ABSTRACT: Proteome profiles of isolated peripheral human neutrophils pretreated with purified Tamm-Horsfall protein and stimulated to undergo NETosis with PMA.
Project description:To elucidate changes in macrophage subpopulations in the presence of non-polymerizing Tamm-Horsfall Protein, we performed single cell RNAseq on mouse bone marrow derived macrophages following 18 hour treatment with either a non-polymerizing form of Tamm-Horsfall Protein or vehicle. After treatment, cells were harvested and dead cells removed prior to sorting on a 10X Chromium platform version 3 and generation of libraries. Sequencing was perfomred on an Illlumina NovaSeq 6000.
Project description:MOLM13 cells and patient-derived de novo AML cells with MLL1 rearrangement were treated with 100 nM of FHD-286 or 100 nM of AU15330, a dual BRG1/BRM protein degrader for 48 hours. The goal was to determine the global protein expression alterations that correlate with the cell cycle, growth inhibitory and/or lethal effects of treatment with a chromatin remodeling inhibitor, FHD-286, or a BRG1/BRM protein degrader in MLL1-rearranged AML cells.
Project description:We treated patient-derived mutant NPM1-and FLT3-ITD expressing AML cells with a chromatin remodeling inhibitor, FHD-286, at a dose of 100 nM, for 48 hours to determine FHD-286-mediated changes to the AML proteome.
Project description:We treated MLL1-rearranged AML MOLM13 cells with a chromatin remodeling inhibitor, FHD-286, at a dose of 100 nM, for different time intervals (up to 48 hours) to measure the time-dependent changes to the AML proteome
Project description:TcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. In situ NETosis of kidneys from pyelonephritis mouse model induced by TcpC-secreting wild-type CFT073 (CFT073wt) and LPS-induced in vitro NETosis in CFT073wt- or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses suggest that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion.
Project description:Label-free quantitative proteomics was employed to compare the protein content of extracellular vesicles isolated by various differential centrifugation-based approaches from expressed prostatic secretions in urine (EPS-urine) from men with prostate cancer. The developed optimized approach improved EV purity by depleting the high-abundance urine protein Tamm-Horsfall protein (THP) and other common contaminants and achieved relative enrichment of prostate cancer-associated EV-resident proteins.
Project description:NETosis, a novel cell death leads to neutrophil extracellular trap (NET) formation, is involved in both infectious and noninfectious disease. However, mechanisms underlying NETosis remain unclear. To explore the underlying molecular mechanisms and common factors associated with both NOX-dependent and NOX-independent NETosis, we conducted global proteomics and phospho-proteomics analyses of phorbol 12-myristate 13-acetate (PMA)-, ionomycin-, and monosodium urate (MSU) - induced early stage NETosis. Global proteomic analyses identified 64, 97, and 141 proteins differentially regulated in the PMA, ionomycin, and MSU comparisons with the control, respectively. Next, phospho-proteomic analyses identified: 931, 565 and 201 phosphorylation sites differentially regulated in the PMA, ionomycin, and MSU comparisons with the control, respectively. Furthermore, overlap analysis of the three comparisons identified 9 proteins and 49 phosphorylation sites derived from 41 phosphoproteins. Among the 41 differentially regulated phosphoproteins, 23 were associated with the nucleus, 5 with chromatin binding and 13 with poly(A) RNA binding according to GO annotation. Of which, DEK, Methyl-CpG-binding protein 2 (MECP2) and structure-specific recognition protein 1 (SSRP1) involved in both chromatin and poly(A) RNA binding. In conclusion, our study provides insight into molecular mechanisms of NETosis and this dataset will be benefit for further elucidation.
Project description:Type 1 diabetes (T1D) is a chronic T-cell-mediated autoimmune disease, leading to the destruction of the pancreatic insulin-producing beta cells. Little is known about the involvement of neutrophils that exert multifaceted functions like phagocytosis, degranulation, production of cytokines and the formation of neutrophil extracellular traps (NETosis), in the pathogenesis of T1D. Our aim was to gain insight into the proteome of neutrophils undergoing NETosis from patients with long-standing T1D compared to age- and sex-matched healthy controls under both resting and stimulated conditions (phorbol-myristate acetate, PMA, 100nM; ionomycin, 20µM; 3hours) by LC/MS-MS.