A method to selectively degrade proteins of the secretory pathway

Proteins that enter the secretory pathway, either soluble or membrane bound, are subjected to ER quality control mechanisms: if protein folding is aberrant or delayed, proteins are subjected to additional folding cycles or selected for ERassociated degradation (ERAD). In this process, proteins that fail to reach their terminal folding, and thus selected for ERAD, are recognised and retrotranslocated to the cytosol for ubiquitinylation and subsequent degradation by the proteasome. The most characterised mammalian ubiquitin ligase is HRD1. For its activity HRD1 needs the interaction with SEL1L an ER resident type I transmembrane glycoprotein. SEL1L has been defined as an adaptor protein because, beside its interaction with the ubiquitin ligase HRD1, it interacts either with proteins involved in substrate recruitment or in the retro-translocation process. To induce degradation of specific targets we designed and constructed a new class of fusion molecules, termed degradins, where the NH2-terminal part consists of a target-specific binding domain, such as an antibody-derived scFv or a ligand of a receptor, fused to the COOH-terminal 372 aminoacids of human SEL1L. The SEL1L moiety lacks the first NH2-terminal 401 amino acids probably involved in identification and recognition of ERAD substrates, but retains the luminal portion, homologous to the yeast protein Hrd3p and involved in the interaction with the ubiquitin ligase HRD1, and the transmembrane and cytosolic domains. By testing the system on two different models (FcεRI-α chain and TGEV spike protein S) we found that degradins were able to induce retro-translocation and proteasome-mediated degradation of the target. To study the retro-translocation induced by degradins, we developed a system based on the specific in vivo biotinylation of a reporter engineered by the addition of a biotin acceptor peptide (BAP). The BAP peptide is recognised by the cyt-BirA enzyme that catalyses the addition of a biotin molecule in the cytosol. Proteins normally residing in the ER lumen can get biotinylated only after their dislocation to the cytosol. As a model, the MHC-Iα was chosen because target of retrotranslocation induced by US2 or US11 immunoevasins of HCMV; the system demonstrated to be very specific and efficient in detecting all types of intermediates of retro-translocation or completely retro-translocated molecules. Altogheter, my results strongly suggest that the degradin system can be efficiently used to degrade specific target proteins depending on the target recognition moiety, moreover our in vivo biotinylation represents a novel and unique method for the study of retro-translocation process.