Protein Serine/Threonine Phosphatases: Keys to Unlocking Regulators and Substrates.

Protein serine/threonine phosphatases (PPPs) are ancient enzymes, with distinct types conserved across eukaryotic evolution. PPPs are segregated into types primarily on the basis of the unique interactions of PPP catalytic subunits with regulatory proteins. The resulting holoenzymes dock substrates distal to the active site to enhance specificity. This review focuses on the subunit and substrate interactions for PPP that depend on short linear motifs. Insights about these motifs from structures of holoenzymes open new opportunities for computational biology approaches to elucidate PPP networks. There is an expanding knowledge base of posttranslational modifications of PPP catalytic and regulatory subunits, as well as of their substrates, including phosphorylation, acetylation, and ubiquitination. Cross talk between these posttranslational modifications creates PPP-based signaling. Knowledge of PPP complexes, signaling clusters, as well as how PPPs communicate with each other in response to cellular signals should unlock the doors to PPP networks and signaling "clouds" that orchestrate and coordinate different aspects of cell physiology.

VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

Ti4+ functionalized ß-cyclodextrin covalent organic framework as a new immobilized metal ion affinity chromatography platform for selective capture of phosphorylated peptides and exosomes.

A Ti4+ functionalized ß-cyclodextrin covalent organic framework nanoparticle (named as ß-CD-COF@Ti4+) was synthesized using a one-pot method successfully realizing the enrichment of phosphorylated peptides and exosomes based on the immobilized metal ion affinity chromatography strategy. The functionalized ß-CD-COF@Ti4+ exhibited superior performance on the enrichment of phosphopeptides, including high selectivity (1:1000), low detection limit (0.5 fmol), and loading capacity for phosphopeptides (100 mg·g-1). After treatment with ß-CD-COF@Ti4+, 9 phosphopeptides from defatted milk, 29 phosphopeptides related to 23 phosphoproteins from normal group serum, and 24 phosphopeptides related to 22 phosphoproteins from the serum of uremia patients were captured. Through the analysis of Gene Ontology, the captured phosphoprotein is closely related to kidney disease, including lipoprotein metabolism, very-low-density lipoprotein particle, high-density lipoprotein particle, and lipid binding activity process. Furthermore, western blot verification showed that this nanoparticle could successfully capture exosomes from human serum. This study demonstrates great prospects for the enrichment of phosphopeptides and exosomes from actual bio-samples.

Phospho-DIGE Identified Phosphoproteins Involved in Pathways Related to Tumour Growth in Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth.

Protein Phosphorylation Alterations in Myotonic Dystrophy Type 1: A Systematic Review.

Among the most common muscular dystrophies in adults is Myotonic Dystrophy type 1 (DM1), an autosomal dominant disorder characterized by myotonia, muscle wasting and weakness, and multisystemic dysfunctions. This disorder is caused by an abnormal expansion of the CTG triplet at the DMPK gene that, when transcribed to expanded mRNA, can lead to RNA toxic gain of function, alternative splicing impairments, and dysfunction of different signaling pathways, many regulated by protein phosphorylation. In order to deeply characterize the protein phosphorylation alterations in DM1, a systematic review was conducted through PubMed and Web of Science databases. From a total of 962 articles screened, 41 were included for qualitative analysis, where we retrieved information about total and phosphorylated levels of protein kinases, protein phosphatases, and phosphoproteins in DM1 human samples and animal and cell models. Twenty-nine kinases, 3 phosphatases, and 17 phosphoproteins were reported altered in DM1. Signaling pathways that regulate cell functions such as glucose metabolism, cell cycle, myogenesis, and apoptosis were impaired, as seen by significant alterations to pathways such as AKT/mTOR, MEK/ERK, PKC/CUGBP1, AMPK, and others in DM1 samples. This explains the complexity of DM1 and its different manifestations and symptoms, such as increased insulin resistance and cancer risk. Further studies can be done to complement and explore in detail specific pathways and how their regulation is altered in DM1, to find what key phosphorylation alterations are responsible for these manifestations, and ultimately to find therapeutic targets for future treatments.

Phosphoproteomic Approaches to Discover Novel Substrates of Mycobacterial Ser/Thr Protein Kinases.

Mycobacterial Ser/Thr protein kinases (STPKs) play a critical role in signal transduction pathways that ultimately determine mycobacterial growth and metabolic adaptation. Identification of key physiological substrates of these protein kinases is, therefore, crucial to better understand how Ser/Thr phosphorylation contributes to mycobacterial environmental adaptation, including response to stress, cell division, and host-pathogen interactions. Various substrate detection methods have been employed with limited success, with direct targets of STPKs remaining elusive. Recently developed mass spectrometry (MS)-based phosphoproteomic approaches have expanded the list of potential STPK substrate identifications, yet further investigation is required to define the most functionally significant phosphosites and their physiological importance. Prior to the application of MS workflows, for instance, GarA was the only known and validated physiological substrate for protein kinase G (PknG) from pathogenic mycobacteria. A subsequent list of at least 28 candidate PknG substrates has since been reported with the use of MS-based analyses. Herein, we integrate and critically review MS-generated datasets available on novel STPK substrates and report new functional and subcellular localization enrichment analyses on novel candidate protein kinase A (PknA), protein kinase B (PknB) and PknG substrates to deduce the possible physiological roles of these kinases. In addition, we assess substrate specificity patterns across different mycobacterial STPKs by analyzing reported sets of phosphopeptides, in order to determine whether novel motifs or consensus regions exist for mycobacterial Ser/Thr phosphorylation sites. This review focuses on MS-based techniques employed for STPK substrate identification in mycobacteria, while highlighting the advantages and challenges of the various applications.

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping.

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

Regulatory role of phosphoproteins in the development of bovine small intestine during early life.

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.

Phosphorylated Proteins from Serum: A Promising Potential Diagnostic Biomarker of Cancer.

Cancer is a fatal disease worldwide. Each year ten million people are diagnosed around the world, and more than half of patients eventually die from it in many countries. A majority of cancer remains asymptomatic in the earlier stages, with specific symptoms appearing in the advanced stages when the chances of adequate treatment are low. Cancer screening is generally executed by different imaging techniques like ultrasonography (USG), mammography, CT-scan, and magnetic resonance imaging (MRI). Imaging techniques, however, fail to distinguish between cancerous and non-cancerous cells for early diagnosis. To confirm the imaging result, solid and liquid biopsies are done which have certain limitations such as invasive (in case of solid biopsy) or missed early diagnosis due to extremely low concentrations of circulating tumor DNA (in case of liquid biopsy). Therefore, it is essential to detect certain biomarkers by a noninvasive approach. One approach is a proteomic or glycoproteomic study which mostly identifies proteins and glycoproteins present in tissues and serum. Some of these studies are approved by the Food and Drug Administration (FDA). Another non-expensive and comparatively easier method to detect glycoprotein biomarkers is by ELISA, which uses lectins of diverse specificities. Several of the FDA approved proteins used as cancer biomarkers do not show optimal sensitivities for precise diagnosis of the diseases. In this regard, expression of phosphoproteins is associated with a more specific stage of a particular disease with high sensitivity and specificity. In this review, we discuss the expression of different serum phosphoproteins in various cancers. These phosphoproteins are detected either by phosphoprotein enrichment by immunoprecipitation using phosphospecific antibody and metal oxide affinity chromatography followed by LC-MS/MS or by 2D gel electrophoresis followed by MALDI-ToF/MS analysis. The updated knowledge on phosphorylated proteins in clinical samples from various cancer patients would help to develop these serum phophoproteins as potential diagnostic/prognostic biomarkers of cancer.

Evolutionary Divergence of Phosphorylation to Regulate Interactive Protein Networks in Lower and Higher Species.

The phosphorylation of proteins affects their functions in extensively documented circumstances. However, the role of phosphorylation in many interactive networks of proteins remains very elusive due to the experimental limits of exploring the transient interaction in a large complex of assembled proteins induced by stimulation. Previous studies have suggested that phosphorylation is a recent evolutionary process that differently regulates ortholog proteins in numerous lineages of living organisms to create new functions. Despite the fact that numerous phospho-proteins have been compared between species, little is known about the organization of the full phospho-proteome, the role of phosphorylation to orchestrate large interactive networks of proteins, and the intertwined phospho-landscape in these networks. In this report, we aimed to investigate the acquired role of phosphate addition in the phenomenon of protein networking in different orders of living organisms. Our data highlighted the acquired status of phosphorylation in organizing large, connected assemblages in Homo sapiens. The protein networking guided by phosphorylation turned out to be prominent in humans, chaotic in yeast, and weak in flies. Furthermore, the molecular functions of GO annotation enrichment regulated by phosphorylation were found to be drastically different between flies, yeast, and humans, suggesting an evolutionary drift specific to each species.

Phosphoproteomic analysis of duck egg yolk provides novel insights into its characteristics and biofunctions.

BACKGROUND: Although the importance of phosphorylation in the function of proteins is known, investigation of the protein phosphorylation of duck egg yolk (DEY) is still very limited. This study aimed to conduct a detailed phosphoproteomic study of DEY using immobilized metal affinity chromatography and ultra-high liquid chromatography tandem mass spectrometry. RESULTS: A total of 253 phosphorylation sites assigned to 66 phosphoproteins were identified in DEY, of which VTG-1, VTG-2, and fibrinogen alpha chain were found to be the highly phosphorylated proteins in DEY. The biological functions of the identified phosphoproteins were illuminated through gene ontology analysis, which showed that they were mainly involved in binding, catalytic, immune response, and metabolic activity. S-X-E and S-X-S were found to be the most conserved serine motifs of phosphorylation in DEY. The comparison of DEY phosphoproteins with those of chicken egg yolk (CEY) revealed that differences mostly involved molecular functions and biological processes. The comparison also revealed a higher phosphorylation level in DEY proteins. CONCLUSION: The higher phosphorylation level in DEY proteins than that in CEY proteins are supposed to help enhance duck growth performance and biological activities (e.g. antibacterial and antioxidant ability) for better adapting the humid environment the duck lived. © 2021 Society of Chemical Industry.

Gamma Visual Stimulation Induces a Neuroimmune Signaling Profile Distinct from Acute Neuroinflammation.

Many neurodegenerative and neurological diseases are rooted in dysfunction of the neuroimmune system; therefore, manipulating this system has strong therapeutic potential. Prior work has shown that exposing mice to flickering lights at 40 Hz drives gamma frequency (∼40 Hz) neural activity and recruits microglia, the primary immune cells of the brain, revealing a novel method to manipulate the neuroimmune system. However, the biochemical signaling mechanisms between 40 Hz neural activity and immune recruitment remain unknown. Here, we exposed wild-type male mice to 5-60 min of 40 Hz or control flicker and assessed cytokine and phosphoprotein networks known to play a role in immune function. We found that 40 Hz flicker leads to increases in the expression of cytokines which promote microglial phagocytic states, such as IL-6 and IL-4, and increased expression of microglial chemokines, such as macrophage-colony-stimulating factor and monokine induced by interferon-γ. Interestingly, cytokine effects differed as a function of stimulation frequency, revealing a range of neuroimmune effects of stimulation. To identify possible mechanisms underlying cytokine expression, we quantified the effect of the flicker on intracellular signaling pathways known to regulate cytokine levels. We found that a 40 Hz flicker upregulates phospho-signaling within the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. While cytokine expression increased after 1 h of 40 Hz flicker stimulation, protein phosphorylation in the NF-κB pathway was upregulated within minutes. Importantly, the cytokine expression profile induced by 40 Hz flicker was different from cytokine changes in response to acute neuroinflammation induced by lipopolysaccharides. These results are the first, to our knowledge, to show how visual stimulation rapidly induces critical neuroimmune signaling in healthy animals.SIGNIFICANCE STATEMENT Prior work has shown that exposing mice to lights flickering at 40 Hz induces neural spiking activity at 40 Hz (within the gamma frequency) and recruits microglia, the primary immune cells of the brain. However, the immediate effect of 40 Hz flicker on neuroimmune biochemical signaling was unknown. We found that 40 Hz flicker leads to significant increases in the expression of cytokines, key immune signals known to recruit microglia. Furthermore, we found that 40 Hz flicker rapidly changes the phosphorylation of proteins in the NF-κB and MAPK pathways, both known to regulate cytokine expression. Our findings are the first to delineate a specific rapid immune signaling response following 40 Hz visual stimulation, highlighting both the unique nature and therapeutic potential of this treatment.

Improved cell signaling analysis by biofunctionalized nanospheres and imaging flow cytometry.

The analysis of immune cell signaling is critical for the understanding of the biology and pathology of the immune system, and thus a mandatory step for the development of efficient biomarkers and targeted therapies. Phosflow, which has progressively replaced the traditional western blot approach, relies on flow cytometry to analyze various signaling pathways at a single-cell level. This technique however suffers a lack of sensitivity largely due to the low signal/noise ratio that characterizes cell signaling analysis. In this study, we describe a new technique, which combines the use of biofunctionalized nanospheres (i.e., synthetic particulate antigens, SPAg) to stimulate the immune cells in suspension and imaging flow cytometry to identify homogenously-stimulated cells and quantify the activity of the chosen signaling pathway in selected subcellular regions of interest. Using BCR signaling as model, we demonstrate that SIBERIAN (SPAg-assIsted suB-cEllulaR sIgnaling ANalysis) allows assessing immune cell signaling with unprecedented sensitivity and specificity.

Facile synthesis of titanium(IV) ion-immobilized arsenate-modified poly(glycidyl methacrylate) microparticles and the application to the specific enrichment of phosphoproteins.

Selective separation and enrichment of phosphoproteins possess the distinct clinical and biological importance in the diagnosis, treatment, and management of several fatal human diseases. In this study, a facile synthesis of titanium(IV) ion-immobilized arsenate-modified poly(glycidyl methacrylate) microparticles (denoted as Ti4+-arsenate-PGMA-MPs) was developed for the efficient enrichment of intact phosphoproteins found in biologically complex protein samples. By virtue of the strong interaction between the titanium ions immobilized on the surface of Ti4+-arsenate-PGMA-MPs and phosphate groups of phosphoproteins, Ti4+-arsenate-PGMA-MPs had a high saturated adsorption capacity for phosphoproteins (901 mg/g for ß-casein), which was much higher than that of non-phosphoproteins (73.5 mg/g for BSA). Ti4+-arsenate-PGMA-MPs were characterized by SEM, TEM, and FT-IR, and the average particle diameter was about 2.5 µm with good dispersibility. Besides, the application of Ti4+-arsenate-PGMA-MPs in real biological samples was investigated by SDS-PAGE analysis, and the results showed that Ti4+-arsenate-PGMA-MPs were able to enrich phosphoproteins efficiently.

Titanium dioxide-functionalized dendritic mesoporous silica nanoparticles for highly selective isolation of phosphoproteins.

Selective isolation of phosphoproteins is of great significance in biological applications. Herein, titanium dioxide-functionalized dendritic mesoporous silica nanoparticles are prepared via a post-grafting method for selective capture of phosphoproteins. The fabricated nanoparticles possess a unique central-radial pore structure with a surface area of 666.66 m2 /g and a pore size of 22.2 nm. The high-binding affinity of TiO2 with the phosphate groups facilitates the selective adsorption of phosphoproteins. Moreover, the open central-radial pore structure endows the dendritic mesoporous nanoparticles with better adsorption performance toward phosphoproteins with respect to the commercial titanium dioxide nanoparticles and titanium dioxide-functionalized conventional mesoporous silica nanoparticles by providing more accessible affinity sites. At pH 2, an adsorption capacity of 157.2 mg/g is derived for ß-casein. The feasibility of the as-prepared dendritic material in real biological sample assay is demonstrated by the selective isolation of phosphoproteins from defatted milk, as illustrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay.

Insights into Impact of DNA Copy Number Alteration and Methylation on the Proteogenomic Landscape of Human Ovarian Cancer via a Multi-omics Integrative Analysis.

In this work, we propose iProFun, an integrative analysis tool to screen for proteogenomic functional traits perturbed by DNA copy number alterations (CNAs) and DNA methylations. The goal is to characterize functional consequences of DNA copy number and methylation alterations in tumors and to facilitate screening for cancer drivers contributing to tumor initiation and progression. Specifically, we consider three functional molecular quantitative traits: mRNA expression levels, global protein abundances, and phosphoprotein abundances. We aim to identify those genes whose CNAs and/or DNA methylations have cis-associations with either some or all three types of molecular traits. Compared with analyzing each molecular trait separately, the joint modeling of multi-omics data enjoys several benefits: iProFun experienced enhanced power for detecting significant cis-associations shared across different omics data types, and it also achieved better accuracy in inferring cis-associations unique to certain type(s) of molecular trait(s). For example, unique associations of CNAs/methylations to global/phospho protein abundances may imply posttranslational regulations.We applied iProFun to ovarian high-grade serous carcinoma tumor data from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium and identified CNAs and methylations of 500 and 121 genes, respectively, affecting the cis-functional molecular quantitative traits of the corresponding genes. We observed substantial power gain via the joint analysis of iProFun. For example, iProFun identified 117 genes whose CNAs were associated with phosphoprotein abundances by leveraging mRNA expression levels and global protein abundances. By comparison, analyses based on phosphoprotein data alone identified none. A network analysis of these 117 genes revealed the known oncogene AKT1 as a key hub node interacting with many of the rest. In addition, iProFun identified one gene, BIN2, whose DNA methylation has cis-associations with its mRNA expression, global protein, and phosphoprotein abundances. These and other genes identified by iProFun could serve as potential drug targets for ovarian cancer.

Phosphoproteomic Comparison of Four Eimeria tenella Life Cycle Stages.

Protein phosphorylation is an important post-translational modification (PTM) involved in diverse cellular functions. It is the most prevalent PTM in both Toxoplasma gondii and Plasmodium falciparum, but its status in Eimeria tenella has not been reported. Herein, we performed a comprehensive, quantitative phosphoproteomic profile analysis of four stages of the E. tenella life cycle: unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), and sporozoites (S). A total of 15,247 phosphorylation sites on 9514 phosphopeptides corresponding to 2897 phosphoproteins were identified across the four stages. In addition, 456, 479, and 198 differentially expressed phosphoproteins (DEPPs) were identified in the comparisons SO7h vs. USO, SO vs. SO7h, and S vs. SO, respectively. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEPPs suggested that they were involved in diverse functions. For SO7h vs. USO, DEPPs were mainly involved in cell division, actin cytoskeleton organization, positive regulation of transport, and pyruvate metabolism. For SO vs. SO7h, they were related to the peptide metabolic process, translation, and RNA transport. DEPPs in the S vs. SO comparison were associated with the tricarboxylic acid metabolic process, positive regulation of ATPase activity, and calcium ion binding. Time course sequencing data analysis (TCseq) identified six clusters with similar expression change characteristics related to carbohydrate metabolism, cytoskeleton organization, and calcium ion transport, demonstrating different regulatory profiles across the life cycle of E. tenella. The results revealed significant changes in the abundance of phosphoproteins during E. tenella development. The findings shed light on the key roles of protein phosphorylation and dephosphorylation in the E. tenella life cycle.

A Bioinformatics Systems Biology Analysis of the Current Oral Proteomic Biomarkers and Implications for Diagnosis and Treatment of External Root Resorption.

External root resorption (ERR) is a silent destructive phenomenon detrimental to dental health. ERR may have multiple etiologies such as infection, inflammation, traumatic injuries, pressure, mechanical stimulations, neoplastic conditions, systemic disorders, or idiopathic causes. Often, if undiagnosed and untreated, ERR can lead to the loss of the tooth or multiple teeth. Traditionally, clinicians have relied on radiographs and cone beam computed tomography (CBCT) images for the diagnosis of ERR; however, these techniques are not often precise or definitive and may require exposure of patients to more ionizing radiation than necessary. To overcome these shortcomings, there is an immense need to develop non-invasive approaches such as biomarker screening methods for rapid and precise diagnosis for ERR. In this review, we performed a literature survey for potential salivary or gingival crevicular fluid (GCF) proteomic biomarkers associated with ERR and analyzed the potential pathways leading to ERR. To the best of our knowledge, this is the first proteomics biomarker survey that connects ERR to body biofluids which represents a novel approach to diagnose and even monitor treatment progress for ERR.

IPK2019: David Shugar and the genesis of the IPK conferences.

A short biographical sketch of Professor David Shugar, the "father of the IPK conferences," is presented, focusing on the growing interest of this eminent scientist for protein kinases and his farsighted perception of the extraordinary therapeutic potential of protein kinase inhibitors, after his discovery in 1986 that 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole effects are mediated by inhibition of protein kinase CK2. This led David Shugar to conceive the idea of organizing a periodic international conference on protein kinase inhibitors ("IPK conference"). The first conference was held in 1998 and the 10th one under the auspices of International Union of Biochemistry and Molecular Biology in September 2019. David Shugar died at the age of 100 in 2015, shortly after having organized the eight IPK conference.