Clostridioides difficile Phosphoproteomics Shows an Expansion of Phosphorylated Proteins in Stationary Growth Phase

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile . To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile .

A study of the phosphorylation proteomic skin characteristics of Tan sheep during the newborn and er-mao stages

In this experiment, in order to study the formation mechanism of the lamb fur of Tan sheep, skin samples were collected from Tan sheep at the newborn and er-mao stages. Then, the phosphorylated proteomes of the skin samples of Tan sheep at the two different stages were compared and analyzed using a TMT labeled quantitative phosphorylation proteomic technique. A total of 2806 phosphorylated proteins were identified, including 8184 phosphorylation sites. The results of this study’s quantitative analysis showed that when compared with the skin samples at the er-mao stage, the phosphorylation levels of 171 sites had been upregulated in the skin samples at newborn stage. Meanwhile, 125 sites had been downregulated at the same stage. As shown by the results of the functional enrichment analysis of the differentially phosphorylated proteins, they had been mainly enriched in the cysteine and methionine metabolism. In addition, the phosphorylation levels of KAP4.7 and KAP13.1 had also varied during the different skin stages. These results indicated that the cysteine metabolism pathways, as well as the phosphorylation modifications of the keratin associated proteins in the skin, played important roles in the formation of the er-mao stage fur of the Tan sheep. Therefore, the findings of this study provided a new angle for interpreting the formation mechanism of er-mao stage fur properties.

Identification of Abscisic Acid-Dependent Phosphorylated Basic Helix-Loop-Helix Transcription Factors in Guard Cells of Vicia faba by Mass Spectrometry

An unknown 61 kDa protein is phosphorylated by abscisic acid (ABA)-activated protein kinase in response to ABA and binds to 14-3-3 protein in a phosphorylation-dependent manner in guard-cell protoplasts (GCPs) from Vicia faba. Subsequently, ABA-dependent phosphorylated proteins were identified as basic helix–loop–helix transcription factors, named ABA-responsive kinase substrates (AKSs) in GCPs from Arabidopsis thaliana. However, whether the 61 kDa protein in Vicia GCPs is an AKS is unclear. We performed immunoprecipitation of ABA-treated Vicia GCPs using anti-14-3-3 protein antibodies and identified several AKS isoforms in V. faba (VfAKSs) by mass spectrometry. The 61 kDa protein was identified as VfAKS1. Phosphoproteomic analysis revealed that VfAKSs are phosphorylated at Ser residues, which are important for 14-3-3 protein binding and monomerisation, in response to ABA in GCPs. Orthologs of AtABCG40, an ABA importer in guard cells, and CHC1, a clathrin heavy chain and a regulator of stomatal movement, also co-immunoprecipitated with 14-3-3 protein from guard cells.

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the media, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylations state of proteins when heterologously expressed, since their biochemical and physiological properties are dependent on post-translational modification.

Proteomic analysis of early phosphorylated proteins in acute pancreatitis model

Background and Objective: The exact mechanism of acute pancreatitis (AP), which is an inflammation of the pancreas, still remains unclear. In this study, we examined the protein phosphorylation changes during the early stage of AP in mice using proteomic analysis. Methods: AP model in mice was constructed using an intraperitoneal injection of cerulein. Blood samples and pancreas were collected at 1, 3, 6, 9h after the final injection (n=3 at each time point). Samples collected 3h after the final injection were separately mixed and named S (saline group) and C1 (cerulein group); samples collected 6h after the final injection from the cerulein group were mixed and named C2. Proteins from S, C1, and C2 were extracted, digested by trypsin, and subjected to LC-MS/MS analysis, bioinformatics analysis, and Western blotting. Results: A total of 549 sites (426 proteins) were upregulated, and 501 sites (367 proteins) were downregulated in C1 compared to S; while 491 phosphorylation sites (377 proteins) were upregulated and 367 sites (274 proteins) were downregulated in C2 compared to S. Motif analysis showed that proline-directed kinase and basophilic kinase had a key role during early AP. During an early AP stage, the cellular distributions of proteins slightly changed. The types of domains changed with the development of AP. Phosphorylation proteins associated with calcium signaling, especially IP3R mediated calcium release, lysosome and autophagosome pathway, pancreatic digestive activation, and secretion, were found to be involved in the development of early AP independent of NF-kB activation. Moreover, the MAPK family was found to have a greater impact at the early stage of AP. We also found differentially expressed phosphorylations of amylase and trypsinogen and increased phosphorylation of MAPK6 S189 in early AP. Conclusion: IP3R mediated calcium release and activation of MAPK family are key events promoting the development of early AP.

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this "PermaPhosSer" system — an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins — we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3-β phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli.

Chronic heat stress promotes liver inflammation in broilers via enhancing NF-κB and NLRP3 signaling pathway

Background The study was to investigate the effects of chronic heat stress on liver inflammatory injury and its potential mechanisms in broilers. Chickens were randomly assigned to 1-week control group (Control 1), 1-week heat stress group (HS1), 2-week control group (Control 2), and a 2-week heat stress group (HS2) with 15 replicates per group. Broilers in heat stress groups exposed to heat stress (35 ± 2°C) for 8 h/d with 7 or 14 consecutive days. Growth performance and liver inflammation injury were examined for the analysis of liver injury.ResultsThe results showed that heat stress decreased the growth performance, showed obvious blutpunkte, lowered liver weight and liver index, which resulted in significant liver damage of broilers. Both the gene and protein expressions of HSP70, TLR4 and NF-κB in the liver were significantly enhanced by heat stress. Furthermore, heat stress obviously enhanced IL-6, TNF-α, NF-κB P65, IκB and their phosphorylated proteins expressions in the liver of broilers. In addition, heat stress promoted the activation of NLRP3 with increased NLRP3, caspase-1 and IL-1β. ConclusionsThese results suggested that heat stress can cause the liver inflammation via activation of TLR4-NF-κB and NLRP3 signaling pathway in broilers.

Phosphoproteome analysis reveals an extensive phosphorylation of proteins associated with bast fiber growth in ramie

Background Phosphorylation modification, one of the most common post-translational modifications of proteins, widely participates in the regulation of plant growth and development. Fibers extracted from the stem bark of ramie are important natural textile fibers; however, the role of phosphorylation modification in the growth of ramie fibers is largely unknown. Results Here, we report a phosphoproteome analysis for the barks from the top and middle section of ramie stems, in which the fiber grows at different stages. A total of 10,320 phosphorylation sites from 9,170 unique phosphopeptides that were assigned to 3,506 proteins was identified, and 458 differentially phosphorylated sites from 323 proteins were detected in the fiber developmental barks. Twelve differentially phosphorylated proteins were the homologs of Arabidopsis fiber growth-related proteins. We further focused on the function of the differentially phosphorylated KNOX protein whole_GLEAN_10029667, and found that this protein dramatically repressed the fiber formation in Arabidopsis. Additionally, using a yeast two-hybridization assay, we identified a kinase and a phosphatase that interact with whole_GLEAN_10029667, indicating that they potentially target this KNOX protein to regulate its phosphorylation level. Conclusion The finding of this study provided insights into the involvement of phosphorylation modification in ramie fiber growth, and our functional characterization of whole_GLEAN_10029667 provide the first evidence to indicate the involvement of phosphorylation modification in the regulation of KNOX protein function in plants.

Combination of urinary fibrinogen β-chain and tyrosine-phosphorylated proteins for the detection of bladder cancer

Aim: to evaluate the performance of urinary fibrinogen β-chain (FBC) – either alone or associated with urinary tyrosine-phosphorylated proteins (UPY) – as bladder cancer (BCa) diagnostic biomarker. Materials & methods: 164 subjects were tested. Results: significantly different FBC and UPY levels were found between BCa patients and controls, as well as between low-grade and high-grade cancers. The diagnostic accuracy was 0.84 for FBC and 0.87 for UPY. The combination of FBC and UPY improved the accuracy to 0.91. The addition of clinical variables (age, gender, and smoking habit) to FBC and UPY into a model for BCa prediction significantly improved the accuracy to 0.99. The combination of FBC and UPY adjusted for clinical variables associates with the highest sensitivity and good specificity. Conclusion: urinary FBC and UPY could be used as biomarkers for BCa diagnosis.