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Abstract: Aster proteins (Aster-A, -B, and -C) are crucial for transporting cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER). Asters are expressed in a cell type–specific manner across tissues. Their global disruption leads to varied physiological outcomes given the diverse roles of cholesterol throughout the body. We previously identified sterol analogs, such AI-3d, that inhibit all three Aster proteins. However, their utility is limited by toxicity and off-target effects. Here, we report the development of nonsteroidal Aster inhibitors that are active in cells and in vivo, using binding-guided design to generate compounds with isoform-selective affinities. We found that YKJ-124 is a low-toxicity, Aster-A–preferring inhibitor that elevates PM-accessible cholesterol in primary T cells and potentiates store-operated Ca2+ entry in Th17 cells, phenocopying Aster-A deficiency. YKJ-300 and YKJ-305 selectively target Aster-C; cocrystal structures and point mutation studies reveal a Ser477-dependent hydrogen bond (Gly in Aster-A/B) that underlies this specificity. We also explored the in vivo consequences of pharmacologic Aster-C inhibition. YKJ-305 treatment of mice blunted fasting-induced hepatic cholesterol transport and cholesterol ester formation, accompanied by compensatory activation of the SREBP2 pathway. Last, we also identify broader-spectrum inhibitors (YKJ-86) and dual Aster-A/C inhibitors (YKJ-262) that drive PM cholesterol accumulation in fibroblasts and human intestinal enteroids. Together, these chemical probes enable isoform-resolved manipulation of Aster-dependent cholesterol trafficking and provide a foundation for developing Aster-targeted therapies for cholesterol dysregulation.

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Abstract: The Fc region of immunoglobulin E (IgE-Fc) is an important therapeutic target due to the antibody’s critical role in allergic disorders through interactions with high-affinity (FcεRI) and low-affinity (CD23) receptors. IgE-Fc is known to be conformationally flexible, undergoing large-scale structural changes that modulate receptor binding and downstream biological functions. The mechanistic basis for how ligands induce these allosteric changes is not fully understood. In this study, we investigate structure–function relationships of IgE by characterizing four closely related anti-IgE-Fc Fab fragments with highly conserved sequences that recognize a shared epitope at the base of the Cε2 domain. Despite their sequence similarity, these Fabs exhibit markedly different binding characteristics, stoichiometries, and functional activities. Furthermore, the crystal structures of these four Fab complexes reveal that the bound IgE-Fc adopts a wide range of conformations. These structural differences directly dictate the functional outcomes, ranging from conformations that facilitate FcεRI binding to those that prevent or destabilize receptor interaction. Our findings demonstrate that highly similar antibodies binding to the same epitope on a protein that exhibits significant conformational plasticity can elicit distinct and divergent functional outcomes. This work challenges the conventional antibody discovery paradigm, i.e., “same epitope, same functional outcome,” and highlights the importance of accounting for both the target’s structural flexibility and the resulting functional diversity within antibody selection strategies. This is especially critical when targeting dynamic proteins where allosteric mechanisms are fundamental to function.

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Abstract: UFMylation is an important biological process where proteins are post-translationally modified via the covalent attachment of the ubiquitin-like modifier (UBL), ubiquitin-fold modifier-1 (UFM1). UFMylation is analogous to ubiquitination, occurring via the transfer of UFM1 across E1-, E2-, and E3-like enzymes, represented by ubiquitin-like modifier-activating enzyme 5 (UBA5), ubiquitin-fold modifier conjugating enzyme 1 (UFC1), and UFM1-specific ligase 1 (UFL1), respectively. Recent work has shown that dysregulation of UFMylation is associated with several diseases, sparking interest in characterizing its enzymes as potential drug targets. To date, only inhibitors targeting UBA5 have been identified, but no such ligands exist for UFC1. In this study, we present the structure of UFC1 in complex with the sulfonic acid CAPS, which revealed a novel ligand-binding pocket in UFC1. Using biophysical assays, coupled with X-ray crystallographic studies of UFC1 variants, we show that binding of CAPS to UFC1 appears pH-dependent and is enhanced by Tyr42. Further, our TSA data show that other sulfa- and sulfonate-based compounds induce dose-dependent destabilization of UFC1, consistent with weak but direct interactions with the enzyme. Lastly, using a UFMylation assay, we show that CAPS, along with Tyr42, may have a limited influence on UFM1 transfer to UFC1 and, consequently, downstream UFMylation of protein substrates. Nevertheless, our data indicate that the CAPS-binding pocket may serve as a design scaffold for the development of UFC1 modulators. With UFC1 emerging as a drug target, our study provides a possible avenue for the design and development of novel UFC1-specific modulators with therapeutic potential.

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Abstract: HIV infection disrupts gut epithelial barrier integrity and mucosal immunity, driving chronic inflammation and disease progression which are not fully resolved despite anti-retroviral therapy. Here we identify the microbiota-derived octadecanoid-hydroxy-fatty-acid metabolite 10-hydroxystearic acid (10-HSA), produced by Lactiplantibacillus plantarum, as a key mediator of gut epithelial barrier repair in human intestinal epithelial cells in vitro, ex vivo and in the non-human primate model of HIV/AIDS. X-ray crystallography and transcriptomics combined with functional analyses revealed that 10-HSA directly binds PPARα, inducing lipid metabolism, mitochondrial regeneration and subsequent epigenetic histone crotonylation, thereby promoting gut epithelial renewal. Co-administration of 10-HSA with anti-retroviral therapy in SIV-infected macaques accelerated viral suppression, resolved systemic inflammation, repaired gut epithelial integrity and recovered the gut microbiota. These findings identify a microbiota-derived lipid metabolite–PPARα–histone crotonylation axis that activates gut epithelial regeneration. This study defines a host–microbiome metabolic pathway that restores epithelial–immune homeostasis and enhances the efficacy of anti-retroviral therapy.

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Abstract: The nanofabrication of functional protein-based surfaces is challenging due to the chemical complexity of proteins and their unpredictable behavior at the solid–liquid interface. Many proteins of interest–such as antibodies or large enzymatic complexes–lack strong and dynamic protein–protein and protein–surface interactions necessary to drive self-assembly of stable arrays with high surface coverage. Additionally, adsorption-induced conformational changes at the solid–liquid interface could lead to a loss of activity and increase the risk of undesirable interfacial processes. Here we introduce SAKe, a Kelch-like designer protein, as a versatile platform to address these challenges. Ancestral sequence reconstruction led to high thermal stability, and the high symmetry allowed modularity of the protein’s core. Rational engineering of the bottom side allowed SAKe to form large (up to 5 μm in length), well-defined and pH-dependent two-dimensional assemblies while maintaining structural integrity, which is key for further development of functional materials. SAKe self-assembly was investigated through in-liquid atomic force microscopy on muscovite mica. High resolution imaging confirmed the integrity of the SAKe protein upon adsorption on the solid–liquid interface. These results showcase the SAKe protein as a platform for the further engineering of functional protein-based two-dimensional materials.