Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes. Loss-of-function methods for reducing target protein levels target DNA, RNA or protein 1. Auxin-inducible degron (AID) technology allows rapid targeted protein depletion with the smallmolecule auxin 2. To apply AID, an auxin-inducible destabilizing domain, or 'degron' , is fused to the target protein. In addition, an auxin receptor F-box protein TIR1/AFB is exogenously expressed, forming a chimeric SKP1-CUL1-F-Box (SCF) ubiquitin E3 ligase with endogenous components in eukaryotic cells. The chimeric E3 ligase recruits the degron in an auxin-dependent manner 3. Addition of indole-3-acetic acid (IAA) or other small molecules of the auxin class to the culture medium thus induces the polyubiquitination and rapid proteasomal degradation of the degron-fused protein (Fig. 1a). This approach has been used as a powerful tool to acutely deplete target proteins in studying their functions 4-7. However, current AID systems can severely degrade target proteins before IAA addition (known as 'basal degradation') and suffer from inefficient auxin-inducible depletion in a context-and target-specific manner 2,8-10. These pitfalls have substantially limited the applications of AID technology. An AID system with both minimal basal degradation and rapid auxin-inducible depletion would be desirable. In current AID systems, Oryza sativa TIR1 (OsTIR1) is the most commonly used auxin receptor F-box protein in combination with different degrons deriving from Arabidopsis thaliana IAA17 (AtIAA17) 2,4,5,7-14. We initially aimed to acutely deplete endogenous human seipin, a transmembrane protein involved in lipid droplet (LD) biogenesis. However, seipin tagged with a degron termed miniAID (composed of AtIAA17 amino acid (aa) 68-132) was severely degraded in cells expressing OsTIR1 without IAA addition. Consequently, cells exhibited defective LD biogenesis already before IAA addition, resembling a seipin knockout phenotype 15 (Supplementary Fig. 1a-c). To search for an improved AID system, we established a pipeline in human A431 cells for screening various auxin receptor F-box proteins and degrons, through co-integration into the adenoassociated virus integration site 1 (AAVS1) safe harbor locus 16 (Fig. 1b and Supplementary Note 1). We first compared OsTIR1 to five other auxin receptor F-box proteins, using miniAID as the degron. A. thaliana AFB2 (AtAFB2) was identified as the best hit: compared to OsTIR1, it displayed minimal basal depletion
The β2-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β2-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β2-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.
Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.
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