Fernandez-Ballester, G. | bioRxiv Science

bioRxiv2025

Druggability of Phospholipase C-β Isoforms with Small Peptides Patterned after the Autoinhibitory XY Linker

The phospholipase C-{beta} (PLC{beta}) signaling pathway plays a pivotal role in peripheral nociception, particularly during inflammation and pain transduction. Despite their validation as important therapeutic targets, PLC{beta} isoforms are yet undruggable due to the difficulties to identify potent and selective modulators. Here, we addressed this question and used the autoinhibitory XY linker present in these enzymes as a source of peptide inhibitors of PLC{beta} activity. We report that peptides patterned after this motif inhibited PIP2 hydrolysis and the consequent calcium release from endoplasmic reticulum. In primary nociceptor cultures, active peptides notably attenuated bradykinin-induced electrogenesis and TRPV1 sensitization, thus reducing nociceptor hyperexcitability. Noteworthy, intraplantar administration of a lead peptide prevented inflammation and hypersensitivity in a mouse model of inflammatory pain. Collectively, our findings indicate that peptides patterned after the autoinhibitory XY linker act as selective PLC{beta} inhibitors with in vivo anti-inflammatory and antinociceptive activity, providing pharmacological tools for this enzyme family. SIGNIFICANCEPhospholipases C (PLC) are intracellular signaling proteins, with PLC{beta} isoforms crucial in somatosensory neuron signaling. These enzymes interact with G-protein coupled receptors for pro-inflammatory and algesic agents, sensitizing nociceptors by increasing their excitability. Despite their importance, selective PLC{beta} modulators remain limited; U73122 is widely used, though it lacks specificity and has off-target effects. Here, we introduce peptide inhibitors based on the XY autoinhibitory motif that selectively block PLC{beta} activity, reduce bradykinin-induced neuronal responses and TRPV1 sensitization, and do not affect other PLC isoforms. In a murine inflammatory pain model, local administration of our lead peptide showed both anti-inflammatory and antinociceptive effects, highlighting its therapeutic potential. This approach expands the toolkit of PLC-isoform selective modulators for drug development.

molecular biology↗

bioRxiv2021

Conformational buffering underlies functional selection in intrinsically disordered protein regions

Many disordered proteins conserve essential functions in the face of extensive sequence variation. This makes it challenging to identify the forces responsible for functional selection. Viruses are robust model systems to investigate functional selection and they take advantage of protein disorder to acquire novel traits. Here, we combine structural and computational biophysics with evolutionary analysis to determine the molecular basis for functional selection in the intrinsically disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, triggering early S-phase entry and disrupting normal cellular proliferation. We show that the ability to outcompete host factors depends on the picomolar binding affinity of E1A for Rb, which is driven by two binding motifs tethered by a hypervariable disordered linker. Binding affinity is determined by the spatial dimensions of the linker, which constrain the relative position of the two binding motifs. Despite substantial sequence variation across evolution, the linker dimensions are finely optimized through compensatory changes in amino acid sequence and sequence length, leading to conserved linker dimensions and maximal affinity. We refer to the mechanism that conserves spatial dimensions despite large-scale variations in sequence as conformational buffering. Conformational buffering explains how variable disordered proteins encode functions and could be a general mechanism for functional selection within disordered protein regions.

biophysics↗