Comparative Phosphoproteomics Research Articles

PTMoreR-enabled cross-species PTM mapping and comparative phosphoproteomics across mammals

To support PTM proteomic analysis and annotation in different species, we developed PTMoreR, a user-friendly tool that considers the surrounding amino acid sequences of PTM sites during BLAST, enabling a motif-centric analysis across species. By controlling sequence window similarity, PTMoreR can map phosphoproteomic results between any two species, perform site-level functional enrichment analysis, and generate kinase-substrate networks. We demonstrate that the majority of real P-sites in mice can be inferred from experimentally derived human P-sites with PTMoreR mapping. Furthermore, the compositions of 129 mammalian phosphoproteomes can also be predicted using PTMoreR. The method also identifies cross-species phosphorylation events that occur on proteins with an increased tendency to respond to the environmental factors. Moreover, the classic kinase motifs can be extracted across mammalian species, offering an evolutionary angle for refining current motifs. PTMoreR supports PTM proteomics in non-human species and facilitates quantitative phosphoproteomic analysis.

A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal and pericentral axis. How the mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combine intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We find that periportal and pericentral mitochondria are morphologically and functionally distinct; beta-oxidation is elevated in periportal regions, while lipid synthesis is predominant in the pericentral mitochondria. In addition, comparative phosphoproteomics reveals spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifts mitochondrial phenotypes in the periportal and pericentral regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

Identification of Reduced mTOR T1262 Phosphorylation as a Novel Mechanism and Therapeutic Target of Apoptosis in Senescent Cardiomyocytes: Aging and Cardiomyocyte Apoptosis.

The mechanisms through which aging increases heart injury remain partially understood. Protein phosphorylation plays a critical regulatory role in cell survival and death. Using an unbiased phosphoproteomics approach, we aimed to identify the proteins whose phosphorylation could be causatively related to aging-related cardiomyocyte apoptosis and elucidate the underlying mechanisms. Comparative phosphoproteomics was conducted on cardiac tissues obtained from young (8 weeks) and aged (24 months) mice. Our findings revealed that the Mammalian Target of Rapamycin phosphorylation at T1262 (mTORT1262) was reduced in the aging heart. Immunohistochemical and Western blot analyses confirmed these findings in aging myocardia and D-galactose-induced senescent AC16 cardiomyocytes. In hypoxia/reoxygenation cardiomyocytes, mTORT1262 phosphorylation deficiency (mTORT1262A, lentivirus-mediated transfection) inhibited AKT1, suppressed NF-κB, activated FOXO1/3a signaling, and ultimately exacerbated apoptosis. Conversely, mTORT1262 pseudophosphorylation (mTORT1262E) exhibited opposite effects. Through bioinformatics and CO-IP, purinergic receptor P2X4 (P2X4R) was found to be the possible receptor responsible for mTORT1262 phosphorylation. Knockdown of P2X4R increased apoptosis, whereas its overexpression decreased it. In senescent cardiomyocytes, P2X4R expression and mTORT1262 and AKT1S473 phosphorylation were reduced, NF-κB signaling was suppressed, and FOXO1/3a signaling was activated. We demonstrated that P2X4R downregulation and the subsequent reduction of mTORT1262 phosphorylation is a novel mechanism contributing to cardiomyocyte apoptosis in aging hearts. The P2X4R-mTOR-AKT1 signaling pathway represents a potential therapeutic target against accelerated cardiac injury in aging.

Comparative phosphoproteome analyses provide new insight into ethephon stimulated latex yield in rubber tree

Comparative phosphoproteome analyses provide new insight into ethephon stimulated latex yield in rubber tree

Protein kinase PfPK2 mediated signalling is critical for host erythrocyte invasion by malaria parasite.

Signalling pathways in malaria parasite remain poorly defined and major reason for this is the lack of understanding of the function of majority of parasite protein kinases and phosphatases in parasite signalling and its biology. In the present study, we have elucidated the function of Protein Kinase 2 (PfPK2), which is known to be indispensable for the survival of human malaria parasite Plasmodium falciparum. We demonstrate that it is involved in the invasion of host erythrocytes, which is critical for establishing infection. In addition, PfPK2 may also be involved in the maturation of the parasite post-invasion. PfPK2 regulates the release of microneme proteins like Apical Membrane Antigen 1 (AMA1), which facilitates the formation of Tight Junction between the merozoite and host erythrocyte- a key step in the process of invasion. Comparative phosphoproteomics studies revealed that PfPK2 may be involved in regulation of several key proteins involved in invasion and signalling. Furthermore, PfPK2 regulates the generation of cGMP and the release of calcium in the parasite, which are key second messengers for the process of invasion. These and other studies have shed light on a novel signalling pathway in which PfPK2 acts as an upstream regulator of important cGMP-calcium signalling, which plays an important role in parasite invasion.

New insights into the function of lipid droplet-related proteins and lipid metabolism of salt-stimulated porcine biceps femoris: Label-free quantitative phosphoproteomics, morphometry and bioinformatics.

Lipid droplets (LD) are important multifunctional organelles responsible for lipid metabolism of postmortem muscle. However, the dynamics in their building blocks (cores and layers) and phosphorylation of lipid droplet-related proteins (LDRP) regulating meat lipolysis remain unknown at salt-stimulated conditions. LDRP extracted from cured porcine biceps femoris (1% and 3% salt) were subjected to label-free quantitative phosphoproteomic analysis and LD morphological validation. Results indicated that 3% salt curing significantly decreased triglyceride (TG) content with increase in glycerol and decrease in LD fluorescence compared to 1% salt curing. Comparative phosphoproteomics showed that there were significant changes in phosphorylation at 386 sites on 174 LDRP between assayed groups (p < 0.05). These differential proteins were mainly involved in lipid and carbohydrate metabolism. Curing of 3% salt induced more site-specific phosphorylation of perilipin 1 (PLIN1, at Ser81) and adipose triglyceride lipase (ATGL, at Ser399) than 1%, whereas the phosphorylation (at Ser600) of hormone-sensitive lipase (HSL) was up-regulated. Ultrastructure imaging showed that LD were mostly associated with mitochondria, and the average diameter of LD decreased from 2.34 μm (1% salt) to 1.73 μm (3% salt). Phosphoproteomics unraveled salt-stimulated LDRP phosphorylation of cured porcine meat provoked intensified lipolysis. Curing of 3% salt allowed an enhanced lipolysis than 1% by up-regulating the phosphorylation sites of LDRP and recruited lipases. The visible splitting of LD, together with sarcoplasmic disorganization, supporting the lipolysis robustness following 3% salt curing. The finding provides optimization ideas for high-quality production of cured meat products. This article is protected by copyright. All rights reserved.

Phosphoproteomic analysis highlights phosphorylation of primary metabolism-related proteins in response to postharvest cold storage and subsequent shelf-life in tomato fruit

Tomato fruit are susceptible to postharvest chilling injuries when stored at low temperatures, resulting in quality deterioration and economic losses worldwide. Protein phosphorylation is an important post-translational modification that has been proven to be involved in plant cold tolerance. However, the changes of protein phosphorylation underlying cold stress responses in tomatoes remain poorly understood. In this study, microscopic analyses showed that endomembranes and intracellular organelles were broken down after cold storage, and the breakdown became more pronounced when the fruit was restored to room temperature. Primary metabolomic analysis revealed that large amounts of lipids, amino acids, sugars, and organic acids were altered following cold treatment. Additionally, we performed comparative phosphoproteomics and determined the overall phosphoprotein profile of tomato fruit after postharvest cold storage and subsequent shelf life. A total of 10260 phosphopeptides corresponding to 9640 phosphosites in 3740 phosphoproteins were identified. Differentially phosphorylated proteins were mainly involved in primary metabolism, such as glycerolipid metabolism, glycolysis/gluconeogenesis, and the tricarboxylic acid cycle. Correspondingly, the phosphorylation of large amounts of important primary metabolic enzymes, such as fatty acid desaturase, sucrose synthase, and alcohol dehydrogenase, was upregulated after cold treatment. The present study revealed the involvement of phosphorylation of primary metabolism-related proteins in the response to postharvest cold storage in tomato fruit.

Live Cells Imaging and Comparative Phosphoproteomics Uncover Proteins from the Mechanobiome in Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite and the causative agent of amoebiasis in humans. This amoeba invades human tissues by taking advantage of its actin-rich cytoskeleton to move, enter the tissue matrix, kill and phagocyte the human cells. During tissue invasion, E. histolytica moves from the intestinal lumen across the mucus layer and enters the epithelial parenchyma. Faced with the chemical and physical constraints of these diverse environments, E. histolytica has developed sophisticated systems to integrate internal and external signals and to coordinate cell shape changes and motility. Cell signalling circuits are driven by interactions between the parasite and extracellular matrix, combined with rapid responses from the mechanobiome in which protein phosphorylation plays an important role. To understand the role of phosphorylation events and related signalling mechanisms, we targeted phosphatidylinositol 3-kinases followed by live cell imaging and phosphoproteomics. The results highlight 1150 proteins, out of the 7966 proteins within the amoebic proteome, as members of the phosphoproteome, including signalling and structural molecules involved in cytoskeletal activities. Inhibition of phosphatidylinositol 3-kinases alters phosphorylation in important members of these categories; a finding that correlates with changes in amoeba motility and morphology, as well as a decrease in actin-rich adhesive structures.

Comparative phosphoproteomics analysis provides insights into the responses of Chilo suppressalis to sublethal chlorantraniliprole exposure.

Sublethal exposure to insecticides causes changes in insect behaviors and physiologies including feeding, mobility, communication, hormone homeostasis, development and fecundity, however, the underlying molecular mechanisms were largely unclear. Our previous studies revealed that sublethal chlorantraniliprole exposure disturbed the hormone homeostasis, reduced the weight and longevity and prolonged the developmental duration of Chilo suppressalis. In the present study, the potential phosphorylation modification regulation mechanisms in C. suppressalis in response to sublethal chlorantraniliprole exposure were explored using comparative and quantitative phosphoproteomics. A total of 2640 phosphopeptides belonging to 1144 phosphoproteins were identified, among which 446 phosphopeptides derived from 303 unique phosphoproteins were differentially phosphorylated between the chlorantraniliprole-treated and control larvae. The phosphorylation levels of differentially phosphorylated phosphopeptides were further validated using parallel reaction monitoring (PRM). Functional classification and protein-protein interaction of the differentially phosphorylated proteins (DPPs) were analyzed. Generalized analysis of the DPPs and the differentially expressed genes (DEGs) identified in our previous study showed that sublethal chlorantraniliprole exposure significantly changed the transcription and phosphorylation levels of genes/proteins associated with carbohydrate and lipid metabolism, cytoskeleton, signal transduction, transcription, translation and post-translational modification, leading to the dysfunctions of energy metabolism, transcription regulation, protein synthesis and modification, and signal transduction in C. suppressalis. Further analysis of the phosphorylation motifs in DPPs revealed that the MAPKs, CDKs, CaMK II, PKA, PKC and CK II protein kinases might be directly responsible for the phosphoproteomics response of C. suppressalis to chlorantraniliprole treatment. Our results provide abundant phosphorylation information for characterizing the protein modification in insects, and also provide valuable insights into the molecular mechanisms of insect post-translational modifications in response to sublethal insecticide exposure. © 2023 Society of Chemical Industry.

Abstract 3074: (Phospho)proteomics profiling of patient-derived colon cancer organoids for the discovery of putative drug targets

Abstract Introduction: Colorectal cancer is the second cause of cancer related deaths worldwide, with the poor survival outcomes attributable to the presence of metastases. To tackle this problem, (phospho)proteomics analysis enables valuable insights into changes of protein expression and signalling in cancer that can be perturbed by drugs. To study mechanisms driving metastasis and perform subsequent drug testing, patient derived organoids (PDOs) are in development as preclinical models. PDOs are obtained by 3D culture of tumour tissue ex-vivo, which enables them to retain the heterogeneity and architecture of the tumor source. To identify putative drug targets for colon cancer metastasis, we performed comparative (phospho)proteomics analysis of metastatic colon tissues and their matched PDOs. Methods: (Phospho)proteomics profiling was performed on PDOs generated from 10 patients with colon metastases to the liver, 10 matched tumour tissues and 4 normal colon mucosa. Unsupervised analysis of the (phospho)proteomic landscape was used to identify differentially expressed genes and pathways in colon metastases and their matched PDOs, compared to normal colon mucosa. We focused on phosphosites, proteins and pathways showing consistently altered expression in tumour tissues and PDOs. Kinase-substrate enrichment analysis was used to identify aberrantly activated kinases, based on the abundance of phosphorylation on the substrates. Putative drugs were selected using the consensus expression response to multiple small molecule drugs across cell lines and conditions from the Library of Integrated Network-Based Cellular Signatures (LINCS) database. Drug treatment of drug candidates was performed using the MTT Cell Proliferation Assay. Results: Using the approach described above we identified 103 differentially expressed proteins and 236 phosphosites between tumour tissues and normals, which were also detected in PDOs, with overall high correlation of t statistics (0.6 Spearman’s rho) between tumor tissues and PDOs. MYC-targets, G2M checkpoints and E2F-targets were amongst the top positively enriched pathways in common, and the LINCS analysis identified multi-kinase inhibitor Nintedanib and NFKB pathway-targeting KIN0-260 as putative drugs for upregulated proteins. The kinase CSNK2A1 was overactivated in both groups, pointing towards the use of CK2 inhibitors for drug testing. Treatment at 5 days with Nintedanib and KIN001-260 on the PDOs resulted in significant reduction of cell viability compared with 5-FU. Conclusion: Our study provides evidence that PDOs recapitulate relevant tumor features at the proteomic and phosphoproteomic levels, supporting the utility of this ex-vivo model as a tool for drug sensitivity testing for metastatic colon cancer. Citation Format: Gina Faye Boot, Federica Panebianco, John Gallon, Charlotte Kiu Yan Ng, Salvatore Piscuoglio. (Phospho)proteomics profiling of patient-derived colon cancer organoids for the discovery of putative drug targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3074.

Comparative phosphoproteomics reveal new candidates in the regulation of spermatogenesis of Drosophila melanogaster.

The most common phenotype induced by the endosymbiont Wolbachia in insects is cytoplasmic incompatibility, where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females. This suggests that some modifications are induced in host sperms during spermatogenesis by Wolbachia. To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster, we applied isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic strategy combined with titanium dioxide (TiO2 ) enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male reproductive systems in D. melanogaster. We identified 182 phosphopeptides, defining 140 phosphoproteins, that have at least a 1.2 fold change in abundance with a P-value of <0.05. Most of the differentially abundant phosphoproteins (DAPPs) were associated with microtubule cytoskeleton organization and spermatid differentiation. The DAPPs included proteins already known to be associated with spermatogenesis, as well as many not previously studied during this process. Six genes coding for DAPPs were knocked down, respectively, in Wolbachia-free fly testes. Among them, Slmap knockdown caused the most severe damage in spermatogenesis, with no mature sperm observed in seminal vesicles. Immunofluorescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted. These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction. By identifying phospho-modulated proteins we also provide a significant candidate set for future studies on their roles in spermatogenesis.

Phosphorylation of CAD1, PLDdelta, NDT1, RPM1 Proteins Induce Resistance in Tomatoes Infected by Ralstonia solanacearum.

Ralstonia solanacaerum is one of the most devastating bacteria causing bacterial wilt disease in more than 200 species of plants, especially those belonging to the family Solanaceae. To cope with this pathogen, plants have evolved different resistance mechanisms depending on signal transduction after perception. Phosphorylation is the central regulatory component of the signal transduction pathway. We investigated a comparative phosphoproteomics analysis of the stems of resistant and susceptible tomatoes at 15 min and 30 min after inoculation with Ralstonia solanacearum to determine the phosphorylated proteins involved in induced resistance. Phosphoprotein profiling analyses led to the identification of 969 phosphoproteins classified into 10 functional categories. Among these, six phosphoproteins were uniquely identified in resistant plants including cinnamyl alcohol dehydrogenase 1 (CAD1), mitogen-activated protein kinase kinase kinase 18 (MAPKKK18), phospholipase D delta (PLDDELTA), nicotinamide adenine dinucleotide transporter 1 (NDT1), B3 domain-containing transcription factor VRN1, and disease resistance protein RPM1 (RPM1). These proteins are typically involved in defense mechanisms across different plant species. qRT-PCR analyses were performed to evaluate the level of expression of these genes in resistant and susceptible tomatoes. This study provides useful data, leading to an understanding of the early defense mechanisms of tomatoes against R. solanacearum.

Comparative Phenotypic, Proteomic, and Phosphoproteomic Analysis Reveals Different Roles of Serine/Threonine Phosphatase and Kinase in the Growth, Cell Division, and Pathogenicity of Streptococcus suis.

Eukaryote-like serine/threonine kinases (STKs) and cognate phosphatases (STPs) comprise an important regulatory system in many bacterial pathogens. The complexity of this regulatory system has not been fully understood due to the presence of multiple STKs/STPs in many bacteria and their multiple substrates involved in many different physiological and pathogenetic processes. Streptococci are the best materials for the study due to a single copy of the gene encoding STK and its cognate STP. Although several studies have been done to investigate the roles of STK and STP in zoonotic Streptococcus suis, respectively, few studies were performed on the coordinated regulatory roles of this system. In this study, we carried out a systemic study on STK/STP in S. suis by using a comparative phenotypic, proteomic, and phosphoproteomic analysis. Mouse infection assays revealed that STK played a much more important role in S. suis pathogenesis than STP. The ∆stk and ∆stp∆stk strains, but not ∆stp, showed severe growth retardation. Moreover, both ∆stp and ∆stk strains displayed defects in cell division, but they were abnormal in different ways. The comparative proteomics and phosphoproteomics revealed that deletion of stk or stp had a significant influence on protein expression. Interestingly, more virulence factors were found to be downregulated in ∆stk than ∆stp. In ∆stk strain, a substantial number of the proteins with a reduced phosphorylation level were involved in cell division, energy metabolism, and protein translation. However, only a few proteins showed increased phosphorylation in ∆stp, which also included some proteins related to cell division. Collectively, our results show that both STP and STK are critical regulatory proteins for S. suis and that STK seems to play more important roles in growth, cell division, and pathogenesis.

The genetic deletion of the Dual Specificity Phosphatase 3 (DUSP3) attenuates kidney damage and inflammation following ischaemia/reperfusion injury in mouse.

Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischaemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30min/48hours). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischaemic vs non-ischaemic kidneys in WT vs Dusp3-/- . At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs WT ischaemic kidneys. Dusp3-/- ischaemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1β and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs WT kidneys post-I/R. Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.

Quantitative Phosphoproteomic Comparison of Lens Proteins in Highly Myopic Cataract and Age-Related Cataract.

Purpose To investigate and compare the lens phosphoproteomes in patients with highly myopic cataract (HMC) or age-related cataract (ARC). Methods In this study, we undertook a comparative phosphoproteome analysis of the lenses from patients with HMC or ARC. Intact lenses from ARC and HMC patients were separated into the cortex and nucleus. After protein digestion, the phosphopeptides were quantitatively analyzed with TiO2 enrichment and liquid chromatography-mass spectrometry. The potential functions of different phosphopeptides were assessed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Results In total, 522 phosphorylation sites in 164 phosphoproteins were identified. The number of phosphorylation sites was significantly higher in the cortex than in the nucleus, in both ARC and HMC lenses. The differentially phosphorylated peptides in the lens cortex and nucleus in HMC eyes were significantly involved in the glutathione metabolism pathway. The KEGG pathway enrichment analysis indicated that the differences in phosphosignaling mediators between the ARC and HMC lenses were associated with glycolysis and the level of phosphorylated phosphoglycerate kinase 1 was lower in HMC lenses than in ARC lenses. Conclusions We provide an overview of the differential phosphoproteomes of HMC and ARC lenses that can be used to clarify the molecular mechanisms underlying their different phenotypes.

Comparative Phosphoproteomics of Classical Bordetellae Elucidates the Potential Role of Serine, Threonine and Tyrosine Phosphorylation in Bordetella Biology and Virulence.

The Bordetella genus is divided into two groups: classical and non-classical. Bordetella pertussis, Bordetella bronchiseptica and Bordetella parapertussis are known as classical bordetellae, a group of important human pathogens causing whooping cough or whooping cough-like disease and hypothesized to have evolved from environmental non-classical bordetellae. Bordetella infections have increased globally driving the need to better understand these pathogens for the development of new treatments and vaccines. One unexplored component in Bordetella is the role of serine, threonine and tyrosine phosphorylation. Therefore, this study characterized the phosphoproteome of classical bordetellae and examined its potential role in Bordetella biology and virulence. Applying strict identification of localization criteria, this study identified 70 unique phosphorylated proteins in the classical bordetellae group with a high degree of conservation. Phosphorylation was a key regulator of Bordetella metabolism with proteins involved in gluconeogenesis, TCA cycle, amino acid and nucleotide synthesis significantly enriched. Three key virulence pathways were also phosphorylated including type III secretion system, alcaligin synthesis and the BvgAS master transcriptional regulatory system for virulence genes in Bordetella. Seven new phosphosites were identified in BvgA with 6 located in the DNA binding domain. Of the 7, 4 were not present in non-classical bordetellae. This suggests that serine/threonine phosphorylation may play an important role in stabilizing/destabilizing BvgA binding to DNA for fine-tuning of virulence gene expression and that BvgA phosphorylation may be an important factor separating classical from non-classical bordetellae. This study provides the first insight into the phosphoproteome of classical Bordetella species and the role that Ser/Thr/Tyr phosphorylation may play in Bordetella biology and virulence.

Comparative phosphoproteomic analysis unravels MAPK1 regulated phosphoproteins in Leishmania donovani

Mitogen Activated Protein Kinase1 (MAPK1) of Leishmania donovani functions as key regulators of various cellular activities, which seem to be imperative for parasite survival, infectivity, drug resistance and post-translational modification of chaperones/co-chaperones. However, very less is known about LdMAPK1 target proteins. With recent advancements in proteomics, we aimed to identify phosphoproteins which were differentially expressed in LdMAPK1 overexpressing (Dd8++/++) and single replacement mutants (Dd8+/) as compared to wild type (Dd8+/+) parasites, utilizing LC-MS/MS approach. An in-depth label-free phospoproteomic analysis revealed that modulation of LdMAPK1 expression significantly modulates expression levels of miscellaneous phosphoproteins which may act as its targets/substrates. Out of 1974 quantified phosphoproteins in parasite, 140 were significantly differentially expressed in MAPK1 overexpressing and single replacement mutants. These differentially expressed phosphoproteins are majorly associated with metabolism, signal transduction, replication, transcription, translation, transporters and cytoskeleton/motor proteins, hence suggested that MAPK1 may act in concert to modulate global biological processes. The study further implicated possible role of LdMAPK1 in regulation and management of stress machinery in parasite through post translational modifications. Precisely, comparative phosphoproteomics study has elucidated significant role of LdMAPK1 in regulating various pathways contributing in parasite biology with relevance to future drug development. SignificanceMAPKinase1, the downstream kinase of MAPK signal transduction pathway, has drawn much attention as potential therapeutic drug target due to their indispensable role in survival and infectivity of Leishmania donovani. However, limited information is available about its downstream effector proteins/signaling networks. Utilizing label free LC-MS/MS analysis, phosphoproteome of LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single replacement mutants (Dd8+/−) with wild type (Dd8+/+) parasites was compared and identified 140 LdMAPK1 modulated phosphoproteins, mainly involved in pathways like signal transduction, metabolism, transcriptional, translational, post-translational modification and regulation of heat shock proteins. Interestingly, LdMAPK1 interacts directly with only six phosphoproteins i.e. casein kinase, casein kinase II, HSP83/HSP90, LACK, protein kinase and serine/threonine protein kinase. Thus, the study elucidates significant role of LdMAPK1 in Leishmania biology which may drive drug-discovery efforts against visceral leishmaniasis.

Phosphoproteome and Biological Evidence Revealed Abnormal Calcium Homeostasis in Keloid Fibroblasts and Induction of Aberrant Platelet Aggregation.

Keloid is a benign tumor characterized by persistent inflammation, increased fibroblast proliferation, and abnormal deposition of collagen in the wound. The etiology of keloid is unclear. Here, we explored the phospho-signaling changes in human keloid fibroblasts via phosphoproteome mass spectrometry analysis. We found that comparative phosphoproteomics could statistically distinguish keloid from control fibroblasts. Differentially expressed phosphoproteins could predict the activation of known keloid-relevant upstream regulators including transforming growth factor-β1, interleukin (IL)-4, and IL-5. With multiple bioinformatics analyses, phosphorylated FLNA, TLN1, and VCL were significantly enriched in terms of calcium homeostasis and platelet aggregation. We biologically verified that keloid fibroblasts had a higher level of Ca2+ influx than the control fibroblasts upon ionomycin stimulation. Via co-cultivation analysis, we found that human keloid fibroblasts could directly promote platelet aggregation. As suggested by PhosphoPath and gene set enrichment analysis, pFLNA was centered as the top phosphoproteins associated with keloid phenotypes. We validated that pFLNA was upregulated both in keloid fibroblasts and keloid tissue section, implicating its biomarker potential. In conclusion, we reported the first phosphoproteome on keloid fibroblasts, based on which we revealed that keloid fibroblasts had aberrant calcium homeostasis and could directly induce platelet aggregation.

Comparative phosphoproteome analysis to identify candidate phosphoproteins involved in blue light-induced brown film formation in Lentinula edodes.

Light plays an important role in the growth and differentiation of Lentinula edodes mycelia, and mycelial morphology is influenced by light wavelengths. The blue light-induced formation of brown film on the vegetative mycelial tissues of L. edodes is an important process. However, the mechanisms of L. edodes’ brown film formation, as induced by blue light, are still unclear. Using a high-resolution liquid chromatography-tandem mass spectrometry integrated with a highly sensitive immune-affinity antibody method, phosphoproteomes of L. edodes mycelia under red- and blue-light conditions were analyzed. A total of 11,224 phosphorylation sites were identified on 2,786 proteins, of which 9,243 sites on 2,579 proteins contained quantitative information. In total, 475 sites were up-regulated and 349 sites were down-regulated in the blue vs red group. To characterize the differentially phosphorylated proteins, systematic bioinformatics analyses, including gene ontology annotations, domain annotations, subcellular localizations, and Kyoto Encyclopedia of Genes and Genomes pathway annotations, were performed. These differentially phosphorylated proteins were correlated with light signal transduction, cell wall degradation, and melanogenesis, suggesting that these processes are involved in the formation of the brown film. Our study provides new insights into the molecular mechanisms of the blue light-induced brown film formation at the post-translational modification level.

Multi-omics analysis revealed crucial genes and pathways associated with black carp antiviral innate immunity

Multi-omics strategy contributes as an indispensable and efficient approach for the investigation of the innate immunity in vertebrates. To explore the crucial genes and pathways involved in the antiviral innate immunity of black carp (Mylopharyngodon piceus), the comparative phosphoproteomics and transcriptomics of Mylopharyngodon piceus kidney (MPK) cells with/without GCRV infection were performed in this manuscript. In phosphoproteomics analysis, 2637 phosphosites corresponding to 1532 proteins were identified and quantified, in which 1372 proteins were identified as differentially expressed proteins (DEPs) with 683 upregulated and 689 downregulated in GCRV infected cells. Functional annotation, enrichment analysis and pathway analysis highlighted that a large number of DEPs were enriched in immune related pathways including TLR pathway and NLR pathway. In transcriptomics analysis, a total of 2936 genes were identified as differentially expressed genes (DEGs), in which 2290 and 646 genes were upregulated and downregulated respectively after GCRV infection. As expected, pathway analysis based on DEGs also showed that a large proportion of DEGs were enriched in immune related pathways including TLR and RLR pathway. A combined list of DEPs and DEGs that enriched in above pathways were imported in Cytoscape for network analysis, reconstruction and visualization. The integrative study suggested that several significant DEPs and DEGs, such as MAP3K7 (TAK1), JUN, MAP2K2, CASP8, IL8 and IRF7 might be functionally crucial in host antiviral innate immunity. Thus, this study contributes as an indispensable reference map for the further investigation of the innate immune system of black carp.