ABSTRACT: Proteome profiles of isolated peripheral human neutrophils pretreated with purified Tamm-Horsfall protein and stimulated to undergo NETosis with PMA.
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: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.
2023-05-15 | PXD033599 | Pride
Project description:Understanding Complex Chromatin Dynamics of Primary Human Neutrophils During PMA Induced NETosis
Project description:Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.
Project description:In order to determine the calcineurin inhibitory effect of CABIN1 peptide, we performed RNA-sequencing in Jurkat T cells expressing negative contorl (HA-mCherry) or HA-mCherry-CABIN1 peptide. Jurkat T cells were activated by treatment 40 nM PMA and 1 μM Ionomycin for 8 hr. 0.5 μM FK506 (Tacrolimus, Tac) was pretreated for 1 hr before treatment with PMA and Ionomycin. Total RNA was extracted from these cells. Extracted RNA was used to prepare an mRNA sequencing library using TruSeq Stranded mRNA sample preparation kit. All samples were sequenced on Illumina NextSeq 500 with a 75 bp paired end read.
Project description:MSCV-GFP-IRES (IRES), MSCV-GFP-Myc-NIC1 (NIC1) and MSCV-GFP-DNMAML (DNMAML) were retrovirally transduced in to THP-1 cell (pCL-Ampho was used as packaging plasmid). GFP positive cells were sorted for selective desired cell. Then IRES, NIC1 and DNMAML overexpressing THP-1 were pretreated with PMA (5ng/ml) for 48 h before stimulation with IL-4 (20 ng/ml) for 3 h.
Project description:Multiplexed, deep-scale LC-MSMS analysis for proteome and phosphoproteome expression profile and the targeted LC-MSMS analysis for kinases in enriched kinome of the ER+ breast cancer patient-derived xenograft tumors.