The Kopito Laboratory
Stanford University
RESEARCH
ENSURING THE INTEGRITY OF THE SECRETORY AND MEMBRANE PROTEOME
Approximately one third of proteins synthesized in cells are destined for integration into or secretion beyond the plasma membrane. Secreted proteins antibodies and signaling molecules such as growth factors and hormones. Integral membrane proteins include ion channels and solute transporters and hormone and growth factor receptors. The endoplasmic reticulum (ER) is the "port of entry" for all proteins destined for the cell surface and beyond. The vast majority of proteins entering the secretory pathway are synthesized on ribosomes docked at ER translocons and are co-transationally translocated into the membrane or the lumen of the ER. Integral membrane are threaded co-translationally into the lipid bilayer and often adopt complex topologies. This highly complex "protein biogenesis" process is assisted by a diverse network of folding catalysts and protein-modifying enzymes and is scrutinized by molecular chaperones. "Quality control" factors monitor this process to ensure that only correctly folded and assembled proteins exit the ER and proceed to plasma membrane. Our goal is to elucidate the functional networks that coordinate protein synthesis and quality control in the secretory pathway. Currently the lab is focused on two specific systems: Triage of folding-defective multipass proteins and the role of ribosome UFMylation in regulating the ribosome-translocon juction.
ER-associated degradation (ERAD)
Folding begins during translocation and often persists after the polypeptide chain has been released from the ribosome. Folding intermediates are not competent for export out of the ER and may undergo multiple rounds of folding attempts, with different proteins folding at different rates. Some proteins are unable to achieve a native conformation because of the presence of mutations, amino acid mis-incorporation, or unavailability of oligomeric partners. Triage refers to the process by which folding-defective proteins are destroyed prior to being deployed to their final destinations. Because protein degradation is mediated by the ubiquitin-proteasome system in the cytosol, in order to be degraded, proteins that were translocated into the
ER must be dislocated back across the ER bilayer, through a proteinaceous membrane pore called a dislocon. This process requires metabolic energy and covalent modification by ubiquitin. Because ERAD clients are often very hydrophobic, the dislocation process is thought to be tightly coupled to proteolysis in order to avoid the release of aggregation-prone intermediates to the cytosol.
Functional genomic analysis of corrector-resistant cystic fibrosis
Cystic fibrosis (CF) is the most common fatal genetic disorder in the US, affecting ~32,000 Americans. CF is caused by loss-of-function mutations in the gene encoding CFTR, a Cl– channel expressed at the plasma membrane (PM) of epithelial cells. Approximately 85% of people with CF (pwCF) carry at least one copy of the F508del mutation, which impairs CFTR folding at the ER, leading to the degradation of CFTR by ERAD. Since 2019 pwCF now have access to Trikafta®, a CFTR modulator drug that contains small molecule correctors, which act as 'pharmacological chaperones' to promote folding of mutant CFTR, allowing it to escape ERAD. This drug has revolutionized treatment of CF, increasing lifespan and quality of life for thousands of pwCF.
Despite the remarkable clinical success of Trikafta, ~15-20% of pwCF with eligible mutations do not respond fully to Trikafta in the clinic while other pwCF have ineligible mutations that do not qualify due to a lack of response in vitro. Consequently, there is a pressing unmet medical need to develop new therapeutic strategies for pwCF. In collaboration with the Porteus (https://med.stanford.edu/porteuslab.html) and Milla (https://med.stanford.edu/profiles/carlos-milla) laboratories, we are using CRISPR loss-of-function and gain-of-function screens to identify drug targets that work through different mechanisms than the current FDA-approved therapies and validate that modulating these targets rescue CFTR function in airway epithelia derived from upper airway basal stem cells from pwCF.
RIBOSOME UFMYLATION AT THE RIBOSOME-TRANSLOCON JUNCTION
UFM1 is a ubiquitin-like protein modifier that shares with ubiquitin a common structural fold, analogous chemistry and enzymology. Like ubiquitin, UFM1 is covalently conjugated to lysine residues on target protein by a canonical E1-E2-E3 cascade and removed by a dedicated deUFMylase (DUF). We identified the ribosomal protein RPL26 as the principal client of UFMylation. RPL26 is positioned adjacent to the polypeptide exit site of the ribosome and UFMylated at two lysine residues on a helical extension of RPL26. Both UFMylation and de-UFMylation of RPL26 occur exclusively at the ER, owing to to physical restriction of the E3 and DUF, respectively to the ER membrane. UFMylation is stimulated by conditions that cause ribosomes to stall and collide while co-translationally synthesizing secretory and membrane proteins at ER translocons.
We solved a series of cryoEM snapshots of the UFM1 E3 ligase as it engages a terminated 60S ribosomal subunit still docked at the SEC61 translocon in the ER membrane. Remarkably, the E3 binds more tightly to the product of the reaction it catalyzes, ie UFMylated 60S than to its substrate, 60S. The structures support a model in which UFM1 conjugation promotes a conformational change that repositions the long alpha-helix on the DDRGK1 subunit (magenta) at the 60S polypeptide tunnel exit, displacing SEC61. Because the E3 is tethered to the ER membrane, final release of 60S from the membrane requires deUFMylation.