| Project Detail |
Summary of the research planBackground-The endoplasmic reticulum (ER) is the major biosynthetic organelle of nucleated cells. It produces lipids, oligosaccharides and about 40% of the eukaryotic cell’s proteome. The ER contains molecular chaperones and folding enzymes that welcome nascent polypeptide chains and assist their conformational maturation. The structure and assembly of newly synthesized proteins is checked by quality control factors that prolong ER retention and folding attempts of proteins exposing misfolded domains, hydrophobic patches, charged residues in intra-membrane domains, unpaired cysteines or peptidyl-prolyl bonds in the wrong configuration. Terminally misfolded polypeptides are degraded to prevent ER clogging with aberrant gene products. Most of the polypeptides entering the ER are covalently modified at asparagine side chains with a conserved (from yeast to humans) oligosaccharide composed of 2 N-acetylglucosamine, 9 mannose and 3 glucose residues. In the last two decades, it has been established that sequential removal of terminal glucose and mannose residues from protein-bound oligosaccharides determines the engagement of molecular chaperones and folding enzymes such as oxidoreductases and peptidyl-prolyl cis/trans isomerases that catalyze rate-limiting steps of protein folding programs. Oligosaccharide processing also determines interruption of futile folding attempts and selection of terminally misfolded proteins for clearance from the ER, which requires translocation into the cytosol for degradation by the ubiquitin proteasome system (UPS) via ER-associated degradation (ERAD), or segregation in ER subdomains that do eventually vesiculate and are removed from cells via ER-to-lysosome-associated degradation (ERLAD).Objectives-We aim at dissecting the pathways activated by mammalian cells to remove polypeptides that fail attainment of the native structure in the ER. Emphasis will be given on establishing the mechanisms that regulate segregation of misfolded polypeptides within specialized ER subdomains that are eventually subject to lysosomal clearance. Methods-The mechanisms of selection of misfolded proteins for clearance will be investigated with a series of model proteins derived from disease-causing mutant variants of alpha1-antitrypsin and will be validated by testing the fate of other folding-defective polypeptides such as disease-causing beta-secretase and pro-collagen variants. Model proteins are designed to explore the role of protein-bound oligosaccharides, polymerization and aggregation propensities, membrane anchoring and localization in sub-compartments of the early secretory pathway including the ER itself, ER exit sites, intermediate compartment. Experiments will be performed in cultured mammalian cells, including patient-derived cell lines. Genome editing (CRISPR/Cas technology) will be used to generate cells lacking genes-of-interest or expressing tagged versions of endogenous proteins-of-interest. The vast expertise and technological knowledge available in the lab, crucial tools such as conformation and polymer-specific antibodies specific for the model proteins under investigation, new-generation protein trafficking reporters developed in our lab for steady state and time-resolved visualization of intracellular transport routes in confocal light scanning microscopy and in transmission electron microscopy, will be implemented with deep learning approaches, also developed in our lab, for unbiased and automated segmentation and classification of fluorescence and electron microscopy images, which are instrumental for quantitative assessment of experimental results. Expected results and impact-We will investigate the features of defective gene products that determine engagement of ERAD or ERLAD for destruction. We will mechanistically characterize the ERLAD pathways engaged by clients with different topology, localization, chemico-physical features. Client-specific pathways likely rely on different ER-phagy receptors and likely use different transport routes for delivery to the endolysosomal compartments (i.e., micro- ER-phagy, macro-ER-phagy, LC3-dependent vesicular delivery). We will establish when, and to what extent, ERAD and ERLAD pathways collaborate to control efficient clearance from cells of aberrant gene products. We will establish new technology for time-course monitoring of the events under investigation in light and electron microscopy and for their quantitative assessment. Understanding the mechanisms and identifying the regulators of protein clearance will offer diagnostic and prognostic disease indicators; their modulation is expected to offer therapeutic opportunities in protein misfolding diseases. |
