Current Proteomics - Volume 18, Issue 3, 2021

A proteome is an efficient rendition of a genome, unswervingly controlling various cancer processes. Molecular mechanisms of several cancer processes have been unraveled by the proteomic approach. Thus far, numerous tumors of diverse status have been investigated by two-dimensional electrophoresis. Numerous biomarkers have been recognized and precise categorization of apparent lesions has led to the timely detection of various cancers in persons at peril. Currently used pioneering approaches and technologies in proteomics have led to highly sensitive assays of cancer biomarkers and improved the early diagnosis of various cancers. The discovery of novel and definite biomarker signatures further widened our perceptive of the disease, and novel potent drugs and aimed therapeutic outcomes in persistent cancers have emerged. However, a major limitation, even today, of proteomics is resolving and quantifying the proteins of low abundance. Despite the rapid development of proteomics technologies and their applications in cancer management, annulling the shortcomings of present proteomic technologies and the development of better methods are still desirable. The main objectives of this review are to discuss the developing aspects, merits and demerits of pharmacoproteomics, redox proteomics, novel approaches and therapies being used for various types of cancer-based on proteome studies.

Background: The current study was undertaken with a major goal of evaluating the influence of the heavy metal Copper (Cu) on growth and physiology as well as the proteomic status of Ziziphus spina-christi. Objective: We set a hypothesis that Z. spina-christi could be a potent phytoremediator in Cu-polluted environments and the industrial area of Riyadh was chosen as a model of contaminated sites. Methods: To address our goal, young Z. spina-christi plants were maintained in pots filled with soil comprised of sand and clay in an open area. Cu treatments were performed by subjecting young Z. spina-christi seedlings to different copper treatments [0 - 100 μM] which was supplied to the plants in the form of hydrated copper sulfate (CuSO4.5H2O) and the treatment period lasted 45 days. To monitor plant growth during Cu exposure period, measurements of some morphological and physiological variables were taken regularly every week and the growth rates were determined. Results: Results from proteomics showed 16 proteins participated in expression, 10 proteins showed up regulations and other six showed down regulation. Such expressed proteins are involved in plant photosynthesis process, metabolism, antioxidant enzymes as well as those associated with plant defense response and signal transduction. Conclusion: The study outcomes offered understandings of the molecular systems linked to Cu stress condition, in addition to Cu effect on Z. spina-christi seedlings morphology and physiology. Regarding phytoremediation potential we recommend that, future experiments should be conducted on Z. spina-christi at different developmental stages to better view its Cu accumulative power.

Background: Salinity is a major abiotic stress that limits plant growth and development. Salinity affects several physiological and biochemical characteristics adversely, which results in oxidative stress in plant species. Plants change the protein pattern to cope with salinity stress. The identified salt-responsive proteins in alfalfa are involved in energy and metabolism, photosynthesis, regulation of carbohydrates, transcription/translation, signal transduction, stress/redox homeostasis, ion binding, and stress and defense with ROS scavenging and detoxification. Objective: The present research aimed to study the response of two contrasting alfalfa varieties to salt stress, and to identify the altered leaf proteins by two-dimensional gel electrophoresis and MALDI-TOF/TOF/MS spectrometry. Methods: Salinity stress significantly decreased shoot fresh and dry weights in both Synthetic II (salt-tolerant) and Khajeh (salt-sensitive) varieties, and K+/Na+ ratio in Khajeh, while it significantly increased K+/Na+ ratio, soluble sugars, chlorophyll a and catalase activity in Synthetic II, and peroxidase activity in Khajeh. Results: In the proteome analysis, a total of 188 spots, including 104 spots in the Synthetic II variety (salt-tolerant) and 84 spots in the Khajeh variety (salt-sensitive), were identified under salinity stress. Among the identified protein spots, 15 common spots had a significant change in expression between normal and salt stress conditions in leaves of the two alfalfa varieties. These proteins were grouped into nine functional categories: proteins involved in photosynthesis electron transfer (13.33%), energy metabolism (20%), transcription/translation (20%), stress and defense (13.33%), ROS scavenging and phytohormone signaling (6.67%), protein degradation (6.67%), ubiquitination (6.67%), biological and physiological processes (6.67%), and unknown (6.67%). Conclusion: Salinity stress reduced the yield of alfalfa, but the reduction was more pronounced in the sensitive variety of Khajeh. The salinity-tolerant variety, Synthetic II, responded better to salinity stress in terms of K+/Na+ ratio, soluble sugars, chlorophyll a and catalase activity as compared to Khajeh. Proteome analysis showed that the proteins involved in energy metabolism, transcription/- translation, photosynthesis, electron transfer, and defense were more important than other functional categories under salinity stress. These proteins mainly increased in the salt-tolerant variety, whereas they decreased in the salt-sensitive variety.

Background: Cancer is a genetic disorder in which several factors like oncoviruses are involved. Among viruses, Human T-lymphotropic virus 1 (HTLV-1) is a human oncovirus whose long-term infection leads to Adult T-Cell Leukemia/Lymphoma (ATLL). The lack of a vaccine against HTLV-1 and limited efficacy of available treatments for ATLL due to the weakness of the immune system led us to develop novel therapeutic\prophylactic epitope vaccine, which is able to potentiate the immune system against HTLV-1. Methods: In this research, the amino acid sequences of TAX, HBZ, gp62 and NY-ESO-1 were retrieved from the UniProt database. Afterwards, the bioinformatics analyses were performed to select the Cytotoxic T Lymphocytes (CTL) and Helper T Lymphocyte (HTL) epitopes using IEDB, RANKPEP, CTLpred and PA Complex servers. The selected epitopes, along with RS09 protein adjuvant, were connected to each other via proper amino acid linkers. RS09 adjuvant was used as a TLR4 agonist to assure the induction of immune response. Then, the three-dimensional model of the protein vaccine was generated via Phyer2 software. In the next step, the optimization of the final structure of the protein vaccine was performed using GalaxyRefine, Galaxyloop and KOBAMIN servers. Results: Evaluation of 3D protein vaccine with ERRAT, PROSA-web and Ramachandran plots servers showed that the vaccine possesses a high-quality structure; moreover, the vaccine was antigen and non-allergen. Conclusion: We believe that the designed vaccine candidate can stimulate cellular and humoral immunity effectively; however, the potency of the vaccine should confirm via in vitro and in vivo immunological assays.

Aim: Molecular cloning and analysis of Pectin Iyase (PNL) genes from different strains of Fusarium for structural predictions and docking studies. Background: PNLs cleave pectin by β-elimination resulting in the formation of 4,5-unsaturated oligogalacturonates, without affecting the ester content of the polymer chain and hence maintaining the specific aroma of fruits. Several PNL lyase genes from Aspergillus and Penicillium have been cloned, but the molecular biology of that from Fusarium has not been explored. Objective: To obtain an insight into the three-dimensional structure of PNL of Fusarium. Methods: PCR amplification-based molecular cloning of PNL genes from Fusarium strains, sequencing, and sequence analysis using bioinformatics tools for homology search, multiple sequence alignment, motif search, physiochemical characterization, phylogenetic tree construction, 3D structure prediction, and molecular docking were conducted. Results: Five PNL genes were cloned from F. oxysporum MTCC1755, F. monoliforme var. subglutinans MTCC2015, F. avenaceum MTCC10572, and F. solani MTCC3004 using the PCR approach. Many conserved amino acids were found at several positions in all the PNL proteins. Phylogenetic analysis of these proteins with other pectinases revealed two major clusters representing members of lyases and hydrolases. In-silico characterization revealed stable PNL proteins. PNL proteins from different Fusarium strains were similar in structural features and biochemical properties owing to their similar primary sequence. Docking studies revealed that electrostatic forces and van der Waal and hydrogen bonds effectuate the interaction between the ligand and the enzyme. Aspartate, tyrosine, and tryptophan residues in the active site of the enzyme are responsible for ligand binding. Conclusion: PNL from different Fusarium species show similarity at structural as well as biochemical level. PNL protein from F. moniliforme and F. solani was similar in properties except for the variation of single amino acid. Docking studies on the enzyme and different ligands provided an insight into the interacting residues and forces as well as the suitability of the substrate for catalysis.

Background: CCl4 Acute Liver Injury (ALI) is a classical model for experimental research. However, there are few reports involved in the fundamental research of CCl4-induced ALI. Ligustri Lucidi Fructus (LLF) and its prescription have been used to treat hepatitis illness clinically. LLF and its active ingredients displayed anti-hepatitis effects, but the mechanism of . function has not yet been fully clarified. Objective: To investigate the proteomic analysis of CCl4-induced ALI, and examine the effects of active Total Glycosides (TG) from LLF on ALI of mice4, including histopathological survey and proteomic changes of liver tissues, and delineate the possible underlying mechanism. Methods: CCl4 was used to produce the ALI mice model. The model mice were intragastrically administrated with TG and the liver histopathological changes of mice were examined. At the end of the test, mice liver samples were collected, and after protein denaturation, reduction, desalination and enzymatic hydrolysis, identification was carried out by nano LC-ESI-OrbiTrap MS/MS technology. The data was processed by Maxquant software. The differentially-expressed proteins were screened and identified, and their biological information was also analyzed based on GO and KEGG analysis. Key protein expression was validated by Western blot analysis. Results: A total of 705 differentially-expressed proteins were identified in the normal, model and administration groups. 9 significant differential proteins were focused based on the analysis. Liver protein expression changes of CCl4-induced ALI mice were mainly involved in several important signaling channels, namely FoxO signaling pathway, autophagy-animal, insulin signaling pathway. TG has an anti-liver damnification effect in ALI mice, the mechanism of which is related to FoxO1 and autophagy pathways. Conclusion: CCl4 inhibited expression of insulin-Like growth factor 1 (Igf1) and 3-phosphoinositide- dependent protein kinase 1 (Pdpk1) in liver cells and induced insulin resistance, thus interfering with mitochondrial autophagy and regeneration of liver cells and the metabolism of glucose and lipid, and causing hepatic necrosis in mice. TG resisted liver injury in mice. TG adjusted the expression level of key proteins Igf1 and Pdpk1 after liver injury and improved insulin resistance, thus promoting autophagy and resistance to liver damage.

Background: Amyloids are a class of ordered protein aggregates which have been implicated in the onset of several degenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Type II diabetes, and so on. Despite extensive research, the exact mechanism and the driving factors for the amyloidogenesis process remain elusive. Identifying molecules which can effectively inhibit and/or disaggregate the fibrils may be one effective therapeutic strategy against amyloidosis. Objectives: In the current study, few hydroxy-benzoic phytochemicals were selected to study their effects on the formation as well as disaggregation of Hen Egg-White Lysozyme (HEWL) amyloids, namely gallic acid, syringic acid, vanillic acid, and iso-vanillic acid. Methods: Amyloidogenesis was monitored using methods like the thioflavin T assay, Field Emission Scanning Electron Microscopy (FESEM), and dynamic light scattering (DLS) studies. Further protein conformational changes were monitored using methods like 8-Anilino-Naphthalene-1-Sulfonate (ANS) fluorescence, Circular Dichroism (CD) spectroscopy, and guanidine hydrochloride mediated stability studies. Computational approach was also employed to get an insight on the interaction( s) between the selected compounds and HEWL using docking studies. Results: The selected compounds exhibited significant inhibitory as well as disaggregation effects on HEWL amyloids. Interaction with the phytochemicals was also associated with considerable conformational changes in HEWL. Docking studies show the role of hydrogen bonding between HEWL and the phytochemicals. Conclusion: Thus, the current study throws light on the key factors that drive amyloid formation and hence will be helpful in the development of effective therapeutics against amyloidosis.

Background: “Angiogenesis”, a major oncogenic signaling pathway, has been termed to be the most fascinating area of cancer therapy. Viewing this, the molecular chaperone Hsp90 has surfaced as a potential molecular target due to being vitally engrossed in sustaining stability, integrity, and functions of crucial proteins involved in multiple signaling pathways of tumor progression and metastasis. Objective: The study set sights on virtual screening (molecular docking and PreADMET study), MD simulation, and metabolism study of compounds from Crucifereae family along with the intensive structure-activity relationship studies in search of potent lead targeting HSP90. Methods: All the chemical structures were drawn using ChemDraw and converted into suitable 3D-structures. The target protein, HSP90 was retrieved from RCSB PDB. All the compounds of Crucifereae family and analogs were subjected to Lipinski’s rule of five and ADMET prediction using Molinspiration and PreADMET software respectively. The screened compounds were further exposed to MD simulation and metabolism studies. Conclusion: The docking results showed the promising inhibitory potential of Ana51 against Hsp90 with the binding energy of -11.32 kcal/mol as compared to its parent compound ‘spirobrassinin’ and a known inhibitor ‘Ganetespib’ exhibiting binding energy of -7.57 kcal/mol and -9.83 kcal/mol respectively. Optimization, flexibility prediction, and ascertaining the stability of Hsp90 in complex with the ligands were done by means of Molecular Dynamics (MD) simulations for 50 ns. The Hsp90-Ana51 complex exhibited stability with an RMSD value of 0.15 nm and Rg value to be 1.62 nm. The investigation further extends towards the SOM analysis of Ana51 to forecast the probable toxic and non-toxic in vivo metabolites via in silico tools (SMARTCyp, Xenosite Web, and PASS online server). Results: Ana 51 came out to be metabolically stable withstanding phase I metabolism and producing non-toxic metabolites compared to the parent compound and the standard drug. Obtained results propose Ana51 as a novel anti-Hsp90 lead compound with exceptional antiangiogenic capability.

Background: Haptoglobin (Hp), an acute-phase protein, is known as a potential diagnostic biomarker in human diseases. Two alleles of Hp (Hp¹ and Hp² ) exist in humans allowing three phenotypes (Hp1-1, Hp2-1, and Hp2-2), which influence the biophysical and biological properties of Hp. Objective: This work aimed to investigate the variation of serum level and fucosylation change among Hp phenotypes in patients with lung cancer compared to healthy donors. Methods: 44 patients with lung cancer and 26 healthy blood donors who lived in the Northern-Thailand region were investigated by the glycoproteomic procedure. Results: The phenotypic distribution of the Hp (Hp1-1:Hp2-1:Hp2-2) in healthy donors was 0.04:0.38:0.58, while the patient group was 0.09:0.52:0.39. The Hp¹ allele frequency of patients with lung cancer (0.34) was higher than the healthy donor (0.23). Glycoprotein blotting technique represented that the level of serum Hp and its fucosylation were significantly higher among lung cancer patients compared to those of the healthy donors. However, a downward trend in the fucosylation level from Hp1-1 to Hp2-1, Hp2-2, was seen in the patient group, but varying in the serum Hp level. An N-linked glycan was enzymatically released from discrete Hp multimers of Hp2-1 and Hp2-2 samples. Analysis of glycan profiling by MALDI-TOF-MS showed that reduction of the fucosylated glycan was associated with the size of Hp multimers, resulting in the lower level of fucosylation in Hp2-1 and Hp2-2, respectively. Conclusion: Our finding demonstrates that the Hp phenotype is a dependent risk factor for lung cancer and should be incorporated into further clinical and biochemical investigations of diseases, including lung cancer.

Background: Escherichia coli host has been the workhorse for the production of heterologous proteins due to simplicity of use, low cost, availability of various expression vectors, and a plethora of knowledge on its genetic characteristics, but without a suitable signal sequence, this host cannot be used for the production of secretory proteins. Humulin is a form of insulin used to treat hyperglycemia caused by types 1 and 2 diabetes. To improve expression and make a straightforward production of Humulin protein, we chose a series of signal peptides. Objective: The aim of this study was to predict the most excellent signal peptides to express secretory Humulin in E. coli organism. Methods: Therefore, to forecast the most excellent signal peptides for expression of Humulin in Escherichia coli, 47 signal sequences from bacteria organisms were elected and the most imperative elements of them were studied. Hence, signal peptide probability along with physicochemical features was evaluated by signal 4.1, and Portparam, PROSO II servers respectively. Later, the in- -silico cloning in a known pET28a plasmid system also estimated the possibility of best signal peptide+ Humulin expression in E. coli. Results: The outcomes demonstrated that among 47 signal peptides only 2 signal peptides can be considered as suitable signal peptides. Conclusion: Ultimately protein yebF precursor (YEBF_ECOLI) and protein yebF precursor (YEBF_YERP3) were suggested as the most excellent signal peptides to express Humulin (With D scores 0.812 and 0.623, respectively). Although verification of these results warrants experimental analysis.

Aims: This study aimed to perform a quantitative analysis of the proteomic changes in neural cells under H2O2 exposure and N-acetylcysteine (NAC) treatment. Background: NAC is a potent antioxidant and an effective free radical scavenger that has been used as a potential treatment of several neurological disorders. Although the molecular mechanisms of neuroprotective action by NAC have been studied, more efforts are still needed for the elucidation of the mechanisms through a quantitative proteomic analysis. Objective: This study aimed to identify differentially expressed proteins among control, H2O2-treated group, and NAC+H2O2-treated group as well as reveal proteins involved in the protection of neural cells from H2O2-induced toxicity. Methods: SK-N-MC cells were untreated (control), treated with H2O2 (disease group), pretreated with NAC, and then treated with H2O2 (NAC group). Proteins were digested to peptides and analyzed using liquid chromatography-tandem mass spectrometry with the data-independent acquisition. Skyline was used to quantify peptides and proteins. MSstats was used for statistical analysis. Gene ontology and protein-protein interactions were performed using the Differentially Expressed Proteins (DEPs). Results: Cytoprotective effects of NAC on the cell against H2O2-induced toxicity were first proven using a cell viability study and lactate dehydrogenase assay. The proteomic analysis found 93 DEPs in three comparisons. Among them, 37 proteins were differentially expressed under H2O2 exposure. Only 10 DEPs were rescued in the case of NAC pretreatment. Aspartate aminotransferase and L-lactate dehydrogenase B chain were two DEPs involved in the cysteine and methionine metabolism pathway, which might relate to the mechanisms of NAC protective effects. Conclusion: The findings of cell studies and proteomic analysis were in agreement with previous results, confirming the cytoprotective effects of NAC on neural cells against oxidative stress.

Background: The human serotonin transporter is an important drug target for the treatment of various medical conditions of which depression is the most important, but also include attention deficit hyperactivity disorder, schizophrenia, social anxiety disorder and irritable bowel syndrome, among others. The transmembrane portion of the human transporter has been studied extensively and was first crystalized in 2016. However, the dynamical nature of the N-terminal segment of protein and its post-translational modifications remain insufficiently explored. Objective: The present study aims to evaluate the structure and dynamics of the N-terminal segment of the human serotonin transporter and the presence and stability of possible secondary structure elements along with its post-translational modifications and disorder propensity. Methods: The segment was investigated using a combination of bioinformatics tools for physicochemical characterization, secondary structure prediction, post-translational modifications and disorder prediction, followed by ab initio modeling and microsecond long explicit solvent molecular dynamics. Results: Our study reveals the presence of metastable secondary structure elements, namely two alpha helices and a beta-sheet, throughout the molecular dynamics run and identifies numerous sites with high probability for post-translational modifications. Conclusion: Our results show that, despite the intrinsically unstructured nature, the N-terminus adopts a stable conformation with stable secondary structure elements, that could indicate an important functional role for the segment. Also, there is a high probability that the segment undergoes multiple post-translational modifications.

Aim: The purpose of this study was to conduct in silico analysis of the Lampyroidea maculata luciferase enzyme. Background: Bioluminescence is the production and emission of light by the luciferase enzymes in a living organism. The luciferase enzyme has been widely used in biotechnology due to its excellent properties. Objective: Recently, the new gene of the luciferase from the Lampyroidea maculata, has been cloned and characterized. Methods: In the following, in silico analysis of this enzyme were conducted by structural modeling in the I-TASSER web server. Finally, the binding site properties were studied using the AutoDock Vina. In the following, the codon usage bias parameters as the CAI, CBI, ENC, and the base composition of this sequence were studied. After analysis of the base composition, it was found that the coding DNA sequence is rich in AT. Moreover, the indices GC1, GC2, and GC3 were computed to establish the relationship among three codon positions. On the other hand, the GC2 has a much lower frequency. Results: By molecular modeling, some rare codons were identified that may have a critical role in the structure and function of this luciferase. The GC3% of the CDs was 17/304 and GC3 Skewness was 0.115. The GC content at the first codon position (GC1) and second codon position (GC2) was compared with that of the third codon position (GC3). AutoDock Vina was used in the molecular docking that recognizes some residues that yield closely related to the ADSL binding site. This bioinformatics analyzes play an important role in the design of new drugs. Conclusion: Previous studies show that mutation pressure and natural selection reasons for codon usage variation among different genes. If the mutational pressure was the only effective reason for the codon usage bias, then the frequency of nucleotides C and G should be equal to A and T at the synonymous third codon position. By these analyses, a new understanding of the sequence and structure of this enzyme was created, and our findings can be used in some fields of clinical and industrial biotechnology.