Krol, A. | bioRxiv Science

bioRxiv2025

Exploration of chemical probes and conformational flexibility of GID4 - the substrate receptor of human CTLH E3 ligase complex

The application of targeted protein degradation (TPD) is currently constrained by the limited availability of low-molecular-weight molecules that can recruit E3 ligases other than CRBN (Cereblon) or VHL (Von Hippel-Lindau ligase). In this study, we present the structure-based drug design (SBDD) of high-affinity ligands that engage E3 ligase GID4 (Glucose-induced degradation protein 4) in biophysical and cellular experiments. Through structural studies and molecular modeling, we identified three clusters of compounds that induce distinct conformations of GID4. We characterized potential exit vectors and used the most promising ligand as a building block to prepare bifunctional degraders in the form of proteolysis-targeting chimeras (PROTACs). Although ternary complex formation was successful in vitro, degradation of BRD4 was not observed, highlighting the need for further optimization of the degraders. Finally, we theoretically investigated the likelihood of the identified GID4 conformations participating in protein-protein interactions mediated by molecular glue mechanisms. We believe the expanded ligand diversity discovered in this study may pave the way for tuning the selectivity and efficacy of interactions involving GID4 and its neosubstrates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/662521v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@19187e8org.highwire.dtl.DTLVardef@171abd1org.highwire.dtl.DTLVardef@1c75a5forg.highwire.dtl.DTLVardef@fe17fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Failed Metabolic Adaptation to Stress Underlies Pathogenesis in a Heterozygous Genetic Disorder

Disorders linked to heterozygous variants occupy a continuum in terms of the timing and severity of phenotypic emergence. An important question regarding this variability entails the effect stress has on the residual protein function. Using Darier disease (DD), caused by heterozygous variants of the SERCA2 calcium pump, as a model, we uncovered a potential connection between extrinsic stress and pathogenesis. The skin lesions characteristic of DD entail loss of intercellular adhesion and rarely appear pre-adolescence, suggesting that factors beyond heterozygosity contribute to disease pathogenesis. Testing whether age-related stressors contribute to DD, we show that DD patient-derived keratinocytes subjected to stress yield twice the reactive oxygen species of controls, accompanied by greater disruption of intercellular adhesion. Metabolic analysis of DD cells revealed perturbation of the pentose phosphate pathway (PPP), a stress response system responsible for regenerating antioxidants like glutathione. At baseline, DD cells had less free glutathione but an increase in protective glutathione-based modifications of SERCA2, a reversible form of protein oxidation. With stress, DD cells form an aberrant, heavily glutathionylated perinuclear halo consisting of keratin and the intercellular adhesion component, desmoplakin. We propose a model whereby SERCA2 heterozygosity causes mild oxidative stress that under homeostatic conditions can be buffered by glutathionylation. When stressed, the depleted glutathione store is shunted towards the desmoplakin-intermediate filament system at the expense of SERCA2, rendering it vulnerable to damage. A lesional flare, then, would represent a case of more complete SERCA2 inhibition and a novel example of how heterozygous disorders interact with stress to disrupt intercellular adhesion.

cell biology↗