Michael J. Bollong

The differentiation of naïve CD4 ⁺ T cells into effector T cell subsets such as T _H 1, T _H 2, T _H 17 and T _REG cells is governed by tightly coordinated activation programmes dependent on T cell receptor (TCR) engagement, co-stimulation, and cytokine signalling. This differentiation process involves regulation of stress-responsive signalling pathways such as the unfolded protein response (UPR) and the oxidative stress response (OSR) to adapt to the physiologic demands specific to distinct T cell subset functions. This suggests that pharmacologically targeting stress-responsive pathways offers a unique opportunity to selectively remodel the differentiation of these different T cell subsets. We previously identified compound AA147 as a metabolically activated proteostasis regulator that can induce both the ATF6 signalling arm of the UPR and, in certain cell types, the NRF2-regulated arm of the OSR.(Paxman et al., 2018; Plate et al., 2016) Here, we show that treatment with AA147 selectively reduces differentiation of pro-inflammatory T _H 17 cells by promoting degradation of the lineage-specifying transcription factor RORγt, without impacting the transcription factors of other CD4 ⁺ T cell subsets. We find that AA147-dependent reduction in T _H 17 differentiation is independent of ATF6 activation but involves NRF2 activation and reductions in intracellular reactive oxygen species (ROS). Apart from RORγt, we further show that AA147 decreases expression of the TCR-responsive factor IRF4, thus suppressing production of select effector cytokines across effector T cells, revealing additional ways in which this compound reshapes the activities of these essential T cell subsets. Our results demonstrate the potential for metabolically activated proteostasis regulators such as AA147 to selectively reshape T _H 17 cell identity while broadly dampening effector cytokine responses across effector T cell subsets.

The air–water interface (AWI) remains the primary barrier to routine high-resolution cryo-EM structure determination, driving protein adsorption, structural denaturation, and restricted particle orientations during vitrification. Here, we describe a simple and broadly applicable strategy to mitigate these effects using the mild non-ionic detergent n-decyl-β-D-maltopyranoside (DM). Addition of DM at low millimolar concentrations immediately prior to vitrification consistently suppresses AWI-driven artifacts, resulting in improved angular sampling, reduced structural damage, and enhanced reconstruction quality across diverse macromolecular systems. Using this approach, we obtained a high-resolution reconstruction of the 65 kDa Nucleophosmin 1 pentamer, a target previously limited by severe preferred orientation issues. We further show that DM promotes isotropic particle distributions for high-resolution reconstruction of hemagglutinin, transthyretin, as well as suppressing denaturation of aldolase while stabilizing its C-terminus. Our results indicate that DM effectively passivates deleterious air-water interface interactions without compromising particle integrity. These results establish DM as an effective additive for improving the robustness of single-particle cryo-EM sample preparation. Abstract Figure:

The NLRP3 inflammasome is a protein complex that promotes pro-inflammatory signaling as part of the innate immune response. Hyperactivation of the NLRP3 inflammasome has been implicated in many inflammatory and neurodegenerative diseases, leading to significant effort in developing strategies to limit its activation to intervene in these disorders. We previously showed that pharmacologic inhibition of endoplasmic reticulum (ER)-localized protein disulfide isomerase PDIA1 suppresses NLRP3 activation and activity, identifying PDIA1 as a potential therapeutic target to mitigate hyperactive NLRP3 activity. Herein, we screen PDIA1 inhibitors to identify highly potent compounds, including P1 and PACMA31, that pharmacologically target PDIA1 and block NLRP3 inflammasome assembly and activity. While sustained treatment with these PDIA1 inhibitors reduces THP1 viability, we show that acute treatment with these compounds is sufficient to both fully modify PDIA1 and inhibit NLRP3 inflammasome activity independent of any overt cellular toxicity. These results establish a treatment paradigm that can be exploited to develop highly selective PDIA1 inhibitors to mitigate hyperactive NLRP3 inflammasome activity implicated in etiologically diverse diseases.

The functionally and evolutionarily conserved Hippo-YAP signaling pathway plays a critical role in regulating cellular proliferation, organ size control, and regeneration. Accordingly, activators of YAP-driven transcription hold therapeutic promise for treating disease states driven by insufficient proliferative repair, yet only a handful of pharmacological mechanisms exist for augmenting YAP activity. Here we report the discovery of sCMF231, a small molecule activator of YAP discovered from high throughput screening that acts by targeting the poorly characterized ubiquitin-like protein FUBI. Using canonical ubiquitin conjugation machinery—UBA1, UBE2C, and APC/C—FUBI covalently modifies the Hippo pathway protein Annexin A2, reinforcing its YAP suppressive role at the plasma membrane. Binding of sCMF231 to FUBI discourages its conjugation to Annexin A2, resulting in the membrane delocalization of Annexin A2 and a liberated, transcriptionally active form of YAP. This work provides the first definitive evidence of covalent modification of proteins by FUBI, a post-translational modification termed fubylation, and defines how fubylation regulates the activity of a central growth pathway.

Proteolysis-targeting chimeras (PROTACs) enable the selective and sub-stoichiometric elimination of pathological proteins, yet only two E3 ligases are routinely used for this purpose. Here, we expand the repertoire of PROTAC-compatible E3 ligases by identifying a novel small molecule scaffold targeting the ubiquitin E3 ligase KLHDC2 using a fluorescence polarization-based high-throughput screen. We highlight the utility of this ligand with the synthesis of PROTACs capable of potently degrading BRD4 in cells. This work affords additional chemical matter for targeting KLHDC2 and suggests a practical approach for identifying novel E3 binders by high-throughput screening.

The NF-κB family of transcription factor complexes are central regulators of inflammation, and their dysregulation contributes to the pathology of multiple inflammatory disease conditions. Accordingly, identifying pharmacological mechanisms that restrain NF-κB overactivation remains an area of key importance. Here, we demonstrate that inhibition of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) with the small molecule inhibitor CBR-470-2 results in attenuated NF-κB signaling, decreasing transcriptional output in response to several canonical NF-κB activating stimuli. Mechanistically, PGK1 inhibition promotes the accumulation of the glycolytic metabolite methylglyoxal, which crosslinks and inactivates NF-κB proteins, limiting the phosphorylation and nuclear translocation of these transcription factor complexes. This work establishes a key connection between central carbon metabolism and immune signaling and further supports the notion that PGK1 inhibition may be a viable strategy to increase cellular survival and dampen inflammation in disease.

Proteolysis targeting chimeras (PROTACs) enable the selective and sub-stoichiometric elimination of pathological proteins, yet only two E3 ligases are routinely used for this purpose. Here, we expand the repertoire of PROTAC compatible E3 ligases by identifying a novel small molecule scaffold targeting the ubiquitin E3 ligase KLHDC2 using a fluorescence polarization-based high throughput screen. We highlight the utility of this ligand with the synthesis of PROTACs capable of potently degrading BRD4 in cells. This work affords additional chemical matter for targeting KLHDC2 and suggests a practical approach for identifying novel E3 binders by high throughput screening.

Activation of the YAP-TEAD transcriptional complex drives the growth of several cancer types and is a key resistance mechanism to targeted therapies. Accordingly, a host of pharmacological inhibitors to TEAD family paralogs have been developed, yet little is known as to the resistance mechanisms that might arise against this emerging therapeutic class. Here, we report that genetic augmentation of de novo coenzyme A biosynthesis desensitizes YAP-dependent cancer cells to treatment with TEAD inhibitors, an effect driven by increased levels of palmitoyl-CoA that outcompete drug for engagement of the lipid-binding pocket. This work uncovers a potential therapeutic resistance mechanism to TEAD palmitoylation site inhibition with implications for future combinatorial treatments in the clinic.

Proteolysis targeting chimeras (PROTACs) enable the selective and sub-stoichiometric elimination of pathological proteins, yet only two E3 ligases are routinely used for this purpose. Here, we expand the repertoire of PROTAC compatible E3 ligases by identifying a novel small molecule scaffold targeting the ubiquitin E3 ligase KLHDC2 using a fluorescence polarization-based high throughput screen. We highlight the utility of this ligand with the synthesis of PROTACs capable of potently degrading BRD4 in cells. This work affords additional chemical matter for targeting KLHDC2 and suggests a practical approach for identifying novel E3 binders by high throughput screening.