability analysis based on a variety of factors, including but not limited to %G+C content and minimum free energy (-ΔG), as a filter to remove pseudo pre-miRNAs. Results: This study identified 30 unique miRNAs of Agave deserti harboring 14 different categories of precursors. Phylogenetic analysis revealed evolutionary relationship between newly identified pre-miRNAs with corresponding pre-miRNA homologues. Target genes of miRNAs were predicted subsequently, and possible functions were defined by functional annotation analysis. Conclusion: The results of this study will pave the way for further research, exploring the molecular mechanisms in Agave deserti and the role of miRNAs in gene regulation under abiotic stresses.