Researchers discover previously unknown link between protein and fatty acids

Study contributes to a better understanding of a protein essential to the survival and rapid proliferation of tumor cells.

By Karina Toledo

Agência FAPESP – A study by the Tumor Metabolism Research Group at Brazil’s National Bioscience Laboratory (LNBio) has contributed to a better understanding of the action of one of the proteins that are essential to the survival and rapid proliferation of tumor cells.

The group’s investigation of the structure of the hypoxia-inducible transcription factor 3α (HIF-3α) protein showed that it alone is capable of binding to fatty acids present in the extracellular fluid, unlike all other proteins in the HIF family.

The results of the research project, supported by FAPESP, were recently published in the journal Scientific Reports.

“It’s possible that when the fatty acid molecule binds to HIF-3α, it switches the protein on or off. Our next step is to try to understand the effects of this interaction in the cell,” said Andre Ambrosio, who coordinated the project along with Sandra Dias, another researcher at LNBio.

To grow and multiply rapidly, tumor cells must undergo a process of metabolic adaptation that enables them to capture larger amounts of nutrients and oxygen, Ambrosio explained. This entails the production of alternative proteins, including HIF, which modify cells’ gene expression profile during the adaptation process.

“HIF proteins regulate the expression of some 150 genes, or 5% of the human genome,” Ambrosio said. “They’re produced whenever cells become oxygen deficient and need to form new blood vessels, for example. This can happen in physiological situations such as embryo development or in pathological situations such as cancer or cardiovascular disease.”

HIF proteins can only act in pairs, which consist of the α (alpha) and β (beta) isoforms, Ambrosio added. Previous studies have shown that there are at least three variants of HIF-α (HIF-1α, HIF-2α, and HIF-3α) and that they all bind to the only type of HIF-β known to scientists so far.

“We decided to focus on HIF-3α in this research project because it has been discovered only recently and is less well known from a functional standpoint,” Ambrosio said.

Previous research has associated variants 1 and 2 with the regulation of genes encoding molecules such as erythropoietin (a hormone secreted by the kidneys that increases the production of red blood cells in response to falling levels of oxygen in the tissues), glucose transporters, vascular endothelial growth factor (VEGF), and other molecules that increase oxygen capture and facilitate metabolic adaptation to hypoxia.

When Ambrosio evaluated the three-dimensional (3D) structures of variants 1 and 2 described by other research groups, he concluded that they are incapable of binding to fatty acids, unlike variant 3. Moreover, this is the first time that an endogenous non-covalent ligand has been described for the HIF family.

Accidental discovery

The initial purpose of the project was to investigate how HIF-3α interacts with the β isoform and to look for clues to its function in the cell. To do this, the team at LNBio developed a system for producing the protein in the laboratory: the gene that encodes human HIF-3α was inserted into bacteria of the species Escherichia coli, which then expressed the molecule.

Next, the researchers decided to study the protein’s 3D structure using X-ray crystallography. A crystal formed from a concentrated solution of the purified protein was placed in an intense beam of X-rays so that the crystal’s atoms would diffract the X-rays.

This study was part of Angela Maria Fala’s research for her master’s degree and her early PhD research, performed under Ambrosio’s supervision and supported by scholarships from FAPESP.

“Our analysis of the X-ray diffraction data showed that a fatty acid molecule originating in E. coli binds to the protein in a highly organized way. This was completely unexpected,” Ambrosio said.

Through in vitro assays, the group discovered that when the protein interacts with the fatty acid, the bond with the β isoform becomes more stable. What they do not yet know is how this affects cell functioning.

According to Ambrosio, fatty acids are present in the phospholipids that form cell membranes and organelles. They are also key components of triacylglycerol (TAG), one of the sources of cellular energy.

“In other words, fatty acids are associated both with structural events in cells, and especially the control of nutrients entering and exiting via the membrane, and with energy events,” Dias said. “The fact that they bind to HIF-3α suggests that the protein may somehow be involved in the regulation of these processes.”

According to the researchers, the results of the in vitro experiments also suggest that the fatty acid in question plays an important role in maintaining the structure of HIF-3α, as if it were a sort of spinal column. “Every time we removed the fatty acid, the protein precipitated out of solution. That means either the protein unraveled, losing its 3D structure, or protein aggregation occurred. In both cases, it loses function,” Ambrosio said.

Human cell context

In partnership with Dias, Ambrosio plans to perform new experiments at LNBio, which is affiliated with the National Energy & Materials Research Center (CNPEM), to investigate how the interaction between HIF-3α and fatty acids affects the functioning of human cells.

“The first step is to identify the cellular models in which this protein is important. Nothing is known about this at present. Then, we’ll have to learn how to isolate the protein and find out which lipids it interacts with in human cells. Only then will we be able to investigate the importance of these lipids to the functions of the protein,” Ambrosio said.