Bunnmccaffrey1936

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that cause severe infections in immunocompromised individuals and cystic fibrosis (CF) patients. Both P. aeruginosa and S. aureus require iron to infect the mammalian host. To obtain iron, these pathogens may rely on siderophore mediated ferric [Fe(III)] iron uptake, ferrous [Fe(II)] iron uptake, or heme uptake at different points during infection. The preferred iron source depends on environmental conditions, including the presence of iron-sequestering host defense proteins. Here, we investigate how the presence of heme, a highly relevant iron source during infection, affects bacterial responses to iron withholding by the innate immune protein calprotectin (CP). Prior work has shown that P. aeruginosa is starved of iron in the presence of CP. We report that P. aeruginosa upregulates expression of heme uptake machinery in response to CP. Furthermore, we show that heme protects P. aeruginosa from CP-mediated inhibition of iron uptake and induction of iron starvation responses. We extend our study to a second bacterial pathogen, S. aureus, and demonstrate that CP also inhibits iron uptake and induces iron starvation responses by this pathogen. Similarly to P. aeruginosa, we show that heme protects S. aureus from CP-mediated inhibition of iron uptake and iron starvation responses. These findings expand our understanding of microbial responses to iron sequestration by CP and highlight the importance of heme utilization for bacterial adaptation to host iron withholding strategies.Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturinprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.Meningiomas (MN) arise from the arachnoid/meningeal layer and are non-responsive to chemotherapies, with ~50-60% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 signaling, leading to clinical trials for NF2 and meningioma. Recently our 'omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in both NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly NRG1/neuregulin 1, and confirm increased NRG1 secretion and activation of ERBB3 receptor tyrosine kinase to which NRG1 binds. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2 and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated basal mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a PDK1-dependent signaling mechanism independent of NRG1-ERBB3, but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of another receptor tyrosine kinase, IGF1R Drug treatment co-targeting mTORC1/2 and IGF1R/IR in NF2-null cells attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion of NRG1 and activation of ERBB3. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/IR, suggesting that co-targeting these pathways may prove effective for treatment of NF2-deficient meningioma.We address the role of enzyme conformational dynamics in specificity for a high-fidelity DNA polymerase responsible for genome replication. We present the complete characterization of the conformational dynamics during the correct nucleotide incorporation forward and reverse reactions using stopped-flow and rapid-quench methods with a T7 DNA polymerase variant containing a fluorescent unnatural amino acid, (7-hydroxy-4-coumarin-yl) ethylglycine, which provides a signal for enzyme conformational changes. We show that the forward conformational change (> 6000 s-1) is much faster than chemistry (300 s-1) while the enzyme opening to allow release of bound nucleotide (1.7 s-1) is much slower than chemistry. find more These parameters show that the conformational change selects a correct nucleotide for incorporation through an induced-fit mechanism. We also measured conformational changes occurring after chemistry and during pyrophosphorolysis, providing new insights into processive polymerization. Pyrophosphorolysis follows a conformational selection mechanism as the pyrophosphate binds to a rare pre-translocation state of the enzyme-DNA complex. Global data fitting was achieved by including experiments in the forward and reverse directions to correlate conformational changes with chemical reaction steps. This analysis provided well constrained values for nine rate constants to establish a complete free energy profile including the rates of DNA translocation during processive synthesis. Translocation does not follow Brownian ratchet or power stroke models invoking nucleotide binding as the driving force. Rather, translocation is rapid and thermodynamically favorable after enzyme opening and pyrophosphate release, and it appears to limit the rate of processive synthesis at 4° C.