Scientists at the Scripps Research Institute have developed a set of clickable, photoreactive sterol probes and used them for proteome-wide profiling of cholesterol-binding proteins in HeLa cells.
Using a chemoproteomic approach detailed in a paper published this week in Nature Methods, the researchers identified more than 250 cholesterol-binding proteins, among them a number of proteins not previously known to interact with cholesterol, said Jonathan Hulce, first author on the study and a graduate student in the lab of Scripps professor Benjamin Cravatt, who led the effort.
As an important element in cellular membranes and a precursor for a variety of signaling molecules, cholesterol plays a significant role in a number of physiological functions. However, Hulce told ProteoMonitor, scientists have lacked a convenient method for broadly identifying proteins that interact with the molecule.
Previously, "the evaluation of whether or not a protein interacts with cholesterol could really only be done on a case-by-case basis," he said. A researcher "would have to hypothesize at the outset that a given protein might be interacting with cholesterol … and then do some straightforward binding assays in vitro with, say, radiolabeled cholesterol."
To achieve their global profiling approach, the Scripps researchers used a set of three synthetic sterol probes – a cis-sterol, trans-sterol, and epi-sterol – that included a photoreactive group to allow for cross-linking to interacting proteins and an alkyne group that enabled them to purify the probes and their bound proteins via click chemistry.
Photoreactive sterol probes like those used in the study have existed for more than a decade, he noted, but no one had previously incorporated into them the click chemistry functionality.
This, Hulce suggested, wasn't due to any technical difficulties involved in incorporating such a chemistry, but stemmed more likely from a lack of good methods to analyze the large collections of proteins it would generate. The increased adoption in recent years of mass spec by life science researchers made such approaches more appealing, he said.
"With the appropriately high-powered mass spec instruments, it now becomes feasible to do these sorts of large-scale, proteomic profiling type of experiments," Hulce said. "Now the chemical tools match the technology in [terms of] the complexity of the data and what we can analyze."
The Scripps team used a Thermo Fisher Scientific Orbitrap Velos for their analysis, identifying roughly 850 proteins that interacted with the trans-sterol probe.
To establish a more stringent set of cholesterol-interacting proteins, they performed competition assays, treating light and heavy SILAC-labeled HeLa cells with the trans-sterol probe and then treating the heavy-labeled cells with excess cholesterol. Those proteins – a set of 265 – that exhibited decreased binding to the probe in the presence of excess cholesterol were determined to be cholesterol-interacting.
Among these proteins were a number of known cholesterol interactors, which, Hulce noted, encouraged the team regarding the validity of their findings. These proteins included Scap, a well-known cholesterol-sensing protein in the endoplasmic reticulum; and HMG-CoA reductase, which plays a role in cholesterol production in the liver and is the target of statin drugs.
Perhaps more interesting, though, Hulce said, were the proteins they identified that had not previously been known to interact with cholesterols, including hexokinase 1 and 2 and ADP-dependent glucokinase, all of which are involved in regulating glycolysis and the pentose phosphate pathway.
Additionally, the researchers observed "a number of proteins regulating protein modifications in terms of lipidation as well as glycosylation," he said. "Taken all together, we identified what I believe to be certain nodes and biochemical pathways that indicate a level of regulation that sterols exhibit outside their own biosynthesis."
Hulce and his colleagues experimentally validated six of these previously unobserved interactions, expressing recombinant target proteins in a HeLa cell, treating the cell with the trans-sterol probe, and detecting the probe-labeled proteins via in-gel fluorescence. They also repeated the competition assays, determining that these probe-protein interactions were inhibited by excess cholesterol.
The Scripps group noted several potential limitations of the probes, in particular the possibility that the alkyne modification enabling the click chemistry could impair some sterol-protein interactions. They also said that native esterase could cleave the alkyne's ester linkage, potentially reducing the assay's sensitivity. Both issues, they suggested, could be resolved by modifications to the probe's chemistry.
The researchers now aim to apply their method to specific research questions and have launched two collaborations: One with University of California, San Diego, scientist Paul Mischel investigating the role of cholesterol in glioma with the goal of identifying novel therapeutic targets; the other with Scripps researcher Phil Baran on competitive profiling of steroidal natural products.
Hulce said that the team is also in talks with several companies to make the probes commercially available as a research tool, although he declined to name them. He said the researchers currently had no plans to patent the probes.
"We're going to try to keep it an open tool," he said. "I think it will find the most utility that way."