This study employed systems biology and high-throughput technologies to analyze complex molecular components of MS pathophysiology, combining data from multiple omics sources to identify potential biomarkers and propose therapeutic targets and repurposed drugs for MS treatment. This study analyzed GEO microarray datasets and MS proteomics data using geWorkbench, CTD, and COREMINE to identify differentially expressed genes associated with MS disease. Protein-protein interaction networks were constructed using Cytoscape and its plugins, and functional enrichment analysis was performed to identify crucial molecules. A drug-gene interaction network was also created using DGIdb to propose medications. This study identified 592 differentially expressed genes (DEGs) associated with MS disease using GEO, proteomics, and text-mining datasets. 37 DEGs were found to be important by topographical network studies, and 6 were identified as the most significant for MS pathophysiology. Additionally, we proposed six drugs that target these key genes. Crucial molecules identified in this study were dysregulated in MS and likely play a key role in the disease mechanism, warranting further research. Additionally, we proposed repurposing certain FDA-approved drugs for MS treatment. Our in silico results were supported by previous experimental research on some of the target genes and drugs. SIGNIFICANCE: As the long-lasting investigations continue to discover new pathological territories in neurodegeneration, here we apply a systems biology approach to determine multiple sclerosis's molecular and pathophysiological origin and identify multiple sclerosis crucial genes that contribute to candidating new biomarkers and proposing new medications.

Protein lysine succinylation is a recently discovered posttranslational modification. This study examined the role of protein lysine succinylation in the pathogenesis of aortic aneurysm and dissection (AAD). 4D label-free LC-MS/MS analysis was used to perform the global profiles of succinylation in aortas obtained from 5 heart transplant donors, 5 patients with thoracic aortic aneurysm (TAA), and 5 patients with thoracic aortic dissection (TAD). In comparison to normal controls, we detected 1138 succinylated sites from 314 proteins in TAA, and 1499 sites from 381 proteins in TAD. Among these, 120 differentially succinylated sites from 76 proteins overlapped between TAA and TAD (|log2FC| > 0.585, p < 0.05). These differentially modified proteins were mainly localized in the mitochondria and cytoplasm, and were primarily involved in diverse energy metabolic processes, including carbon metabolism, amino acid catabolism, and β-oxidation of fatty acids. By establishing an in vitro model of lysine succinylation in vascular smooth muscle cells, we observed changes in the activities of three key metabolic enzymes (PKM, LDHA, and SDHA). These findings suggest that succinylation potentially contributes to the pathogenesis of aortic diseases, and presents a valuable resource for investigating the functional roles and regulatory mechanisms of succinylation in AAD. SIGNIFICANCE: AAD are interrelated life-threatening diseases associated with high morbidity and mortality. Although we discovered that lysine succinylation was significantly up-regulated in the aorta tissues of patients with AAD, its role in the progression of aortic diseases is largely unknown. We conducted a 4D label-free LC-MS/MS analysis and identified 120 differentially succinylated sites on 76 proteins that overlapped between TAA and TAD as compared to normal controls. Lysine succinylation may contribute to the pathogenesis of AAD by regulating energy metabolism pathways. The proteins containing succinylated sites could be served as potential diagnostic markers and therapeutic targets for aortic diseases.

N-glycosylation is an important post-translational modification necessary to maintain the structural and functional properties of proteins. Impaired N-glycosylation has been observed in several diseases. It is significantly modified by the state of cells and is used as a diagnostic or prognostic indicator for multiple human diseases, including cancer and osteoarthritis (OA). Aim of the study was to explore the N-glycosylation levels of subchondral bone proteins in patients with primary knee OA (KOA) and screen for potential biological markers for the diagnosis and treatment of primary KOA. A comparative analysis of total protein N-glycosylation under the cartilage was performed in medial subchondral bone (MSB, N = 5) and lateral subchondral bone (LSB, N = 5) specimens from female patients with primary KOA. To analyse the N-glycosylation sites of the proteins, non-labelled quantitative proteomic and N-glycoproteomic analyses were performed based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) data. Parallel reaction monitoring (PRM) validation experiments were carried out on differential N-glycosylation sites of proteins in selected specimens, including MSB (N = 5) and LSB (N = 5), from patients with primary KOA. In total, 1149 proteins with 1369 unique N-chain glycopeptides were detected, and 1215 N-glycosylation sites were found, in which ptmRS scores for 1163 N-glycosylation sites were ≥ 0.9. In addition, N-glycosylation of the total protein in MSB compared to that in LSB was identified, in which 295 N-glycosylation sites were significantly different, including 75 upregulated and 220 downregulated N-glycosylation sites in MSB samples. Importantly, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses of proteins with differential N-glycosylation sites showed that they were primarily associated with metabolic pathways including ECM-receptor interactions, focal adhesion, protein digestion and absorption, amoebiasis, and complement and coagulation cascades. Finally, PRM experiments confirmed the N-glycosylation sites of collagen type VI, alpha 3 (COL6A3, VAVVQHAPSESVDN[+3]ASMPPVK), aggrecan core protein (ACAN, FTFQEAAN[+3]EC[+57]R, TVYVHAN[+3]QTGYPDPSSR), laminin subunit gamma-1 (LAMC1, IPAIN[+3]QTITEANEK), matrix-remodelling-associated protein 5 (MXRA5, ITLHEN[+3]R), cDNA, FLJ92775, highly similar to Homo sapiens melanoma cell adhesion molecule (MCAM), mRNA(B2R642, C[+57]VASVPSIPGLN[+3]R), and aminopeptidase fragment (Q59E93, AEFN[+3]ITLIHPK) in the array data of the top 20 N-glycosylation sites. These abnormal N-glycosylation patterns provide reliable insights for the development of diagnostic and therapeutic methods for primary KOA.

The Cancer Proteome Atlas (TCPA) project collects reverse-phase protein arrays (RPPA)-based proteome datasets from nearly 8000 samples across 32 cancer types. This study aims to investigate the pan-cancer proteome signature and identify cancer subtypes of glioma, kidney cancer, and lung cancer based on TCPA data. We first visualized the tumor clustering models using t-distributed stochastic neighbour embedding (t-SNE) and bi-clustering heatmap. Then, three feature selection methods (pyHSICLasso, XGBoost, and Random Forest) were performed to select protein features for classifying cancer subtypes in training dataset, and the LibSVM algorithm was empolyed to test classification accuracy in the validation dataset. Clustering analysis revealed that different kinds of tumors have relatively distinct proteomic profiling based on tissue or origin. We identified 20, 10, and 20 protein features with the highest accuracies in classifying subtypes of glioma, kidney cancer, and lung cancer, respectively. The predictive abilities of the selected proteins were confirmed by receiving operating characteristic (ROC) analysis. Finally, the Bayesian network was utilized to explore the protein biomarkers that have direct causal relationships with cancer subtypes. Overall, we highlight the theoretical and technical applications of machine learning based feature selection approaches in the analysis of high-throughput biological data, particularly for cancer biomarker research. SIGNIFICANCE: Functional proteomics is a powerful approach for characterizing cell signaling pathways and understanding their phenotypic effects on cancer development. The TCPA database provides a platform to explore and analyze TCGA pan-cancer RPPA-based protein expression. With the advent of the RPPA technology, the availability of high-throughput data in TCPA platform has made it possible to use machine learning methods to identify protein biomarkers and further differentiate subtypes of cancer based on proteomic data. In this study, we highlight the role of feature selection and Bayesian network in discovery protein biomarker for classifying cancer subtypes based on functional proteomic data. The application of machine learning methods in the analysis of high-throughput biological data, particularly for cancer biomarker researches, which have potential clinical values in developing individualized treatment strategies.

Genetic variation in phosphorus utilization efficiency (PUE) widely exists among wheat genotypes. However, the underlying mechanisms are still unclear. Two contrasting wheat genotypes, Heng4399 (H4399) and Tanmai98 (TM98), were screened out from 17 bread wheat genotypes based on shoot soluble phosphate (Pi) concentrations. The TM98 had a significantly higher PUE than the H4399, especially under Pi deficiency. The induction of genes in the PHR1-centered Pi signaling pathway was significantly higher in TM98 than in H4399. Collectively, through a label-free quantitative proteomic analysis, 2110 high-confidence proteins were identified in shoots of the two wheat genotypes. Among them, 244 and 133 proteins were differentially accumulated under Pi deficiency in H4399 and TM98, respectively. The abundance of proteins related to nitrogen and phosphorus metabolic processes, small molecule metabolic process, and carboxylic acid metabolic process weas significantly affected by Pi deficiency in the shoots of the two genotypes. The abundance of proteins in energy metabolism, especially photosynthesis, was decreased by Pi deficiency in the shoots of H4399. Inversely, the PUE-efficient genotype TM98 could maintain protein abundance in energy metabolism. Moreover, the proteins involved in pyruvate metabolism, glutathione metabolism, and sulfolipid biosynthesis were significantly accumulated in TM98, which probably contributed to its high PUE. SIGNIFICANCE: Improving the PUE of wheat is urgent and crucial for sustainable agriculture. Genetic variation among wheat genotypes provides materials for exploring the underlying mechanisms for high PUE. This study selected two wheat genotypes with contrasting PUE to reveal the differences in the physiological and proteomic responses to phosphate deficiency. The PUE-efficiency genotype TM98 greatly induced the expression of genes in the PHR1-centered Pi signaling pathway. Subsequently, the TM98 could maintain the abundance of proteins related to energy metabolism and enhance the abundance of proteins involved in pyruvate metabolism, glutathione metabolism, and sulfolipid biosynthesis to increase PUE under Pi deficiency. The differentially expressed genes or proteins between the genotypes with contrasting PUE would provide potential and basis for breeding wheat varieties with improved phosphorus use efficiency.

Controlling Rhipicephalus microplus is among the most significant challenges for livestock production worldwide. The indiscriminate use of acaricides stimulates the selection of resistant tick populations and is therefore ineffective. Understanding the molecular foundations of resistance could help inform the search for new alternatives for tick control. Although the ovary has been suggested as a relevant target organ for tick control, there are few existing studies that focus on tick ovarian tissue. Therefore, we conducted a comparative proteomic analysis on ovaries of R. microplus strains with differential resistance to ivermectin. In resistant ticks, we observed the over-accumulation of proteins involved in several biological processes, including translation, proteolysis, transport, cellular organization, differentiation, and xenobiotic detoxification. We also observed the accumulation of many structural and extracellular proteins such as papilin-like protein, which glycosylation increase its stability-based molecular modeling. Therefore, we propose that ovaries of ivermectin-resistant ticks overcome the negative impact of ivermectin through the activation of detoxification mechanisms and structural proteins associated with the remodeling of the ovary's extracellular matrix. SIGNIFICANCE: Understanding the molecular foundation of ivermectin resistance in Rhipicephalus microplus represents an essential step in cattle farming, which could provide clues and alternatives for tick control. Excessive use of chemicals like ivermectin allows the generation of resistant tick strains in different countries. However, limited molecular information is available concerning the tick's resistance to ivermectin. Detailed proteomics scrutiny in various tick organs will provide more comprehensive molecular information. Thus, we conducted an ovary comparative proteomic-based TMT-SPS-MS3 approach. We highlight in ivermectin-resistant ticks the over-accumulation of structural proteins and enzymes connected to detoxification mechanisms.

Synthetic plastics, like polyethylene terephthalate (PET), have become an essential part of modern life. Many of these products are remarkably persistent in the environment, and the accumulation in the environment is recognised as a major threat. Therefore, an increasing interest has been focusing on the screening for organisms able to degrade and assimilate the plastic. Ideonella sakaiensis originally isolated from a plastisphere has been reported as a bacterium that was solely thriving on the degradation on PET films. The processes affected by the presence of PET and its monomeric substances terephthalic acid, ethylene glycol, ethyl glycolate, and sodium glyoxylate monohydrate were elucidated by analysis of differential protein expression. The exposure of PET and its monomers induced the MHETase and affect two major pathways: the TCA cycle and the β-oxidation pathway. The increased expression of proteins directly or indirectly involved in these pathways suggests their underlying importance in the degradation of PET by I. sakaiensis since these proteins are mechanistically supporting the enzymes involved in the degradation of PET and its monomers.

Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system's inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilized avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions. SIGNIFICANCE: Proximity biotinylation screens using the last generation biotin ligase TurboID represent a powerful approach for the characterisation of transient protein-protein interaction and signaling networks. However, a constant and high basal biotinylation rate and the associated cytotoxicity often preclude the use of this method in proteomic studies. We report a protocol based on modulation of free biotin levels that prevents the deleterious effects of TurboID while allowing inducible biotinylation, even in subcellular compartments such as the endoplasmic reticulum. This optimised protocol greatly expands the applications of TurboID in proteomic screens.

Vibrio parahaemolyticus, a sea-born bacterial pathogen, is a primary inducement of food-borne gastroenteritis. Previous studies have shown that non-coding small RNA plays a vital role in the regulation of multiple biological processes in pathogenic bacteria, especially autoaggregation and growth competition. However, the inherent mechanisms have not yet to be fully understood. As important regulators in Vibrios, the involvement of Qrr sRNAs in V. parahaemolyticus is largely unknown. Here, we carried out the Qrr5 deletion mutant and utilized a proteomic method to describe global proteomic alterations in response to Qrr5 deletion. A total of 297 significantly expressed proteins were determined between the Qrr5 deletion mutant and wild-type strain, among which 137 proteins were upregulated and 160 proteins were downregulated. The upregulated proteins principally participated in membrane transporters and signal transcription, while the downregulated proteins participated in the two-component system and transcription factor binding. Notably, transcriptional regulator LysR, outer membrane protein OmpA, and conjugal transfer protein TraA-related proteins were upregulated, causing the promotion of autoaggregation ability and growth competition ability against E. coli. This study provides insights into the regulatory network of sRNA in this bacterium, which will facilitate further explorations of important biological processes in pathogenic bacteria. SIGNIFICANCE: sRNA Qrr5 is an important regulator involved in bacterial multiple physiological processes, including auto-aggregation and growth competition among food-borne pathogens Vibrio parahaemolyticus. Here, utilizing a TMT-labeling proteomic approach, we identified 137 proteins were upregulated and 160 proteins were downregulated between the Qrr5 deletion mutant and wild-type strain. The upregulated proteins were involved in membrane transporters and signal transcription, while downregulated proteins were involved in the two-component system and transcription factor binding. Moreover, the LysR, OmpA, and TraA proteins were significantly upregulated, causing the promotion of autoaggregation and commensal growth competition ability. The mechanism of how Qrr5 regulates the targeted genes remains unclarified and need great efforts to explore.

The extracellular matrix (ECM) is a three-dimensional set of proteins that binds tissues and defines their biomechanical properties. Among the ECM components known to be involved in beef sensory qualities, authors have often studied fibrillar collagens but also, although less often, proteoglycans and some glycoproteins. The ECM contains many other proteins. Our hypothesis is that to deepen the role of ECM proteins in beef qualities and to identify new ones among the vast amount of data generated by high throughput methods, it is necessary to have a list of proteins of this matrix to refer to for the bovine species. We have therefore defined the Bos taurus matrisome known as the set of genes encoding ECM (core matrisome proteins and matrisome-associated proteins). We have used orthology, as a reference method, and a bioinformatic approach based on a computational pipeline previously published for Homo sapiens, Mus musculus and Danio rerio for the definition of their respective matrisome. We have reported here that the Bos taurus matrisome is composed of 1022 genes that we have classified according to the different matrisome categories. This list is the only matrisome of a livestock species to be defined to date. SIGNIFICANCE: In this study, we provide the first definition of matrisome of a livestock species, the Bos taurus. We believe that the Bos taurus matrisome will be of great interest for several reasons. It comes as a complement to the matrisomes of several other species such as Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster and Caenorabditis elegans previously defined by other authors. It could be used to identify matrisome molecules among the vast amount of data generated by the high throughput methods. It thus can be used in addition to the other matrisomes as a model by the scientific community to study cell behavior and mechanotransduction and could lead to the identification of novel biomarkers for several diseases and cancers in which ECM is involved. Moreover, in the domain of studies on livestock, the dataset that we have provided here can be used in the context of product quality studies, especially meat quality, but also, for example, for lactation studies.

Cyclic dinucleotides (CDNs), such as 2'3'-cGAMP, bind to STING to trigger the production of cytokines and interferons, mainly via activation of TBK1. STING activation by CDN also leads to the release and activation of Nuclear Factor Kappa-light-chain-enhancer of activated B cells (NF-κB) via the phosphorylation of Inhibitor of NF-κB (IκB)-alpha (IκBα) by IκB Kinase (IKK). Beyond the canonical TBK1 or IKK phosphorylations, little is known about how CDNs broadly affect the phosphoproteome and/or other signaling axes. To fill this gap, we performed an unbiased proteome and phosphoproteome analysis of Jurkat T-cell treated with 2'3'-cGAMP or vehicle control to identify proteins and phosphorylation sites that are differentially modulated by 2'3'-cGAMP. We uncovered different classes of kinase signatures associated with cell response to 2'3'-cGAMP. 2'3'-cGAMP upregulated Arginase 2 (Arg2) and the antiviral innate immune response receptor RIG-I as well as proteins involved in ISGylation, E3 ISG15-protein ligase HERC5 and ubiquitin-like protein ISG15, while downregulating ubiquitin-conjugating enzyme UBE2C. Kinases that play a role in DNA double strand break repair, apoptosis, and cell cycle regulation were differentially phosphorylated. Overall, this work demonstrates that 2'3'-cGAMP has a much broader effects on global phosphorylation events than currently appreciated, beyond the canonical TBK1/IKK signaling. SIGNIFICANCE: The host cyclic dinucleotide, 2'3'-cGAMP is known to bind to Stimulator of Interferon Genes (STING) to trigger the production of cytokines and interferons in immune cells via STING-TBK1-IRF3 pathway. Beyond the canonical phosphorelay via the STING-TBK1-IRF3 pathway, little is known about how this second messenger broadly affects the global proteome. Using an unbiased phosphoproteomics, this study identifies several kinases and phosphosites that are modulated by cGAMP. The study expands our knowledge about how cGAMP modulates global proteome and also global phosphorylations.

Aeromonas hydrophila (Ah) is a Gram-negative bacterium and a serious global pathogen causing Motile Aeromonas Septicaemia (MAS) in fish leading to global loss in aquaculture. Investigation of the molecular alterations of host tissues such as liver could be a powerful approach to identify mechanistic and diagnostic immune signatures of disease pathogenesis. We performed a proteomic analysis of Labeo rohita liver tissue to examine the protein dynamics in the host cells during Ah infection. The proteomic data was acquired using two strategies; discovery and targeted proteomics. Label-free quantification was performed between Control and challenged group (AH) to identify the differentially expressed proteins (DEPs). A total of 2525 proteins were identified and 157 were DEPs. DEPs include metabolic enzymes (CS, SUCLG2), antioxidative proteins, cytoskeletal proteins and immune related proteins (TLR3, CLEC4E). Pathways like lysosome pathway, apoptosis, metabolism of xenobiotics by cytochrome P450 were enriched by downregulated proteins. However, upregulated proteins majorly mapped to innate immune system, signaling of B cell receptor, proteosome pathway, ribosome, carbon metabolism and protein processing in ER. Our study would help in exploring the role of Toll-like receptors, C-type lectins and, metabolic intermediates like citrate and succinate in Ah pathogenesis to understand the Ah infection in fish. SIGNIFICANCE: Bacterial diseases such as motile aeromonas septicaemia (MAS) are among the most serious problems in aquaculture industry. Small molecules that target the metabolism of the host have recently emerged as potential treatment possibilities in infectious diseases. However, the ability to develop new therapies is hampered due to lack of knowledge about pathogenesis mechanisms and host-pathogen interactions. We examined alterations in the host proteome during MAS caused by Aeromonas hydrophila (Ah) infection, in Labeo rohita liver tissue to find cellular proteins and processes affected by Ah infection. Upregulated proteins belong to innate immune system, signaling of B cell receptor, proteosome pathway, ribosome, carbon metabolism and protein processing. Our work is an important step towards leveraging host metabolism in targeting the disease by providing a bigger picture on proteome pathology correlation during Ah infection.

Austere environment existing in tank, submarine and vessel has many risk factors including high temperature and humidity, confinement, noise, hypoxia, and high level of carbon dioxide, which may cause depression and cognitive impairment. However, the underlying mechanism is not fully understood yet. We attempt to investigate the effects of austere environment (AE) on emotion and cognitive function in a rodent model. After 21 days of AE stress, the rats exhibit depressive-like behavior and cognitive impairment. Compared with control group, the glucose metabolic level of the hippocampus is significantly decreased using whole-brain positron emission tomography (PET) imaging, and the density of dendritic spines of the hippocampus is remarkably reduced in AE group. Then, we employ a label-free quantitative proteomics strategy to investigate the differentially abundant proteins in rats' hippocampus. It is striking that the differentially abundant proteins annotated by KEGG enrich in oxidative phosphorylation pathway, synaptic vesicle cycle pathway and glutamatergic synapses pathway. The synaptic vesicle transport related proteins (Syntaxin-1A, Synaptogyrin-1 and SV-2) are down-regulated, resulting in the accumulation of intracellular glutamate. Furthermore, the concentration of hydrogen peroxide and malondialdehyde is increased while the activity of superoxide dismutase and complex I and IV of mitochondria is decreased, indicating that oxidative damage to hippocampal synapses is associated with the cognitive decline. The results of this study offer direct evidence, for the first time, that austere environment can substantially cause learning and memory deficits and synaptic dysfunction in a rodent model via behavioral assessments, PET imaging, label-free proteomics, and oxidative stress tests. SIGNIFICANCE: The incidence of depression and cognitive decline in military occupations (for example, tanker and submariner) is significantly higher than that of global population. In the present study, we first established novel model to simulate the coexisting risk factors in the austere environment. The results of this study offer the direct evidences, for the first time, that the austere environment can substantially cause learning and memory deficits by altering plasticity of the synaptic transmission in a rodent model via proteomic strategy, PET imaging, oxidative stress and behavioral assessments. These findings provide valuable information to better understand the mechanisms of cognitive impairment.