Limited proteolysis of neutrophil granule proteins by the bacterial protease RgpB 2 depletes neutrophil antimicrobial capacity
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PROVIDER: PRJNA1170006 | ENA |
REPOSITORIES: ENA
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Project description:Neutrophils are highly abundant in the gingival tissues where they play an essential role in immune homeostasis by preventing microbial invasion. Here, we show that the oral periodontal pathogen Porphyromonas gingivalis utilizes its cysteine proteases (gingipains) to disengage phagosomal antimicrobial capacity without inducing neutrophil apoptosis. Arginine gingipains are a sub-family of trypsin-like proteases produced by P. gingivalis that cleave several host proteins at arginine residues. We found that RgpB action is not limited to the extracellular or plasma membrane-associated host proteins but can proteolytically modify several intracellular proteins in neutrophils. We used 2D-DIGE coupled with mass spectrometry to identify neutrophil proteins susceptible to RgpB-mediated cleavage and determined the impact of these modifications on neutrophil antimicrobial capacity and inflammatory responses. RgpB cleaved several cytoskeletal and cytoplasmic proteins, including metabolic enzymes and antimicrobial proteins such as neutrophil elastase, myeloperoxidase, and proteinase 3 within neutrophil granules. RgpB also directly primed neutrophils, increasing integrin expression and inducing broad transcriptional changes, consistent with proinflammatory reprogramming. However, despite their primed status and augmented inflammatory capacity, RgpB-treated neutrophils were conducive to intracellular bacterial survival due to the reduced activity of granule proteins and oxidative burst. Thus, our data show a previously unknown role for P. gingivalis proteases in the systematic inactivation of neutrophil microbicidal capacity via proteolysis of intracellular proteins.
2024-10-08 | GSE279027 | GEO
Project description:Though limited proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during mammalian differentiation, however the specific genomic sites targeted for H3NT proteolysis and their functional significance of H3NT cleavage remain unknown.We used genome wide Chip-seq (ChIPac-Seq) approaches to an established cell model of osteoclast differentiation. We discovered that H3NT proteolysis is selectively targeted near transcription start sites of a small group of genes and that most of these H3NT-cleaved genes are epigenetically regulated during osteoclastogenesis.We also discovered that the principal H3NT protease of osteoclastogenesis is matrix metalloproteinase 9 (MMP-9).Abrogation of H3NT proteolysis impaired osteoclastogenic gene activation concomitant with defective osteoclast differentiation. In summary our results support the necessity of MMP-9-dependent H3NT proteolysis in the epigenetic reprogramming of gene pathways required for proficient osteoclastogenesis.
2016-02-02 | GSE72957 | GEO
Project description:Though limited proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during mammalian differentiation, however the specific genomic sites targeted for H3NT proteolysis and their functional significance of H3NT cleavage remain unknown.We used genome wide RNA-seq approaches to an established cell model of osteoclast differentiation. We discovered that H3NT proteolysis is selectively targeted near transcription start sites of a small group of genes and that most of these H3NT-cleaved genes are epigenetically regulated during osteoclastogenesis.We have identified that the principal H3NT protease of osteoclastogenesis is matrix metalloproteinase 9 (MMP-9). We next studied genomewide mRNA expression in MMP9 knockout cells and its effect in the epigenetic reprogramming of gene pathways required for proficient osteoclastogenesis.
2016-02-02 | GSE72846 | GEO
Project description:Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm(3)) and small pore diameters in the membrane lead to digestion in <1 s, the low membrane thickness (170 ?m) affords control over residence times at the millisecond level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kDa) that increase the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion). With this approach, we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, Root Hair Defective 3 (RHD3) and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.
| S-EPMC3683959 | biostudies-literature
Project description:PTMs on a mycobacterial DNA-binding protein with a long, lysine-rich, intrinsically disorders region were analyzed by limited proteolysis with trypsin, unveiling a massive methylation of most lysine residues of the protein in quantitative manner, which was lacked in the recombinant version of the protein expressed in E. coli.
2024-06-14 | PXD042969 | JPOST Repository
Project description:Human neutrophil phosphopeptide study using sequential TiO2 and IMAC isolation
2016-06-30 | MSV000079871 | MassIVE
Project description:Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are beta-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.
| S-EPMC404006 | biostudies-other
Project description:The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited.
2023-04-03 | GSE228809 | GEO
Project description:We used a limited proteolysis-coupled mass spectrometry approach to pinpoint binding sites of cortisol on SARS-CoV-2 S1. Binding target identification is based on the principle that small-ligand binding alters (increases or decreases) the protease accessibility of the target protein37,38. The binding of the ligand (cortisol) to the target protein (SARS-CoV-2 S1) induces perturbations/conformational changes at the binding sites which can lead to either facilitating or preventing proteolysis by non-specific proteases (thermolysin and trypsin)37,38. We subjected SARS-CoV-2 S1 incubated with either cortisol or vehicle to proteolysis by thermolysin followed by trypsin digestion and mass spectrometry-based identification of the resultant peptides. We found that cortisol either facilitated or prevented S1 proteolytic cleavage at discrete sites. Six unique peptides were identified that were only present for SARS-CoV-2 S1 incubated with either cortisol or vehicle.
2022-06-10 | PXD032937 | Pride
Project description:The matrix metalloproteinase 9 (MMP-9) is a member of the MMP family and has been recently identified as a nuclear protease capable of clipping histone H3 N-terminal tails (H3NT). This MMP-9-dependent H3NT proteolysis is critical for establishing active state of gene transcription during osteoclast differentiation and melanoma development. However, whether H3NT cleavage by MMP-9 plays a similar role in other cellular events has not been explored. Here we dissect the functional contribution of MMP-9-dependent H3NT clipping to colonic tumorigenesis by using a combination of genome-wide transcriptome data, ChIP/ChIPac-qPCR, CRISPR/dCas9 gene-targeting system, and in vivo xenograft models. We show that MMP-9 is overexpressed in colon cancer cells and catalyzes H3NT proteolysis to drive transcriptional activation of growth stimulatory genes. Our studies using knockdown and inhibition approaches clearly indicate that MMP-9 mediates transcriptional activation and promote colonic tumorigenesis in a manner dependent on its protease activity toward H3NT. Remarkably, artificial H3NT proteolysis at target gene promoters with dCAS9-MMP-9 is sufficient for establishing their transcriptional competence in colon cancer cells, underscoring the importance of MMP-9-dependent H3NT proteolysis per se in transactivation process. Our data establish new functions and mechanisms for MMP-9 in driving oncogenic transcription program in colon cancer through H3NT proteolysis and demonstrate how this epigenetic pathway can be exploited as a potential therapeutic target for cancer treatment.
2024-07-18 | GSE242924 | GEO