A novel tool has been developed to visualize the process of palmitoylation—the post-translational modification that allows peripheral membrane proteins (PMPs) to associate with cellular membranes—in living cells. More specifically, the method investigates the enzymes, palmitoyl acyltransferases (PATs), that catalyze lipid modification so that peripheral membrane proteins can temporarily bind to cell membranes.

The tool not only allows a deeper understanding of how palmitoylation works, but it may also open up its use for therapeutic purposes. Some of the proteins that undergo this process can mutate and become oncogenic, such as the RAS proteins, which are responsible for about one-third of cancers.

The research team has demonstrated that, contrary to what was previously thought, palmitoylation is possible not only in the Golgi apparatus, the usual site for protein processing and delivery, but also at the plasma membrane.

This work is published in Nature Communications in the article, “Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo.”

Palmitoylation consists of the introduction of fatty acids into certain proteins for them to be able to bind to cell membranes. The mechanism is governed by precise rules, which depend on the sequence of each type of protein, and on the presence of specialized enzymes. Until now, the scientific community thought that palmitoylation of peripheral membrane proteins could only take place in one place in the cell—the Golgi apparatus.

“Indeed, these proteins are produced in the cytosol—the cell fluid—and then ‘swim’ to the Golgi apparatus, where they are modified before being transported to where they need to act,” explained Gonzalo Solis, PhD, senior research associate in the department of cell physiology and metabolism at the Université de Genève (UNIGE). “Nevertheless, we hypothesized the possibility of local palmitoylation, without passing through the Golgi apparatus. If this is true, it opens up completely new possibilities for the intervention of this mechanism.”

To test this hypothesis, the research team used a new methodology to focus on the Gαo protein, which is normally located at the plasma membrane and the Golgi apparatus.

More specifically, they describe the “partitioning of Gαo—α-subunit of heterotrimeric Go proteins—to PM and Golgi, independent from Golgi-to-PM transport.”

“We brought the palmitoylating enzymes to a totally different compartment in the cell, the nuclear membrane,” explained Solis. “Gαo was recruited at the nuclear membrane, allowing us to identify the specific enzyme that palmitoylates them. We thus confirmed that this process can take place on the very site the protein is needed.”

The new tool, S-palmitoylation at the outer nuclear membrane assay (“SwissKASH”), probes substrate specificity of PATs in intact cells. It keeps the cell alive and allows for the observation of the process dynamically. “Until now, there was no alternative to destroying the cell,” said Vladimir Katanaev, PhD, professor at the Centre for Translational Research in Onco-haematology and at the department of cell physiology and metabolism at UNIGE. “Our method also makes it possible to determine exactly which protein reacts to which enzyme locally, which is essential if we want to control this mechanism for therapeutic purposes.”

This discovery paves the way for innovative drug discovery strategies to target very precisely the membrane binding—and therefore activity—of oncogenic proteins.

Several peripheral membrane proteins, and in particular Gα subunit proteins and RAS proteins, are susceptible to mutation and thus acquire an aggressive oncogenic potential. Their oncogenicity depends on their ability to bind to the plasma membrane; palmitoylation thus plays a key role in the transformation of a healthy cell into a cancerous one.

“Inhibiting the enzyme that induces palmitoylation, and preventing the oncogenic protein from binding to the plasma membrane, could therefore defuse its pathogenicity,” noted Solis. “We can thus imagine blocking this specific reaction without unbalancing the whole system.” The scientists will now aim at automatizing this methodology to study the effect of a whole series of pharmaceutical products on the palmitoylation of selected oncoproteins, as well as testing their toxicity on the whole cell.

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