Biology subjects

Carmona, E. M.

Publications and source records attributed to Carmona, E. M..

5 recordsLinked to original sources

bioRxiv2026

Measuring protein conformational changes with genetically encoded bipyridylalanine and transition-metal ion FRET

Transition-metal ion Forster resonance energy transfer (tmFRET) measures short-range distances and conformational changes in proteins, but its distance sensitivity depends on the donor-acceptor pair. Here, we establish the metal-chelating noncanonical amino acid bipyridylalanine (bpyA) as a flexible, genetically encoded acceptor platform whose Forster distance (R0) can be tuned without changing protein labeling. Cu2+, Cu2+ complexed with nitrilotriacetic acid (NTA), and Fe2+ form distinct acceptors at the same bpyA site, allowing distance sensitivity to be adjusted by changing the solution conditions. We incorporated bpyA at two sites in maltose binding protein (MBP) and labeled nearby engineered cysteines with the donor fluorophore BODIPY. The resulting [Cu(bpyA)]2+, [CuNTA(bpyA)]2+, and [Fe(bpyA)]2+ acceptors provided R0 values ranging from 13.9 to 21.2 [A]. Analysis of time-resolved tmFRET data recovered donor-acceptor distance distributions and maltose-dependent changes consistent with structural predictions. At the 237/295 site pair, both copper and iron acceptors resolved the maltose-dependent decrease in distance. At the 309/322 site pair, [Cu(bpyA)]2+ did not appreciably resolve the distance increase, whereas adding NTA shifted R0 toward shorter distances and enabled detection of an increase from 12.1 to 14.4 [A]. These results demonstrate that a single bpyA-labeled protein preparation supports multiple tmFRET acceptors, providing a cysteine-independent acceptor-labeling strategy with adjustable distance sensitivity.

biophysics↗

bioRxiv2026

Myeloperoxidase promotes fibrosis by inhibiting cathepsin K to bias the lung toward ECM accumulation

Pulmonary fibrosis (PF) involves excessive collagen accumulation, yet mechanisms shifting the balance of synthesis and degradation toward net deposition remain unclear. Myeloperoxidase (MPO) inversely correlates with survival in PF. Using the bleomycin model, we found MPO knockout (MPOko) mice were protected from fibrosis, and pharmacological MPO inhibition after peak inflammation (day 7) recapitulated this protection. MPO persisted in lung tissue 21 days post-injury despite neutrophil efflux, linking acute inflammation to sustained remodeling. Mechanistically, we identified that MPO inhibits Cathepsin K (CatK), a potent collagenolytic enzyme involved in fibrosis resolution. Notably, CatK gene expression (CTSK) is elevated in PF, suggesting post-translational inhibition of CatK. MPOko and inhibitor-treated mice exhibited elevated CatK activity after bleomycin; exogenous addition of pathophysiologic concentrations of MPO reduced CatK activity in mouse precision-cut lung slices and human fibroblasts. Biochemically, MPO reduced CatK activity to 33% of control. In two distinct cohorts of PF patients, we observed significantly increased MPO protein levels in platelet poor plasma and in lung tissue. In PF patients, 62% had MPO levels in platelet poor plasma exceeding healthy controls, while lung tissue from other PF patients showed significantly elevated MPO staining. Plasma levels were inversely correlated with decreased survival, FVC, and DLCO. These findings establish MPO as a post-translational inhibitor of CatK-mediated collagenolysis, revealing a mechanism linking acute inflammation to sustained fibrosis and suggest a patient subpopulation that may benefit from MPO-targeted therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/713467v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@90789forg.highwire.dtl.DTLVardef@df248dorg.highwire.dtl.DTLVardef@1d8dfe6org.highwire.dtl.DTLVardef@1538145_HPS_FORMAT_FIGEXP M_FIG C_FIG Myeloperoxidase persists in lung tissue after injury and inhibits cathepsin K activity, impairing collagen degradation and promoting extracellular matrix accumulation during pulmonary fibrosis.

pathology↗

bioRxiv2025

Interrogating the structure and function of the human voltage-gated proton channel (hHv1) with a fluorescence noncanonical amino acid.

The human voltage-gated proton channel (hHv1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hHv1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands such as Zn2+. Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hHv1, we developed a Forster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hHv1 purified from E. coli. We used genetic code expansion (GCE) to generate a library of 14 full-length hHv1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hHv1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hHv1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn2+ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.

biophysics↗

bioRxiv2021

Gene Expression in Lung Epithelial Cells Following Interaction with Pneumocystis carinii and its Specific Life Forms Yields Insights into Host Gene Responses to Infection

Pneumocystis spp. interacts with epithelial cells in the alveolar spaces of the lung. It is thought that the binding of Pneumocystis to host cell epithelium is needed for life cycle completion and proliferation. The effect of this interaction on lung epithelial cells have previously shown that the trophic form of this organism greatly inhibits p34cdc2 activity, a serine/threonine kinase required for transition from G2 to M phase in the cell cycle. To gain further insight into the host response during Pneumocystis pneumonia (PCP), we used microarray technology to profile epithelial cell (A549) gene expression patterns following Pneumocystis carinii interaction. Furthermore, we isolated separate populations of cyst and trophic forms of P. carinii, which were then applied to the lung epithelial cells. Differential expression of genes involved in various cellular functions dependent on the specific P. carinii life form in contact with the A549 cell were identified. The reliability of our data was further confirmed by Northern blot analysis on a number of selected up or down regulated transcripts. The transcriptional response to P. carinii was dominated by cytokines, apoptotic, and anti-apoptotic related genes. These results reveal several previously unknown effects of P. carinii on the lung epithelial cell and provide insight into the complex interactions of host and pathogen.

microbiology↗

bioRxiv2021

Preclinical and Toxicology Studies of BRD5529, a Selective Inhibitor of CARD9

BackgroundExuberant inflammation during Pneumocystis pneumonia leads to lung injury. CARD9 is a central mediator of inflammatory signaling mediated by C-type lectin receptors. CARD9 inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis {beta}-glucan-induced proinflammatory signaling and downstream TNF-alpha production, suggesting its viability as a candidate for preliminary drug testing as an anti- inflammatory agent in the rodent Pneumocystis pneumonia model (PCP). MethodsTo assess for potential toxicity, mice were injected intraperitoneally (IP) daily either with vehicle or BRD5529 at 0.1 mg/kg or 1.0 mg/kg for two weeks. Mouse weights were taken daily. At day 14, mice were euthanized, weighed, and analyzed by flexiVent for lung stiffness. Lungs, liver, and kidney were then harvested for H&E staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines via ELISA and extracellular matrix generation via quantitative PCR (q-PCR). Blood collection postmortem was performed for blood chemistry analysis. ResultsBRD5529 at both doses of IP administration resulted in no significant changes in daily or final weight gain. Analysis of lung stiffness by flexiVent showed no significant differences between the control or treated groups. Furthermore, ELISA results for proinflammatory IL-1 Beta, IL-6, and TNF-alpha showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts collagen type I, alpha 1 (Col1a1) and fibronectin (Fn) were statically similar as well in the treated and control groups. Examination and pathology scoring of H&E slides from lung, liver, and kidney from the each of the mice in all groups and subsequent pathology scoring showed no significant change. Blood chemistry analysis revealed similar, non-significant patterns. ConclusionsBRD5529 in our initial general safety and toxicology assessments displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy.

pharmacology and toxicology↗