Novel Protein Quality Checkpoints
Research area: Protein Folding and Quality Control
Group leaders: Maurizio Molinari
Status: In progress
Proteins that reach the native structure are released from the ER and are transported to their site of activity. Misfolded polypeptides are selected for degradation. The stringency of protein quality control in the mammalian ER may lead to the removal of structural-defective polypeptides, independent of their capacity to fulfill their function. This causes loss-of-function proteopathies such as cystic fibrosis, lysosomal storage diseases and many others, where functional polypeptides are inappropriately removed from cells because they display minor structural defects. The development of therapeutic strategies to treat such disorders relies on the characterization, at the molecular level, of the quality checkpoints and pathways operating in mammalian cells. Recently published data in our group show that proteins with native ectodomains presenting an intramembrane defect (an ionizable residue in the transmembrane domain spanning the lipid bilayer) alert a novel post-ER quality control and are retained in a pre-Golgi compartment. This novel checkpoint involves the cytosolic AAA-ATPase p97 and the luminal factor UDP-glucose:glycoprotein glucosyltransferase (UGGT1) and can be by-passed, thus resulting in surface transport of the defective protein, upon p97 inhibition or p97 and UGGT1 silencing (Figure 1). To better characterize this novel protein quality control machinery, we generated mammalian tetracycline-inducible cell lines individually expressing two type I membrane protein chimeras. The first consists in the folding competent ectodomain of human α1-antitrypsin fused with the C-terminal domain of CD3δ, which contains an ionizable aspartic acid at position 6 in the intramembrane sequence (chimera α1ATc). The second in α1-antitrypsin fused with the same domain where the ionizable residue is replaced with an alanine (chimera α1ATcD6A). Ongoing work is focused on the identification of the components of this p97/UGGT1-mediated checkpoint.
A novel retention-based quality checkpoint. (A) Proteins with a misfolded ectodomain are retained in the ER by the conventional quality control relying on UGGT1, CNX and BiP intervention. These proteins are eventually destined to ERAD. (B) A chimera with a native ectodomain, characterized by an ionizable residue in the intramembrane domain (α1ATc) fulfills quality control requirement for release from CNX and BiP, but its transport to the Golgi is halted upon p97 and UGGT1 intervention. This protein quality checkpoint is by-passed upon pharmacologic inhibition of p97 with DBeQ or upon silencing of p97 or UGGT1 expression. (C) Replacement of the ionizable intramembrane residue with an alanine results in efficient transport to the Golgi compartment.
Ferris et al., Mol Biol Cell. 2013; 24:2597-2608.
Merulla et al., Mol Biol Cell. 2015; 26:1532-1542.