P53 upregulation by USP7-engaging molecular glues.

Molecular glues are typically small chemical molecules that act at the interface between a target protein and degradation machinery to trigger ternary complex formation. Identifying molecular glues is challenging. There is a scarcity of target-specific upregulating molecular glues, which are highly anticipated for numerous targets, including P53. P53 is degraded in proteasomes through polyubiquitination by specific E3 ligases, whereas deubiquitinases (DUBs) remove polyubiquitination conjugates to counteract these E3 ligases. Thus, small-molecular glues that enhance P53 anchoring to DUBs may stabilize P53 through deubiquitination. Here, using small-molecule microarray-based technology and unbiased screening, we identified three potential molecular glues that may tether P53 to the DUB, USP7, and elevate the P53 level. Among the molecular glues, bromocriptine (BC) is an FDA-approved drug with the most robust effects. BC was further verified to increase P53 stability via the predicted molecular glue mechanism engaging USP7. Consistent with P53 upregulation in cancer cells, BC was shown to inhibit the proliferation of cancer cells in vitro and suppress tumor growth in a xenograft model. In summary, we established a potential screening platform and identified potential molecular glues upregulating P53. Similar strategies could be applied to the identification of other types of molecular glues that may benefit drug discovery and chemical biology studies.

Glucocorticoids modulate neural activity via a rapid non-genomic effect on Kv2.2 channels in the central nervous system.

Glucocorticoids are primary stress hormones that exert neuronal effects via both genomic and non-genomic signaling pathways. However, their rapid non-genomic effects and underlying mechanisms on neural activities remain elusive. In the present study, we investigated the rapid non-genomic effect of glucocorticoids on Kv2.2 channels in cultured HEK293 cells and acute brain slices including cortical pyramidal neurons and calyx-type synapses in the brain stem. We found that cortisol, the endogenous glucocorticoids, rapidly increased Kv2.2 currents by increasing the single-channel open probability in Kv2.2-expressing HEK293 cells through activation of the membrane-associated glucocorticoid receptor. Bovine serum albumin-conjugated dexamethasone, a membrane-impermeable agonist of the glucocorticoid receptor, could mimic the effect of cortisol on Kv2.2 channels. The cortisol-increased Kv2.2 currents were induced by activation of the extracellular signal-regulated protein kinase (ERK) 1/2 kinase, which could be inhibited by U0126, an antagonist of the ERK signaling pathway. In layer 2 cortical pyramidal neurons and the calyx of Held synapses, cortisol suppressed the action potential firing frequency during depolarization and reduced the successful rate upon high-frequency stimulation by activating Kv2.2 channels. We further examined the postsynaptic responses and found that cortisol did not affect the mEPSC and evoked EPSC, but increased the activity-dependent synaptic depression induced by a high-frequency stimulus train. In conclusion, glucocorticoids can rapidly activate Kv2.2 channels through membrane-associated glucocorticoid receptors via the ERK1/2 signaling pathway, suppress presynaptic action potential firing, and inhibit synaptic transmission and plasticity. This may be a universal mechanism of the glucocorticoid-induced non-genomic effects in the central nervous system.

TMC1 and TMC2 Proteins Are Pore-Forming Subunits of Mechanosensitive Ion Channels.

Transmembrane channel-like (TMC) 1 and 2 are required for the mechanotransduction of mouse inner ear hair cells and localize to the site of mechanotransduction in mouse hair cell stereocilia. However, it remains unclear whether TMC1 and TMC2 are indeed ion channels and whether they can sense mechanical force directly. Here we express TMC1 from the green sea turtle (CmTMC1) and TMC2 from the budgerigar (MuTMC2) in insect cells, purify and reconstitute the proteins, and show that liposome-reconstituted CmTMC1 and MuTMC2 proteins possess ion channel activity. Furthermore, by applying pressure to proteoliposomes, we demonstrate that both CmTMC1 and MuTMC2 proteins can indeed respond to mechanical stimuli. In addition, CmTMC1 mutants corresponding to human hearing loss mutants exhibit reduced or no ion channel activity. Taken together, our results show that the CmTMC1 and MuTMC2 proteins are pore-forming subunits of mechanosensitive ion channels, supporting TMC1 and TMC2 as hair cell transduction channels.

Structure of the mechanosensitive OSCA channels.

Mechanosensitive ion channels convert mechanical stimuli into a flow of ions. These channels are widely distributed from bacteria to higher plants and humans, and are involved in many crucial physiological processes. Here we show that two members of the OSCA protein family in Arabidopsis thaliana, namely AtOSCA1.1 and AtOSCA3.1, belong to a new class of mechanosensitive ion channels. We solve the structure of the AtOSCA1.1 channel at 3.5-Å resolution and AtOSCA3.1 at 4.8-Å resolution by cryo-electron microscopy. OSCA channels are symmetric dimers that are mediated by cytosolic inter-subunit interactions. Strikingly, they have structural similarity to the mammalian TMEM16 family proteins. Our structural analysis accompanied with electrophysiological studies identifies the ion permeation pathway within each subunit and suggests a conformational change model for activation.