Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity
Abstract not provided
Singh, Sunil↗
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Selective posttranslational inhibition of Ca V β 1 -associated voltage-dependent calcium channels with a functionalized nanobody
Ca 2+ influx through high-voltage-activated calcium channels (HVACCs) controls diverse cellular functions. A critical feature enabling a singular signal, Ca 2+ influx, to mediate disparate functions is diversity of HVACC pore-forming α1 and auxiliary Ca V β 1 –Ca V β 4 subunits. Selective Ca V α 1 blockers have enabled deciphering their unique physiological roles. By contrast, the capacity to post-translationally inhibit HVACCs based on Ca V β isoform is non-existent. Conventional gene knockout/shRNA approaches do not adequately address this deficit owing to subunit reshuffling and partially overlapping functions of Ca V β isoforms. Here, we identify a nanobody (nb.E8) that selectively binds Ca V β 1 SH3 domain and inhibits Ca V β 1 -associated HVACCs by reducing channel surface density, decreasing open probability, and speeding inactivation. Functionalizing nb.E8 with Nedd4L HECT domain yielded Chisel-1 which eliminated current through Ca V β 1 -reconstituted Ca V 1/Ca V 2 and native Ca V 1.1 channels in skeletal muscle, strongly suppressed depolarization-evoked Ca 2+ influx and excitation-transcription coupling in hippocampal neurons, but was inert against Ca V β 2 -associated Ca V 1.2 in cardiomyocytes. The results introduce an original method for probing distinctive functions of ion channel auxiliary subunit isoforms, reveal additional dimensions of Ca V β 1 signaling in neurons, and describe a genetically-encoded HVACC inhibitor with unique properties.
60 APPLIED LIFE SCIENCES↗
Structural and functional validation of a highly specific Smurf2 inhibitor
Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor‐β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross‐reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein‐based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2–E3 protein interaction interface.
59 BASIC BIOLOGICAL SCIENCES↗