Group Leader: Maurizio Molinari

Researchers: Tatiana Soldà, Scientist

The aim of the project is to identify new players that intervene in protein folding, quality control and ERAD in the mammalian ER lumen. We generated a collection of human cell lines expressing epitope-tagged folding-competent and folding-defective proteins. The model proteins are used as baits to capture interacting partners in the same immuno-complexes. The proteins co-immunoisolated with the individual baits are subjected to tryptic digestion and fragments are separated by nano-HPLC followed by tandem mass spectrometry. Fragmentation spectra of the samples are matched to a human protein database sequence with the Mascot software. These analytic steps are performed in collaboration with Manfredo Quadroni, coordinator of the Center for Integrative Genomics, University of Lausanne. Involvement in protein quality control of the interacting partners of the model proteins will be validated in 2 steps: i) confirmation of interaction by co-immunoisolation followed by western blot; ii) evaluation of the role of the interactors by monitoring consequences on the substrate fate upon silencing of their expression or upon co-expression with the model substrate of their dominant negative mutants.

Group Leader: Maurizio Molinari

Researchers: Elisa Fasana, Scientist, Tatiana Soldà, Scientist

To maintain ER homeostasis and to ensure the highest efficiency of functional polypeptide production, the quality control machinery operating in the mammalian ER must distinguish non-native intermediates of protein folding programs from terminally misfolded polypeptides. Folding intermediates must be retained in the ER to attain the native structure under the assistance of dedicated molecular chaperones and folding enzymes. Terminally misfolded polypeptides must be rapidly cleared from the ER lumen to avoid interferences with ongoing folding programs. For glycosylated polypeptides, which represent the majority of the cargo entering the secretory pathway, the processing of the N-linked oligosaccharides determines retention in the folding environment (cycles of removal/re-addition of terminal glucose residues) or extraction from the folding environment for disposal. Virtually nothing is known about quality control of non-glycosylated polypeptides. The aim of this project is the identification of ER-resident factors involved in quality control and disposal of both soluble and membrane-bound non-glycosylated variants of model glycopolypeptides generated in our lab.

Tannous et al., Semin Cell Dev Biol. 2015, 41:79-89

The lumen of the ER contains 23 PDI members that insure formation of the correct set of intra- and inter-molecular disulfide bonds as a crucial, rate-limiting reaction of the protein folding process (Figure 1A). The reason for this high redundancy of PDIs remains unclear. Certainly, individual members of the PDI family show tissue-specific distribution or some kind of substrate preference (e.g. ERp57 forms functional complexes with the ER lectins calnexin and calreticulin and acts upon their ligands). The aim of this project is to uncover the role in protein biogenesis of the 5 type I membrane-bound members of the PDI family (TMX1, TMX2, TMX3, TMX4 and TMX5) (Figure 1B). Active PDIs contain the characteristic CXXC active-site motif that engages folding substrates in so-called mixed disulfides (i.e. covalent bonds between a PDI and a substrate cysteine). Mixed disulfides are extremely short living intermediates of the protein folding reaction, which can be stabilized upon replacement of the second (resolving) cysteine residue in the PDIs catalytic site. These so-called PDIs “trapping mutants” have been used to capture endogenous substrates of select ER-resident oxidoreductases such as ERp57, PDI, P5, ERp18, ERp72, ERp46 and ERdj5. The expression of a TMX1 trapping mutant in the living cells and the characterization by mass spectrometry of the polypeptides remaining covalently bound to it revealed a selective association with a series of cysteine-containing membrane-bound proteins. This is in contrast to studies performed with trapping mutants of other PDIs, which were all found to associate both with soluble and membrane-bound endogenous substrates. Studies are ongoing to confirm the substrate topology-dependent specificity of TMX1 and to characterize the role in protein biogenesis of the other TMX proteins (Figure 2).

Misfolded polypeptides produced in the ER are dislocated across the ER membrane to be degraded by cytosolic 26S-proteasomes in processes collectively defined as ERAD. Dislocation across the ER membrane is regulated by multimeric complexes built around one of the several membrane-embedded E3 ubiquitin ligases expressed in the mammalian ER. Physico-chemical features of the misfolded polypeptide (e.g. presence/absence of N-linked oligosaccharides, disulfide bonds, peptidyl-prolyl bonds in the cis conformation, membrane-anchor) may determine the quality control machineries that deliver the misfolded polypeptide at specific dislocation complexes. The definition of the rules that govern protein biogenesis and quality control requires a systematic analysis of appositely designed model folding-competent and folding-defective proteins. We have therefore prepared more than 50 model substrates with select physico-chemical features, whose fate will be monitored in mammalian cultured cells. The model polypeptides recapitulate structural defects found in mutant products of genes causing human disorders such as Alzheimer’s, Parkinson’s, Huntington’s diseases as well as many other rare genetic disorders characterized by gain-of-toxic-function or loss-of-function phenotypes. How the polypeptide’s features determine engagement of specific folding, quality control and degradation pathways will be determined in molecular details.

Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways
Fregno, I. Molinari, M.
in Crit Rev Biochem Mol Biol (2019) Vol. 54, pp153-163

Autophagy is a conserved cellular process in eukaryotes required for degradation of cytoplasm contents into the lysosome/vacuole. Double-membrane vesicles called autophagosomes mediate the engulfment and transport of the cargo to be degraded during autophagy. While this pathway constitutively degrades cytoplasmic targets, it is also up-regulated by different cellular stresses. Starvation-induced autophagy randomly targets bulk cytoplasmic portions. Additionally, it selectively recognizes and degrades cytoplasmic protein aggregates, damaged organelles or invading microorganisms, playing thus a homeostatic and protective role in the cell. Interestingly, accumulation of misfolded proteins within the ER triggers autophagic degradation of portions of this organelle in yeast and mammals, suggesting that ER-phagy might be a conserved mechanism to prevent or overcome ER stress. While ERAD pathway is the classical and best characterized process for protein disposal in the ER, little is known about the mechanisms underlying ER degradation by autophagy. By using series of stable human cell lines created in our lab expressing regulated amounts of folding-competent and folding-defective protein chimeras, we are studying the contribution of autophagy in the degradation of these putative substrates and the molecular mechanisms regulating such a process. These studies will allow us to characterize the conditions for potential preferences in substrate elimination by ERAD and ER-phagy, and the mechanistic crosstalk between these two pathways and ER stress. The information generated by these studies will be validated in pathological model systems expressing disease-causing folding-defective proteins with the final goal of designing pharmacological treatments targeting protein disposal pathways to alleviate the toxicity caused by aberrant protein accumulation.

Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways
Fregno, I. Molinari, M.
in Crit Rev Biochem Mol Biol (2019) Vol. 54, pp153-163

Deep learning approach for quantification of organelles and misfolded polypeptides delivery within degradative compartments
Morone, D. Marazza, A. Bergmann, T. J. Molinari, M.
in Mol Biol Cell (2020) Vol.31 pp1512-1524