Biology subjects

Glasser, E.

Publications and source records attributed to Glasser, E..

4 recordsLinked to original sources

bioRxiv2026

Structural basis for direct NGF/TrkA blockade by an analgesic antibody

The NGF/TrkA signaling axis is a central mediator of inflammatory and chronic pain, where injury-induced NGF binds and activates TrkA on nociceptive neurons to drive peripheral sensitization and persistent pain states. Despite its therapeutic promise, targeting this pathway is limited by adverse effects of systemic NGF sequestration such as rapidly progressive osteoarthritis and poor isoform selectivity of Trk kinase inhibitors leading to off-target neurological effects. Targeting the TrkA extracellular domain (TrkAECD) offers a pathway to achieve high isoform selectivity while avoiding these complications. However, the precise structural basis for selective TrkA neutralization remains poorly understood. Monoclonal antibody (mAb) 42F5-15 inhibits TrkA-mediated signaling and increases pain threshold. Here, we report the high-resolution (2.60 [A]) cryo-EM structure of the TrkAECD in complex with the Fab region of the TrkA-neutralizing mAb 42F5-15. Structural analysis reveals that the antibody epitope overlaps the NGF-binding interface, consistent with orthosteric inhibition and distinct from previously proposed allosteric mechanisms. The epitope includes residues conserved in TrkA but divergent in TrkB and TrkC, providing a structural basis for receptor isoform selectivity. Furthermore, we demonstrate in vivo that the mAb 42F5-15 potently mitigates mechanical allodynia and nociceptive sensitization. These findings establish a structural framework for the development of selective extracellular TrkA-targeted therapies for safer, non-opioid chronic pain management.

neuroscience↗

bioRxiv2022

A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing

In the early stages of spliceosome assembly, the 3 splice site is recognized by sequential complexes of U2AF2 with SF1 followed by the SF3B1 subunit of the U2 small nuclear ribonucleoprotein particle. The U2AF2 - SF1 interface comprises a U2AF homology motif (UHM) of U2AF2 and a well-characterized U2AF ligand motif (ULM)/coiled coil region of SF1. However, the structure of the U2AF2 - SF3B1 interface and its importance for pre-mRNA splicing is unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8 [A] resolution. The trajectory of the SF3B1 ULM across the U2AF2 UHM surface differed from prior UHM/ULM structures. This distinctive structure is expected to modulate the orientations of the fulllength proteins. Using isothermal titration calorimetry, we established similar binding affinities of a minimal U2AF2 UHM - SF3B1 ULM complex and a nearly full-length U2AF2 protein binding the N-terminal SF3B1 region, with or without an auxiliary SF3B6 subunit. We showed that key residues at the U2AF2 UHM - SF3B1 ULM interface are required for high affinity association and co-immunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2 - SF3B1 interface altered splicing of representative human transcripts. Further analysis of these transcripts and genome-wide data sets indicated that the subset of splice sites co-regulated by U2AF2 and SF3B1 are largely distinct from those co-regulated by U2AF2 and SF1. Altogether, these findings support distinct structural and functional roles for the sequential SF1 and SF3B1 complexes with U2AF2 during the pre-mRNA splicing process.

biochemistry↗

bioRxiv2021

Pre-mRNA Splicing Factor U2AF2 Recognizes Distinct Conformations of Nucleotide Variants at the Center of the pre-mRNA Splice Site Signal

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

biophysics↗

bioRxiv2019

A Splice Site-Sensing Conformational Switch in U2AF2 is Modulated by U2AF1 and its Recurrent Myelodysplasia-Associated Mutation

An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3' splice sites. Among myelodysplastic syndromes (MDS), U2AF1 frequently acquires an S34F-encoding mutation. The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Forster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

biochemistry↗