Abstract
The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity.
Highlights
The endoplasmic reticulum-associated degradation (ERAD) is a cellular mechanism to eliminate misfolded proteins
We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a targetspecific binding moiety located on the luminal side of the ER
The SEL1L moiety lacks the first N-terminal 401 amino acids, preserving the portion interacting with ERAD active proteins HRD1, OS9, XTP3-B, p97 ATPase, and Derlin-1 [15]
Summary
The endoplasmic reticulum-associated degradation (ERAD) is a cellular mechanism to eliminate misfolded proteins. Several interactions have been described for the HRD1-SEL1L complex, including association with the E2 ubiquitin-conjugating enzymes UBC6 and UBC7 [17] and the ER-resident lectins OS-9 and XTP3-B, which are responsible of targeting ERAD substrates [15, 18] These lectins seem to be involved in coordinating substrate recognition in the ER lumen with ubiquitin conjugation in the cytosol. We describe the construction and in vivo activities of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety constituted by specific ligands or derived from mAbs. Degradins are able to recognize target proteins within the ER and to induce their degradation following retrotranslocation to the cytosol, resulting in an effective technique to induce protein knock-out in cells with high specificity
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