Novel ubiquitin-dependent quality control in the endoplasmic reticulum

Proteins of the endomembrane system undergo assisted folding in the endoplasmic reticulum (ER), then quality-control and, if misfolded, ER-associated degradation (ERAD). Recent findings on the biogenesis of a type-I membrane protein (an LRP6 mutant) lead us to hypothesize the existence of a novel mechanism promoting folding of membrane proteins from the cytosolic side of the ER. The proposed folding mechanism involves cycles of chaperone binding through mono-ubiquitylation and de-ubiquitylation, followed eventually by poly-ubiquitylation and ERAD. This suggests a novel dual role for ubiquitylation in the ER - dependent on the type of ubiquitin chains involved - in folding and in degradation, and highlights the potential importance of de-ubiquitylating enzymes.

Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6

Michal Feldman Elmalam, Laurence Gouzi Abrami, Béatrice Kunz, Elsa Anne Perrody, Françoise Gisou van der Goot Grunberg

Many membrane proteins fold inefficiently and require the help of enzymes and chaperones. Here we reveal a novel folding assistance system that operates on membrane proteins from the cytosolic side of the endoplasmic reticulum (ER). We show that folding of the Wnt signaling coreceptor LRP6 is promoted by ubiquitination of a specific lysine, retaining it in the ER while avoiding degradation. Subsequent ER exit requires removal of ubiquitin from this lysine by the deubiquitinating enzyme USP19. This ubiquitination-deubiquitination is conceptually reminiscent of the glucosylation-deglucosylation occurring in the ER lumen during the calnexin/calreticulin folding cycle. To avoid infinite futile cycles, folded LRP6 molecules undergo palmitoylation and ER export, while unsuccessfully folded proteins are, with time, polyubiquitinated on other lysines and targeted to degradation. This ubiquitin-dependent folding system also controls the proteostasis of other membrane proteins as CFTR and anthrax toxin receptor 2, two poor folders involved in severe human diseases.

Elife Sciences Publications Ltd2016

Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors

Julie Deuquet Ariosa, Laurence Gouzi Abrami, Nicolas Jean Philippe Guex, Suzanne Salvi, Françoise Gisou van der Goot Grunberg

Hyaline Fibromatosis Syndrome (HIS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized lg-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the lg-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Wiley Open Access2011

Multiscale simulations of protein dynamics

Enrico Spiga

Characteristic timescales associated with the function of biomolecules, like proteins, range from femtoseconds up to minutes, whereas their corresponding spatial extent ranges from few ̊A to μm when associating in large macromolecular complexes. Moreover, biomolecules are functional in a large variety of different physico-chemical conditions strongly dependent on pH, ionic strength, crowding agents, etc. This huge complexity is hard to be studied with an arbitrary level of resolution embracing all these spatial and temporal scales. Molecular simulation is a well established approach to gain mechanistic insights into the function of biomolecular systems, producing atomistically detailed models of in vitro and/or in vivo conditions. I present in this thesis two projects that aim at improving on the current limitations of multiscale molecular simulations, namely (i) the sampling of large systems, and (ii) the detailed representation and description of realistic physiological conditions. Addressing the first issue, I propose a new coarse-grained model for proteins to be used in molecular dynamics simulations. This coarse-grained model is based on a more accurate description of protein electrostatics, which accounts for dipolar contributions. The parameterization of this force field is based on force-matching methods and on the use of a particle swarm optimization heuristic algorithm. The obtained results are encouraging being structural and electrostatic properties accurately reproduced with the coarse-grained model for a variety of protein folds. Moreover, the parameterization procedure can be straightforwardly applied to any protein, and can be extended to a larger dataset to generate a fully transferable coarse-grained force field, to be applied to any protein and any large macromolecular assemblies for which long all-atom simulations are still a challenge. While the development and use of coarse-grained models are important to tackle the limited sampling of large systems, it is still important to use all-atom molecular dynamics simulation to investigate with high accuracy in physiological conditions protein dynamics. For this reason, I present here the results of state-of-art molecular dynamics simulations applied to study the influence of crowding agents on the internal dynamics of the protein ubiquitin. Their analysis allows to describe how ubiquitin dynamics is slaved by crowding agents. This work demonstrates that the description of protein dynamics should take into account its intrinsic multiscale nature. The development and applications of coarse-grained models permit to simulate proteins at low computational cost, the use of atomistic simulations allows to accurately describe proteins in absence and presence of crowding agents, and both of them permit to highlight the essence of protein dynamics.

EPFL2013

Multiscale simulations of protein dynamics

Enrico Spiga

EPFL2013