Proteomic profiling of Serratia marcescens by high-resolution mass spectrometry

Introduction: Serratia marcescens, an opportunistic human pathogen, is reported as an important cause of nosocomial infection and outbreaks. Although the genome of S. marcescens (ATCC 13880) was completely sequenced by 2014, there are no studies on the proteomic profile of the organism. The objective of the present study is to analyze the protein profile of S. marcescens (ATCC 13880) using a high resolution mass spectrometry (MS). Methods: Serratia marcescens ATCC 13880 strain was grown in Luria-Bertani broth and the protein extracted was subjected to trypsin digestion, followed by basic reverse phase liquid chromatography fractionation. The peptide fractions were then analysed using Orbitrap Fusion Mass Spectrometry and the raw MS data were processed in Proteome Discoverer software. Results: The proteomic analysis identified 15 009 unique peptides mapping to 2541 unique protein groups, which corresponds to approximately 54% of the computationally predicted protein-coding genes. Bioinformatic analysis of these identified proteins showed their involvement in biological processes such as cell wall organization, chaperone-mediated protein folding and ATP binding. Pathway analysis revealed that some of these proteins are associated with bacterial chemotaxis and beta-lactam resistance pathway. Conclusion: To the best of our knowledge, this is the first high-throughput proteomics study of S. marcescens (ATCC 13880). These novel observations provide a crucial baseline molecular profile of the S. marcescens proteome which will prove to be helpful for the future research in understanding the host-pathogen interactions during infection, elucidating the mechanism of multidrug resistance, and developing novel diagnostic markers or vaccine for the disease

Phosphotyrosine profiling of human cerebrospinal fluid

Additional file 3: Figure S1. A relative abundance of the tyrosine phosphorylated peptides in the CSF samples

Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B), and catalytic (C) subunit, with PPP2CA being the principal catalytic subunit. The full scope of PP2A substrates in cells remains to be defined. To address this, we employed dTAG proteolysis-targeting chimeras to efficiently and selectively degrade dTAG-PPP2CA in homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins with significantly increased phosphorylation upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. A vast majority of these are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in spliceosome function, cell cycle, RNA transport, and ubiquitin-mediated proteolysis. We identify a pSP/pTP motif as a predominant target for PPP2CA and confirm some of our phospho-proteomic data with immunoblotting. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.</p

Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B), and catalytic (C) subunit, with PPP2CA being the principal catalytic subunit. The full scope of PP2A substrates in cells remains to be defined. To address this, we employed dTAG proteolysis-targeting chimeras to efficiently and selectively degrade dTAG-PPP2CA in homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins with significantly increased phosphorylation upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. A vast majority of these are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in spliceosome function, cell cycle, RNA transport, and ubiquitin-mediated proteolysis. We identify a pSP/pTP motif as a predominant target for PPP2CA and confirm some of our phospho-proteomic data with immunoblotting. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.</p

Integrated stress response and decreased ECM in cultured stromal cells from keratoconus corneas

Purpose: Keratoconus (KC) is a multifactorial disease where progressive thinning and weakening of the cornea leads to loss of visual acuity. Although the underlying etiology is poorly understood, a major endpoint is a dysfunctional stromal connective tissue matrix. Using multiple individual KC corneas, we determined that matrix production by keratocytes is severely impeded due to an altered stress response program. Methods: KC and donor (DN) stromal keratocytes were cultured in low glucose serum-free medium containing insulin, selenium and transferrin. Fibronectin, collagens and proteins related to their chaperone, processing and export, matrix metalloproteinase, and stress response related proteins were investigated by immunoblotting, immunocytochemistry, hydroxyproline quantification, and gelatin zymography. Multiplexed mass spectrometry was used for global proteomic profiling of 5 individual DN and KC cell culture. Transcription of selected proteins was assayed by qPCR. Results: DN and KC cells showed comparable survival and growth. However, immunoblotting of selected ECM proteins and global proteomics showed decreased fibronectin, collagens, PCOLCE, ADAMTS2, BMP1, HSP47, other structural and cytoskeletal proteins in KC. Phosphorylated (p) eIF2α, a translation regulator and its target, ATF4 were increased in KC cultured cells and corneal sections. Conclusions: The profound decrease in structural proteins in cultured KC cells and increase in the p-eIF2α, and ATF4, suggest a stress related blockade in structural proteins not immediately needed for cell survival. Therefore, this cell culture system reveals an intrinsic aggravated stress response with consequent decrease in ECM proteins as potential pathogenic underpinnings in KC

Integrated proteomics and phosphoproteomics revealed druggable kinases in neoadjuvant chemotherapy resistant tongue cancer

Tongue squamous cell carcinoma is an aggressive oral cancer with a high incidence of metastasis and poor prognosis. Most of the oral cavity cancer patients present in clinics with locally advanced unresectable tumors. Neoadjuvant treatment is beneficial for these individuals as it reduces the tumor size aiding complete resection. However, patients develop therapy resistance to the drug regimen. In this study, we explored the differential expression of proteins and altered phosphorylation in the neoadjuvant chemotherapy resistant tongue cancer patients. We integrated the proteomic and phosphoproteomic profiles of resistant (n = 4) and sensitive cohorts (n = 4) and demonstrated the differential expression and phosphorylation of proteins in the primary tissue of the respective subject groups. We observed differential and extensive phosphorylation of keratins such as KRT10 and KRT1 between the two cohorts. Furthermore, our study revealed a kinase signature associated with neoadjuvant chemotherapy resistance. Kinases such as MAPK1, AKT1, and MAPK3 are predicted to regulate the resistance in non-responders. Pathway analysis showed enrichment of Rho GTPase signaling and hyperphosphosphorylation of proteins involved in cell motility, invasion, and drug resistance. Targeting the kinases could help with the clinical management of neoadjuvant chemotherapy-resistant tongue cancer

Phosphoproteomics of retinoblastoma:A pilot study identifies aberrant kinases

Retinoblastoma is a malignant tumour of the retina which most often occurs in children. Earlier studies on retinoblastoma have concentrated on the identification of key players in the disease and have not provided information on activated/inhibited signalling pathways. The dysregulation of protein phosphorylation in cancer provides clues about the affected signalling cascades in cancer. Phosphoproteomics is an ideal tool for the study of phosphorylation changes in proteins. Hence, global phosphoproteomics of retinoblastoma (RB) was carried out to identify signalling events associated with this cancer. Over 350 proteins showed differential phosphorylation in RB compared to control retina. Our study identified stress response proteins to be hyperphosphorylated in RB which included H2A histone family member X (H2AFX) and sirtuin 1. In particular, Ser140 of H2AFX also known as gamma-H2AX was found to be hyperphosphorylated in retinoblastoma, which indicated the activation of DNA damage response pathways. We also observed the activation of anti-apoptosis in retinoblastoma compared to control. These observations showed the activation of survival pathways in retinoblastoma. The identification of hyperphosphorylated protein kinases including Bromodomain containing 4 (BRD4), Lysine deficient protein kinase 1 (WNK1), and Cyclin-dependent kinase 1 (CDK1) in RB opens new avenues for the treatment of RB. These kinases can be considered as probable therapeutic targets for RB, as small-molecule inhibitors for some of these kinases are already in clinical trials for the treatment other cancers

5. 慢性円板状エリテマトーデスに続発せる棘細胞癌の1例について(第434(B)回千葉医学会例会 第15回千葉皮膚科臨床談話会)

Additional file 3: Table S3. Details of proteins identified in the study with reports on whether or not reported in PPD and gene ontology-based classification

Multi-omics analysis of glioblastoma and glioblastoma cell line: Molecular insights into the functional role of GPR56 in mesenchymal transition

G protein-coupled receptor 56 (GPR56/ADGRG1) is an adhesion GPCR with an essential role in brain development and cancer. Elevated expression of GPR56 was observed in the clinical specimens of Glioblastoma (GBM), a highly invasive primary brain tumor. However, we found the expression to be variable across the specimens, presumably due to the intratumor heterogeneity of GBM. Therefore, we re-examined GPR56 expression in public domain spatial gene expression data and single-cell expression data for GBM, which revealed that GPR56 expression was high in cellular tumors, infiltrating tumor cells, and proliferating cells, low in microvascular proliferation and peri-necrotic areas of the tumor, especially in hypoxic mesenchymal-like cells. To gain a better understanding of the consequences of GPR56 downregulation in tumor cells and other molecular changes associated with it, we generated a sh-RNA-mediated GPR56 knockdown in the GBM cell line U373 and performed transcriptomics, proteomics, and phospho-proteomics analysis. Our analysis revealed enrichment of gene signatures, pathways, and phosphorylation of proteins potentially associated with mesenchymal (MES) transition in the tumor and concurrent increase in cell invasion and migration behavior of the GPR56 knockdown GBM cells. Interestingly, our analysis also showed elevated expression of Transglutaminase 2 (TG2) - a known interactor of GPR56, in the knockdown cells. The inverse expression of GPR56 and TG2 was also observed in intratumoral, spatial gene expression data for GBM and in GBM cell lines cultured in vitro under hypoxic conditions. Integrating all these observations, we infer a functional link between the inverse expression of the two proteins, the hypoxic niche, and the mesenchymal status in the tumor. Hypoxia-induced downregulation of GPR56 and activation of TG2 may result in a network of molecular events that contribute to the mesenchymal transition of GBM cells, and we propose a putative model to explain this functional and regulatory relationship of the two proteins