Current Proteomics - Volume 18, Issue 4, 2021

Most clinical related proteomic studies were aimed to screen potential biomarkers for clinical usage. However, it is known that only a few candidates can be validated in later stages. Besides clinical biomarkers, it is also necessary to identify key proteins to better understand the molecular mechanism of human diseases. Here, we updated the connotations of precision and translational proteomics and proposed a refined research framework for clinical proteomics. We abstracted the framework as a three-tier system: to determine research objectives and approaches for achieving a certain purpose, to choose the appropriate methods for optimal results, and to apply a standardized experimental procedure for reliable and reproducible results. Started from the initial discovery stage, we divided the following proteomics-driven translational studies into two directions: biomarker screening and mechanism investigation. We also discussed possible causes that may lead to high rates of experimental variation and low rates of follow-up validation. Moreover, we pointed out that the main bottlenecks for the identification of effective targets are data interpretation and statistical inference. Finally, we emphasized that the transparency of experimental design and results can help to ensure data quality. Conclusively, the refined framework provides constructive recommendations and comprehensive guides for designing and performing clinical related proteomic studies with the aim of obtaining reliable and reproducible results. It can also help to further promote the standardization and integration of proteomic studies.

The world's highest and largest altitude area is called the Qinghai-Tibetan plateau QTP, which harbors unique animal and plant species. Mammals that inhabit the higher altitude regions have adapted well to the hypoxic conditions. One of the main stressor at high altitudes is hypoxia. Metabolic responses to hypoxia play important roles in cell survival strategies and some diseases. However, the homeostatic alterations that equilibrate variations in the demand and supply of energy to maintain organismal function in a prolonged low O2 environment persist partly in understood, making it problematic to differentiate adaptive from maladaptive responses in hypoxia. Tibetans and yaks are two perfect examples innate to the plateau for high altitude adaptation. By the scan of the whole-genome, EPAS1 and EGLN1 were identified as key genes associated with sustained hemoglobin concentration in high altitude mammals for adaptation. The yak is a much more ancient mammal that has existed on QTP longer than humans. It is, therefore, possible that natural selection represented a diverse group of genes/pathways in yaks. Physiological characteristics are extremely informative in revealing molecular networks associated with inherited adaptation, in addition to the whole-genome adaptive changes at the DNA sequence level. Gene-expression can be changed by a variety of signals originating from the environment, and hypoxia is the main factor amongst them. The hypoxia-inducible factors (HIF-1α and EPAS1/HIF-2α) are the main regulators of oxygen in homeostasis, which play a role as maestro regulators of adaptation in the hypoxic reaction of molecular mechanisms. The basis of this review is to present recent information regarding the molecular mechanism involved in hypoxia that regulates candidate genes and proteins. Many transcriptional responses toward hypoxia are facilitated by HIFs that change the number of gene expressions and help in angiogenesis, erythropoiesis, metabolic reprogramming, and metastasis. HIFs also activate several signals highlighting a strong association between hypoxia, the misfolded proteins’ accumulation in the endoplasmic reticulum in stress, and activation of Unfolded Protein Response (UPR). It was observed that at high-altitudes, pregnancies yield a low birth weight 100 g per1000 m of the climb. It may involve variation in the events of energy-demand, like protein synthesis. Prolonged hypobaric hypoxia causes placental ER stress, which, in turn, moderates protein synthesis and reduces proliferation. Further, Cardiac hypertrophy by cytosolic Ca²⁺ raises and Ca²⁺/calmodulin, calcineurin stimulation, NF-AT3 pathway might be caused by an imbalance in Sarcoplasmic reticulum ER Ca²⁺, which might be adaptive in the beginning but severe later on.

Aim: The aim of this systematic review is to identify all the available data on human lens proteomics with a critical role in age-related cataract formation in order to elucidate the physiopathology of the aging lens. Methods: We searched on Medline and Cochrane databases. The search generated 328 manuscripts. We included nine original proteomic studies that investigated human cataractous lenses. Results: Deamidation was the major age-related post-translational modification. There was a significant increase in the amount of αA-crystallin D-isoAsp58 present at all ages, while an increase in the extent of Trp oxidation was apparent in cataract lenses when compared to aged normal lense. During aging, enzymes with oxidized cysteine at critical sites included GAPDH, glutathione synthase, aldehyde dehydrogenase, sorbitol dehydrogenase, and PARK7. Conclusion: D-isoAsp in αA crystallin could be associated with the development of age-related cataract in humans by contributing to the denaturation of a crystallin and decreasing its ability to act as a chaperone. Oxidation of Trp may be associated with nuclear cataract formation in humans, while the role of oxidant stress in age-related cataract formation is dominant.

Objective: Osteoporosis (OP) is mainly characterized by low Bone Mineral Density (BMD) and microarchitectural deterioration of bone tissue. We performed label-free quantitative proteomics to discover novel proteins involved in the pathogenesis of osteoporosis. Methods: We employed an extreme sampling study design to collect subjects with low BMD (Z-score<- 1.30±0.47) and high BMD (Z-score>1.06±0.49). Liquid Chromatography and Mass Spectrometry (LC-MS) technologies were used to identify Peripheral Blood Monocyte (PBM)-expressed proteins significant for OP in Chinese elderly women (Study Sample 1) and men (Study Sample 2), respectively. Results: A total of 131 Differentially Expressed Proteins (DEPs) and 200 DEPs were identified in subjects with low vs. high BMD from the study samples 1 and 2, respectively. Interestingly, three DEPs (WNK1, SHTN1, and DPM1) were significantly and consistently regulated with BMD in both genders. GO analysis showed that these DEPs were significantly enriched in “extracellular exosome”, “protein binding” and “cell-cell adherens junction” (p < 0.05). Pathway enrichment results showed that these DEPs were significantly enriched in “protein ubiquitination”, “ER-Phagosome pathway” and “antigen processing” (p < 0.05). Protein-Protein Interaction (PPI) networks were constructed, pointing out key node proteins, including HSPA8, PKM, AKT1, and ABI1. Mining data from independent -omics studies highlighted that 174 DEPs, as identified from above, were significant for OP in Caucasians as well, including WNK1 and DPM1. Conclusion: The study identified known and novel proteins significant for OP in both genders and/or across ethnicities in both Chinese and Caucasians. Our findings may provide clues for further research on the underlying pathogenic mechanism of OP.

Aim: There are many people with type 2 Diabetes Mellitus (T2DM) worldwide. Rouxen- Y Gastric Bypass (RYGB) is an effective surgery for treating T2DM with beneficial effects on β cell metabolism. However, the mechanism of how RYGB affects the pancreas, is not clear. We focused on metabolic changes in the pancreas of rats following RYGBand to investigate complex postoperative pancreatic metabolic reprogramming and to understand how RYGB improves islet function. Methods: We performed RYGBonstreptozotocin-induced male T2DM rats. Then we measured indicators such as weight, fasting GLU, etc. After 10 weeks, pancreatic tissues were removed to identify and quantify differentially expressed proteins. Furthermore, functional analysis of these proteins and their associated pathways was conducted by bioinformatics methods. Results: After surgery, 451 differentially expressed proteins associated with the mechanism ofRYGB were identified. Protein levels of regenerating islet-derived protein 3 (Reg3A), cell division cycle-associated protein 2 (CDCA2), and ADP-ribosylation factor 1 (Arf1) varied greatly, and Arf1 may be a point target in diabetes. Conclusion: This research shows that RYGB could treat T2DM through changes in the pancreatic proteins. It will give some new insight into the molecular mechanism behind the effect of RYGB.

Background: Inflammation plays a major role in dry eye diseases and is the prime target in clinical treatments of the disease. Objective: To determine whether the inflammatory cytokines in the tear film of patients with dry eye correlate with parameters such as Tear Break-Up Time (TBUT) and Tear Meniscus Height (TMH) and could be used together as predictive biomarkers of disease severity. Methods: This study included 58 eyes (29 female subjects; age, 19–25 years) comprising 20 normal eyes (control), 20 eyes (level 1 dryness), and 18 eyes (level 2 dryness). Dryness level 1 or 2 is keratographic diagnosis according to the tear break-up time cut-off value obtained. After ophthalmic examinations, including determination of non-invasive TBUT average and first second and TMH using Keratograph 4 (to measure dryness level), tears of all participants were collected. Five cytokines, including interleukin 6, 10, 12, interferon-γ, and tumor necrosis factor-α, were measured using ELISA. Results: Significant changes were observed in all measured cytokines and these changes correlated with eye dryness severity scores. Both groups (levels 1 & 2) showed remarkable changes related to ocular surface alterations in clinical examinations. Significantly, shorter TBUT and TMH were recorded in both groups and were positively correlated with the dryness severity. Conclusion: Cytokines could be used as predictive markers for pre-clinical diagnosis of dry eye diseases and as prognostic markers to monitor treatment effectiveness.

Background: In utero exposure to Selective Serotonin Reuptake Inhibitors (SSRI) is considered a risk factor for many neurodevelopmental diseases. Objective: The aim of this study was to investigate whether prenatal SSRI exposure changes newborn brain chemistry. Methods: An animal-based study was designed in which the utero SSRI exposed rat pups were compared to one without drug exposure. Neurochemical changes in the infants were assessed after 2 days of birth by estimating the levels of inflammatory cytokines, neurotransmitters, and caspases in the brain exposed to SSRI at the prenatal stage and compared to normal unexposed newborns. Results: Our results showed significant neurochemical changes in SSRI-exposed newborns. A significant decrease in dopamine, and serotonin levels with a remarkable decrease in noradrenaline in addition to remarkable increase of IFN-γ and caspase-3 levels was observed in the brain tissues of prenatal exposed SSRIs rat pups. Conclusion: The results suggest that prenatal SSRI treatment may affect brain development of newborn hence should be done warily during the gestation period.

Background: Selective Cationic Amphipathic Antibacterial Peptides (SCAAPs) occupy a prominent place in the production of new drugs on account of their high toxicity towards bacteria and low toxicity towards mammalian cells, low hemolytic activity, and contribution to the protection of the human immune system. Aim: their number in nature is very low, and experimental tests are very protracted and costly. Therefore, it would be useful to have bioinformatics tools that would identify them in the existing databases and also propose new synthetic SCAAPs. Methods: In order to reduce the costs of identification and/or chemical synthesis and to know the physicochemical characteristics of SCAAPs at a residues level and to obtain a “bioiformatics fingerprint” suitable for their selection, we have modified the Polarity Index Method® (PIM®) and the α-helical configuration of each sequence is included in determining their individual “PIM® profile”. We have also used a set of the computer program to determine their “Intrinsic Disorder Predisposition”. This information was then compared with other protein groups, such as bacteria, fungi, virus and Cell-Penetrating Peptides (CPP) from the UniProt database and a set of intrinsically disordered proteins. Once the “fingerprint” of SCAAPs was obtained, it was used for searching among the 559228 “reviewed” proteins from the UniProt database and a set of synthetic SCAAPs characterized by the predefined “PIM® profile” selected. Results: Our results showed that the metric named “PIM® profile” can identify, with a high level of accuracy, a group of bacterial SCAAPs. This bioinformatics study was supported at residues level, using the in-house bioinformatics system Polarity Index Method, the commonly used algorithm for predicting intrinsic disorder predisposition, PONDR® FIT. Conclusions: The Polarity Index Method seems highly efficient in identifying SCAAP candidates.

Background: The ability of Staphylococcus aureus to cause severe infections and the difficulty of the treatments due to the multiple antibiotic resistance make this bacterium a lifethreatening human pathogen. This situation necessitates the exploration of novel antimicrobial compounds with known targets on bacteria. Phenolic acids naturally produced in plants as secondary metabolites are good candidates for being alternative antimicrobials for antibiotic-resistant bacteria. Objective: Investigation of protein profile of Methicillin-Resistant S. Aureus (MRSA) in the presence of subinhibitory concentrations of phenolic acids. Methods: MRSA was subjected to subinhibitory concentrations of Vanillic Acid (VA) and 2-Hydroxycinnamic Acid (2-HCA), separately, and the proteomic analyses were carried out by using liquid chromatography coupled to mass spectrometry. Results: Both phenolic acids elicited identification of differently expressed proteins that have roles in DNA replication, repair, RNA processing and transcription, protein synthesis, maintenance of cell homeostasis, several metabolic reactions in energy, carbohydrate and lipid metabolisms and also proteins related with the virulence and the pathogenicity of MRSA when compared with the control group. The numbers of the proteins identified were 444, 375, and 426 for control, VA-treated MRSA, and 2-HCA-treated MRSA, respectively, from which 256 were shared. While VA treatment resulted in 149 unidentified MRSA proteins produced in control, 2-HCA treatment resulted in 126 unidentified proteins. Data are available via ProteomeXchange with identifier PXD016922. Conclusion: The results obtained from this study might indicate the potential targets on bacteria and the effective use of phenolic acids in the battle with antibiotic-resistant pathogens.

Introduction: Controversy exists regarding the impact of Phosphorylation Homology Domain (PePHD) of Hepatitis C Virus (HCV) E2 protein on the interruption of the antiviral signaling pathway. A mechanism by which the virus evades the antiviral effect of interferon (IFN) alpha involves protein kinase (PKR) eukaryotic Initiation Factor 2 alpha (eIF2a) PePHD. By binding to PKR, PePHD inhibits its activity and, therefore, causes the virus to evade the antiviral activity of IFN. This study aimed to clarify the inconsistency of different conclusions reached in previous studies using reliable bioinformatics tools. Methods: Fifty-eight Iranian patients infected with HCV genotypes 1a and 3a and 58 healthy control individuals were examined. Plasma viral RNA was used to amplify and sequence the HCV E2 gene; also, HCV viral load, genotyping, IL-28B genotyping, alanine Aminotransferase (ALT), and aspartate aminotransferase (AST) test were determined. Bioinformatics tools determined the physicochemical properties, B-cell epitopes, post-modification changes, and secondary/tertiary structures, and also evaluated the interactions between E2 and PePHD regions. Results: The results showed a new domain that is responsible for binding to PKR protein and is important to the intrigued antiviral response. Physicochemical features and post-modifications were defined, showing that E2 is highly phosphorylated and there were numerous possible disulfide bonds. Secondary and tertiary structures for E2 protein were constructed. No significant relationship between ALT and AST and treatment failure was detected. Conclusion: Docking analysis showed a new domain in E2 protein that can be involved in the interaction between PKR and E2 protein. This finding may justify our results revealing the non-significant relationship between mutations in PePHD and treatment failure.

Background: Human Papillomavirus (HPV) is the main biological agent causing Sexually Transmitted Diseases (STDs), including precancerous lesions and several types of prevalent cancers. To date, numerous types of vaccines are designed to prevent high-risk HPV. However, their prophylactic effect is not the same and does not clear previous infections. Therefore, there is an urgent need for developing therapeutic vaccines that trigger cell-mediated immune responses for the treatment of HPV. The HPV16 E6 and E7 proteins are ideal targets for vaccine therapy against HPV. Fusion protein vaccines, which include both immunogenic interest protein and an adjuvant for augmenting the immunogenicity effects, are theoretically capable of guaranteeing the power of the immune system against HPV. Methods: A vaccine construct, including HPV16 E6/E7 proteins along with a heat shock protein GP96 (E6/E7-NTGP96 construct), was designed using in silico methods. By the aid of the SWISS-- MODEL server, the optimal 3D model of the designed vaccine was selected, and then the physicochemical and molecular parameters were performed using bioinformatics tools. Docking studies were done to evaluate the binding interaction of the vaccine. Allergenicity, immunogenicity, B, and T cell epitopes of the designed construct were predicted. Results: Immunological and structural computational results illustrated that our designed construct is potentially proper for stimulation of cellular and humoral immune responses against HPV. Conclusion: Computational studies showed that the E6/E7-NTGP96 construct is a promising candidate vaccine that needs further in vitro and in vivo evaluations.

Background: Proteomics facilitates understanding the complexity of molecular and physiological mechanisms involved in the metabolic and biological fungal adaptations to pH changes. Proteomics enables the identification of enzymes and fungal proteins involved in these adaptations. This approach may be used to investigate such fungi as Aspergillus niveus, whose proteome has not yet been analyzed, changes the intra- and extracellular protein profiles in response to extracellular pH. Objective: In the current study, we used two-dimensional gel electrophoresis (2DE) and mass spectrometry to evaluate the response of A. niveus to grow at pH 5, 6, 7, and 8 for 96 hours submerged bioprocess culturing. Methods: This study evaluated the response of A. niveus to grow at pH 5, 6, 7, and 8 for 96 h submerged bioprocess culturing, by analysis of two-dimensional gel electrophoresis (2DE), of the intracellular proteomes and the secretome, protein spots of interest were submitted to tryptic digestion and analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS). Results: This approach revealed substantial differences between the functions of intra- and extracellular proteins of A. niveus. The data suggested that pH-modulated global proteins are involved in important, mainly metabolic, processes, in the pentose phosphate pathway, protein regulation, cell wall maintenance, and others. Moreover, the change in extracellular pH could have altered the availability of nutrients, and induced the production of enzymes that respond to oxidative and other stresses. Conclusion: Proteomic facilitates understanding of the complexity of molecular and physiological mechanisms involved in the metabolic and biological adaptations of fungi to pH changes.

Background: Dysregulated angiogenesis resulting in neovascularization is a critical event in the expansion and progression of Chronic Myeloid Leukemia (CML), hematopoietic cancer. Vascular Endothelial Growth Factor- A (VEGFA), an important angiogenesis mediator, has been a target for treating cancer. Although several anti-VEGFA drugs are available, they are associated with adverse side effects, promoting the need to identify better drugs that may be less toxic. Objective: Our aim was to investigate whether Tyrosine Kinase Inhibitors (TKIs) could be repurposed for use as VEGFA inhibitors via in silico docking software. We also investigated the potential of phytochemicals as VEGFA inhibitors. Methods: We performed molecular docking using Schrödinger Maestro software suite 2014-3 to determine the most potent phytochemical and TKI VEGFA antagonists. Results: Among the TKIs investigated, Bosutinib had the best binding affinity and may be the most potent TKI against VEGFA. The order of binding affinities for the top ten docked ligands was: Ginsenoside Rg3> Bosutinib> Vitamin D> Paclitaxel> Dasatinib> Saponins> Ponatinib> Squalamine> Imatinib> Nilotinib. We found that Ginsenoside Rg3 had the highest binding affinity (MMGBSA score= -99.4 kcal/mol, glide Gscore = -9.16 kcal/mol) to VEGFA. Conclusion: Our study has shown for the first time the binding poses of these TKIs and phytochemicals to VEGFA, using computational methods. We propose that the use of the top scoring ligands, in isolation or a combination of phytochemicals plus TKI, could serve as potent angiogenesis inhibitors via their binding to and inhibiting VEGFA expression to prevent CML progression. We have also profiled the ligand binding residues, which may be explored in designing pharmacophores.

Background: Mycobacterium avium sp. paratuberculosis (MAP) causes Paratuberculosis (pTB) in domestic livestock and has also been associated with auto-immune disorders in humans. Infection leads to huge economic losses to the farmers associated with livestock production system worldwide. Currently, search to find proteins with the potential to develop as vaccine candidates against MAP is underway. Objective: In this study, we aimed to explore the immunogenicity of the proteins of mammalian cell entry (mce) operons of MAP using computational tools. Methods: Genes of mce operons of MAP strain K10 were selected and their orthologs identification was made using VFanalyzer tool. Mce proteins encoded by these operons were analyzed for their antigenicity and sub-cellular localization. Three dimensional structures for Mce proteins were predicted using Phyre2. B cell and T cell epitope analysis was done using methods available at Immune Epitope Database and Analysis Resource. The selection analysis of mce genes was also performed. Results: Eight Mce proteins were predicted with B cell and T cell epitopes. Some of them were reported with overlapping B cell and T cell epitopes. We found positively selected sites within some predicted epitopes that indicated some kind of selection pressure by immune system on these protein regions. Some predicted epitopes also had similarities with experimentally identified epitopes of Mce proteins of M. tuberculosis which further strengthened the immunogenic role of Mce proteins. Conclusion: Our findings may potentially assist in the development of effective vaccine against incurable infection due to MAP bacilli in the domestic livestock species.