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PalmERa Report Summary

Project ID: 340260
Funded under: FP7-IDEAS-ERC
Country: Switzerland

Mid-Term Report Summary - PALMERA (Roles of Palmitoylation networks in ER architecture and functions)

The overall aim of the project is to obtain a better understanding of a poorly characterized post-translational modification, S-palmitoylation, which consists of the addition of an acyl chain, generally palmitate, to cytosolic cysteine residues. The modification is carried out by members of the DHHC family of enzymes. As opposed to other lipid modifications, palmitoylation is reversible and the removal is carried out by Acyl Protein Thioesterases. The number of proteins undergoing palmitoylation is ever increasing and, in the context of this proposal, we have generated a public database, SwissPalm, that assembles proteomics and targeted studies on these proteins.

The focus of the project is on the endoplasmic reticulum (ER) where 18 of the 23 enzymes that mediate palmitoylation are localized. Very little is currently known about these enzymes and their targets. We have focused on DHHC6 since its few known targets are important for ER function and architecture, for example the chaperone calnexin or the ER shaping protein Climp63.
We have shown that DHHC6 is a tightly regulated protein. Regulation occurs through dynamic palmitoylation of 3 cysteine residues in its C-terminal SH3 domain. We identified DHHC16 as the responsible upstream palmitoylatransferase. This is the first evidence for a palmitoylation cascade. We identified APT2 as the depalmitoylating enzyme. We showed that palmitoylation strongly affects the DHHC6 stability, its localization in the ER two-dimensional space and its function. These studies, performed on wild type DHHC6 and single, double and triple cysteine mutants, indicated that the different sites showed vastly different behaviors. To address the site importance and the dynamics of the system, we combined our experimental approaches with mathematical modeling, taking into account the 8 possible DHHC6 species in terms of occupancy of the 3 palmitoylation sites. We generated a model that was first calibrated with a set of experiments and subsequently used to predict a second set. The excellent quality of the predictions indicated that the model adequately captures the system. This analyses showed that palmitoylation of the first site provides the highest activity but renders the protein highly unstable, possibly to avoid excess DHHC6 activity. The presence of the two additional sites allows to regulate DHHC6 activity, i.e. render it possible to increase the activity without inducing massive enzyme degradation. Altogether it appears that DHHC6 is a very potent enzyme, the activity of which requires tightly context-dependent regulation.

The second major focus was aimed at understanding the interplay between palmitoylation and ubiquitination of newly synthesized membrane proteins in the ER. As a model protein we have chosen the Wnt signaling co-receptor LRP6. This led us to identify a novel folding machinery in the ER, which depends on ubiquitination on specific sites. Thus the role of ubiquitination in he ER is not solely to target proteins to the ER associated degradation pathway, but may also serve a role in folding. More specifically we found that newly synthesized LRP6 rapidly acquires ubiquitin on one specific juxtamembrane lysine. This event promotes folding. We found that this ubiquitination is reversed by the ER localized deubiquitinating enzyme USP19, which thus plays a major role in controlling the cellular LRP6 levels in cells. Ubiquitination-deubiquitination may occur several times to provide LRP6 with time to fold. Once folding is adequate, the protein can exit the ER, an event that depends on its palmitoylation. If folding of certain molecules is too slow or deficient, then LRP6 undergoes polyubiquitination on other lysines and is targeted to ER associated degradation. This folding machinery is not restricted to LRP6, and we are currently investigating its relevance to other proteins.

Finally we have started to investigate the role of palmitoylation in regulating the morphology of the ER, which is connected to its function in particular in the balance between protein and lipid synthesis. We found that DHHC6 has a major impact on the formation of ER sheets, which compose the rough ER, and that this effect is to a large extend mediated by its ability to modify the ER shaping protein Climp63.

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