Project description:Most patients with triple negative breast cancer (TNBC) fail to respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression remain elusive. Here, we identify aberrant B7H3 glycosylation and found N-glycosylation of B7H3 at NXT motif sites are responsible for its protein stability and immunosuppression in TNBC tumors. Mechanistically, fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression could be physiologically significant and clinically relevant in TNBC patients. Notably, combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These suggest targeting FUT8-B7H3 axis can be a promising strategy for improving anti-tumor immune responses in TNBC patients. To obtain the direct evidence that B7H3 is N-glycosylated in TNBC cells, we analysed the peptides of purified human B7H3 protein from B7H3-WT re-expressed and B7H3-8NQ re-expressed MDA-MB-231 cell lines by Nanoscale liquid chromatography coupled to tandem MS (nano LC-MS/MS). The result showed that there were eight N-glycosylation sites (Asn positions 91, 104, 189, 215, 309, 322, 407, and 433) in B7H3-WT cells, but not in B7H3-8NQ cells, as determined by Asn to Asp conversion after PNGase F treatment. As B7H3 contains a nearly exact tandem duplication of the IgV-IgC domain, there were four pairs of N-glycosylation sites identified through identical peptide sequence, including N91 and N309, N104 and N322, N189 and N407, and N215 and N433, in each of the IgV-IgC domains. Together, the results indicate that B7H3 is exclusively N-glycosylated at these four pairs of glycosylation sites in TNBC cells.
Project description:Protein phosphorylation on Tyr residues is a key post-translational modification in mammals. In plants, recent studies have identified Tyr-specific protein phosphatase and Tyr-phosphorylated proteins in Arabidopsis by phosphoproteomic screenings, implying that plants have a Tyr phosphorylation signal pathway. However, little is known about the protein kinases (PKs) involved in Tyr phosphorylation in plants. Here, we demonstrate that Arabidopsis calcium-dependent protein kinase (CDPK/CPK)-related PKs (CRKs) have high Tyr autophosphorylation activity and that they can phosphorylate Tyr residue(s) on substrate proteins in Arabidopsis. In order to identify PKs for Tyr phosphorylation, we examined the autophosphorylation activity of 759 PKs using an Arabidopsis protein array based on a wheat cell-free system. In total, we identified 38 PKs with Tyr autophosphorylation activity. The CRK family was a major protein family identified. A cell-free substrate screening revealed that these CRKs phosphorylate beta-tubulin (TBB) 2, TBB7 and certain transcription factors (TFs) such as ethylene response factor 13 (ERF13). All five CRKs tested showed Tyr auto/trans-phosphorylation activity and especially two CRKs, CRK2 and CRK3, showed a high ERF13 Tyr phosphorylation activity. A cell-based transient expression assay revealed that Tyr16/207 sites in ERF13 were phosphorylated by CRK3 and that Tyr phosphorylation of endogenous TBBs occurs in CRK2 overexpressing cells. Furthermore, crk2 and crk3 mutants showed a decrease in the Tyr phosphorylation level of TBBs. These results suggest that CRKs have Tyr kinase activity, and these might be one of the major PKs responsible for protein Tyr-phosphorylation in Arabidopsis plants.
Project description:Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant C. annuum PI (CanPI) at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression upon CanPI feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h).
Project description:We used spontaneously hypertensive rats (SHRs) as an animal model of hypertensive heart disease and treated them with allisartan orally. We continuously monitored the rats' blood pressure levels, measured their body and heart weights, and evaluated their cardiac structure and function using echocardiography. We performed proteome analysis using the tandem mass tag (TMT) technology.
Project description:Here we used non-canonical amino acid labeling and mass-spectrometry based proteomics to perform a quantitative comparison of nascent proteins in fibroblasts from sporadic and G2019S mutation PD patients compared to healthy individuals. We found that several proteins were under-represented among nascent proteins in cells from PD patients. Among these were regulators endo-lysosomal sorting, mRNA processing and translation itself.
Project description:To understand nutrient dynamics during embryonic and cotyledonary photoheterotrophic transition to mature and germinating autotrophic seeds, TiO2-based phosphoproteomics study in three sequential seed developmental phases in chickpea was performed.
Project description:Background: Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Methods: Using an unbiased, discovery and quantitative proteomic approach, we discovered the differentially expressed proteins binding to Na, K-ATPase α1 between LPS-A549 cells and Control-A549 cells. These Na, K-ATPase α1 interacting proteins were screened by co-immunoprecipitation (Co-IP) technology. Among them, some of the differentially expressed proteins with significant performance were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network was constructed by the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Several differentially expressed proteins were validated by Western blot. Results: Of identified 1598 proteins, 89 were differentially expressed proteins between LPS-A549 cells and Control-A549 cells. Intriguingly, protein-protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. The related GO and KEGG analysis found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Conclusions: In conclusion, our proteomic approach revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS.
Project description:In this project, using discovery-based mass spectrometry approach and adopting data-dependent LC-MS/MS, we detected and identified modified peptides in complex clinical plasma samples and developed first-level biomarker verification strategy. Further, using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) based targeted approach which virtually quantifies irreversible oxidation at any targeted cysteine, we quantified cysteine tri-oxidation in HSA in diabetes patients. Site-specific tri-oxidized HSA could be a potential biomarker of diabetes and provide directive of disease mechanism.
Project description:In this study, we evaluated the common proteomic profile, as well as, the exclusively deregulated proteins in ON cells from healthy controls cannabis users (HC/c), SCZ patients non-cannabis users (SCZ/nc) and SCZ patients cannabis users (SCZ/c) as compared to healthy controls non-cannabis users (HC/nc). Moreover, we investigated quantitative and functional differences between HC/c and SCZ, and we characterized the distinct effect of cannabis in SCZ comparing SCZ/nc and SCZ/c.
Project description:Molecular profiling of breast cancer has achieved great depth in defining the mutational landscapes and molecular profiles of primary tumors, though little is still known regarding cancer evolution into a recurrence. Proteogenomic workflows are particularly useful in defining the multi-layered biology of complex diseases by combining genomic, transcriptomic, and proteomic technologies so to inform not only on mutational processes, but also on their repercussion at the effector level, proteins. We employed our recently developed proteogenomic workflow to analyze a cohort of 27 primary breast cancers and their matched loco-regional recurrences by whole genome sequencing, RNA sequencing, and mass spectrometry.