Biology subjects

Bunnett, N. W.

Publications and source records attributed to Bunnett, N. W..

6 recordsLinked to original sources

bioRxiv2025

Probing the activity of cysteine cathepsins in inflammatory bowel diseases

Cathepsin S is a cysteine protease that has been implicated in inflammatory bowel diseases (IBD) for its ability to promote visceral pain. Given its pro-inflammatory roles, we hypothesized that cathepsin S would drive other symptoms associated with IBD. Using activity-based probes, we investigated cysteine cathepsin activation in human and murine colitis. We observed a significant increase in fecal cathepsin S in patients with ulcerative colitis compared to healthy controls, while cathepsin S in mucosal biopsies was unchanged. Mice with experimental colitis exhibited a modest increase in mucosal activity of both cathepsin S and X compared to naive mice. Luminal secretion of cathepsin S was dramatically increased upon colitis induction, although differences between mouse colonies were observed. To investigate the contribution of cathepsin S and cathepsin X to colitis, we induced colitis in cathepsin-deficient mice. Cathepsin X-deficient mice exhibited no clear differences in disease indicators compared to wild-type mice. While cathepsin S-deficient mice exhibited less rectal bleeding, less splenomegaly and marginally improved histological scores, weight loss, diarrhea, colon shortening, and myeloperoxidase activity were not significantly different from wild-type mice. To determine whether pharmacologic inhibition of cathepsin S activity would ameliorate symptoms of colitis, a reversible inhibitor LY3000328 was administered to mice at the initiation of colitis. LY3000328 provoked a clear upregulation of cathepsin S and L activity in the mucosa, most likely through a compensatory mechanism. This increase in protease activity was associated with exacerbated histological scores and splenomegaly. Collectively, these results suggest that cathepsin S, but not cathepsin X, may contribute to some of the symptoms of experimental colitis. While cathepsin S has potential to be a therapeutic target in colitis, improved strategies to sustain its inhibition are required in future.

pathology↗

bioRxiv2025

Designed NGF mimetics with reduced nociceptive signatures in neurons

The clinical use of Nerve Growth Factor (NGF) for neuronal regeneration has been hampered by pain sensitization side effects. NGF signals through the receptor tyrosine kinase TrkA and the co-receptor p75NTR; pain sensitization is thought to involve p75NTR. We sought to overcome this limitation by de novo design of a TrkA agonist that does not bind p75NTR. We designed homodimeric TrkA engaging constructs that dimerize TrkA subunits in a variety of geometries, and identified those eliciting the strongest signaling. The resulting designed agonists are able to stimulate transdifferentiated neurons and neuroblastoma cell lines, leading to neurite outgrowth and neuronal differentiation, with considerably reduced transcription of inflammation and pain related genes. These agonists are promising candidates for promoting neuronal regeneration without adverse side effects. HighlightsO_LIDe novo designed TrkA agonists activate MAPK and PI3K-AKT signaling C_LIO_LIRigid fusions allow for highly tunable signaling signatures C_LIO_LITrkA agonists lead to neurite outgrowth in neuroblastoma cells comparable to retinoic acid C_LIO_LIModulation of the TrkA pathway without co-stimulating p75NTR leads to a downregulation of inflammatory and nociceptive signature in neurons. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/648806v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c48066org.highwire.dtl.DTLVardef@c9c5a7org.highwire.dtl.DTLVardef@cf8ebdorg.highwire.dtl.DTLVardef@a4345a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Identification of a secreted protease from Bacteroides fragilis that induces intestinal pain and inflammation by cleavage of PAR2

Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR2-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

microbiology↗

bioRxiv2024

Nanomedicines targeting signaling of protease-activated receptor 2 in organelles provide sustained analgesia

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of GPCR signaling in organelles are uncertain due to the challenges of measuring organelle-specific signals and of selectively antagonizing receptors in intracellular compartments. Herein, genetically-encoded biosensors targeted to subcellular compartments were used to analyze organelle-specific signaling of protease-activated receptor 2 (PAR2); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to selectively antagonize intracellular PAR2 signaling of pain. PAR2 agonists evoked sustained activation of PAR2, Gq and {beta}-arrestin-1 in early, late and recycling endosomes and the cis- and trans-Golgi apparatus, and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with organelle-targeted biosensors. Dendrimer and core-shell polymeric NPs accumulated in early and late endosomes of HEK293 cells, colonic epithelial cells and nociceptors, detected by confocal imaging of fluorescent NPs. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR2 antagonist. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR2, Gq and {beta}-arrestin-1 activation in endosomes and the Golgi apparatus and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs targeted neurons, sites of PAR2 expression. Both NP-AZ3451 formulations, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. Thus, organelle-specific PAR2 signals in colonocytes and nociceptors mediate pain. Antagonism of PAR2 in organelles, rather than at the plasma membrane, provides effective pain relief. Significance StatementOnce activated at the cell surface, many GPCRs internalize and continue to signal. The mechanisms and physiological relevance of intracellular GPCR signaling are uncertain. By using organelle-targeted biosensors, we detected sustained activation of the GPCR, PAR2, and its effectors in early, late and recycling endosomes, the cis- and trans-Golgi apparatus, and the cytosol and nucleus. NPs that delivered AZ3451, a PAR2 antagonist, to endosomes disrupted these intracellular signals, whereas unencapsulated AZ3451 was minimally effective. After intracolonic administration to mice, NPs accumulated in colonocytes and neurons. NP-encapsulated AZ3451, but not unencapsulated AZ3451, reversed pain in preclinical models of inflammatory bowel disease. Thus, intracellular PAR2 signaling mediates pain and antagonism of intracellular rather than plasma membrane PAR2 provides effective therapy.

physiology↗

bioRxiv2024

Targeting the Schwann Cell EP2/cAMP Nanodomain to Block Pain but not Inflammation

Analgesia by non-steroidal anti-inflammatory drugs (NSAIDs) is ascribed to inhibition of prostaglandin (PG) biosynthesis and ensuing inflammation. However, NSAIDs have life-threatening side effects, and inhibition of inflammation delays pain resolution. Decoupling the mechanisms underlying PG-evoked pain vs. protective inflammation would facilitate pain treatment. Herein, we reveal that selective silencing of the PGE2 EP2 receptor in Schwann cells via an adeno-associated viral vector abrogates the indomethacin-sensitive component of pain-like responses in mice elicited by inflammatory stimuli without affecting inflammation. In human Schwann cells and in mice, EP2 activation and optogenetic stimulation of adenylyl cyclase evokes a plasma membrane-compartmentalized cyclic adenosine monophosphate (cAMP) signal that, via A-kinase anchor protein-associated protein kinase A, sustains inflammatory pain-like responses, but does not delay their resolution. Thus, an unforeseen and druggable EP2 receptor in Schwann cells, via specific cAMP nanodomains, encodes PG-mediated persistent inflammatory pain but not protective inflammation.

pharmacology and toxicology↗

bioRxiv2023

Neuropilin-1 is a co-receptor for NGF and TrkA-evoked pain

Nerve growth factor (NGF) monoclonal antibodies inhibit chronic pain yet, failed to gain approval due to worsened joint damage in osteoarthritis patients. We report that neuropilin-1 (NRP1) is a co-receptor for NGF and tropomyosin-related kinase A (TrkA) pain signaling. NRP1 is coexpressed with TrkA in human and mouse nociceptors. NRP1 inhibitors suppress NGF-stimulated excitation of human and mouse nociceptors and NGF-evoked nociception in mice. NRP1 knockdown inhibits NGF/TrkA signaling, whereas NRP1 overexpression enhances signaling. NGF binds NRP1 with high affinity and interacts with and chaperones TrkA from the biosynthetic pathway to the plasma membrane and endosomes, enhancing TrkA signaling. Molecular modeling suggests that C-terminal R/KXXR/K NGF motif interacts with extracellular "b" NRP1 domain within a plasma membrane NGF/TrkA/NRP1 of 2:2:2 stoichiometry. G Alpha Interacting Protein C-terminus 1 (GIPC1) scaffolds NRP1 and TrkA to myosin VI and colocalizes in nociceptors with NRP1/TrkA. GIPC1 knockdown abrogates NGF-evoked excitation of nociceptors and pain-like behavior. NRP1 is a nociceptor-enriched co-receptor that facilitates NGF/TrkA pain signaling. NRP binds NGF and chaperones TrkA to the plasma membrane and signaling endosomes via the GIPC1 adaptor. NRP1 and GIPC1 antagonism in nociceptors offers a long-awaited non-opioid alternative to systemic antibody NGF sequestration for the treatment of chronic pain. SummaryNeuropilin-1 and G Alpha Interacting Protein C-terminus 1 are necessary for nerve growth factor-evoked pain and are non-opioid therapeutic targets for chronic pain.

neuroscience↗