Sodium proton transport proteins are present in all forms of life. Now researchers understand a little better how they are regulated.
Na+/H+ exchanger 1 (NHE1) is a sodium proton exchanger that regulates the pH (acid-base balance) inside our cells by ejecting acid in the form of protons from the cells.
This protein is one of the very first proteins of life and existed even before the first real cells emerged on Earth.
Many questions about how NHE1 functions have remained unanswered, but now Danish researchers have shed light on how it works.
In the long term, this discovery can be very important for treating people with such diseases as cancer, muscle disorders, cardiovascular diseases and nonalcoholic steatohepatitis.
“NHE1 is important for developing such diseases as cancer or metabolic syndrome that involve metabolic changes, and it is present in virtually all human cells. Further, NHE1 plays a role in all the mechanisms that maintain the function of the proteins in our cells by ensuring that they have the appropriate pH. This means that when NHE1 does not work as it should, the whole body does not work either,” explains a researcher behind the study, Stine Falsig Pedersen, Professor, Department of Biology, University of Copenhagen and group leader of the University’s NHE1 group – one of the world’s leading groups conducting research on this important protein.
The research results were recently published in Nature Communications.
Protein complex controls the activities of many proteins
In addition to regulating the internal pH of cells, NHE1 interacts with another protein, the enzyme calcineurin, which is also present in virtually all cells and regulates the activity of many of the body’s proteins.
Once proteins have been created, they must be able to respond to the condition of a cell so that their activity precisely matches the need.
This is often regulated by adding small phosphorous groups to the proteins or by removing them again. Calcineurin removes them.
When calcineurin is bound to NHE1, it regulates the activity of NHE1, keeping the internal pH tightly regulated.
“We have for the first time mapped the structure of parts of the two proteins together, so we can see how calcineurin can remove just one specific phosphorus group from NHE1, virtually without touching the others,” says Stine Falsig Pedersen.
Collaboration between researchers with different competencies
The researchers used various advanced techniques to elucidate the function and structure of NHE1 and calcineurin, including X-ray crystallography, in which the researchers first crystallized the protein, then froze it before finally sending X-rays through it. The researchers used the scattering of the X-rays to determine the structure of the protein.
Further, the researchers used nuclear magnetic resonance (NMR) imaging to study how the protein complex functions when NHE1 is bound to calcineurin, and they have expressed a wide range of NHE1 mutants in cells and studied the activity of NHE1 using real-time immunofluorescence techniques.
“The research is very interdisciplinary and required the competencies of three research groups, each with its area of expertise,” says Stine Falsig Pedersen.
The research was a collaboration between Stine Falsig Pedersen’s research group, who are experts in NHE1 and cellular pH regulation; Birthe B. Kragelund’s research group, who are experts in using NMR imaging to study the function of proteins; and a research group from the University of Arizona, who are experts in calcineurin and X-ray crystallography.
First to identify specific binding between NHE1 and calcineurin
An interesting basic finding in the new study is that calcineurin is more specific than it might otherwise appear.
Researchers know very little about how calcineurin “selects” the proteins to be dephosphorylated, and based solely on the exact part of the protein that is near the site to be dephosphorylated, calcineurin should non-specifically dephosphorylate most phosphorus groups in NHE1.
But amazingly, the new research shows that only a very specific residue of human NHE1 called T779 is dephosphorylated.
“Besides the association with calcineurin, we are also the first to discover that T779 is important in activating NHE1, and this is essential for understanding how NHE1 functions and affects the regulation of cells and the development of various diseases. We did not know that recognition was so specific, because this has never been described before,” explains Stine Falsig Pedersen.
Mice without NHE1 cannot develop nonalcoholic steatohepatitis
Stine Falsig Pedersen explains that researchers have recently found that knockout mice lacking NHE1 are protected from developing nonalcoholic steatohepatitis.
She recently received a grant to investigate exactly how NHE1 influences the development of nonalcoholic steatohepatitis and whether regulating NHE1 with medicine can counteract it.
“In nonalcoholic steatohepatitis, calcium homeostasis is dysregulated. Calcineurin is also regulated by calcium and dysregulated in nonalcoholic steatohepatitis, which indicates that the entire interaction between NHE1, calcineurin and calcium may be one of the mechanisms behind nonalcoholic steatohepatitis. We are now investigating this further,” says Stine Falsig Pedersen.
NHE1 involved in cancer, muscle disorders and cardiovascular diseases
Discoveries about NHE1 may also mean new ways to investigate or treat people with cancer in the future.
Stine Falsig Pedersen’s research has shown that NHE1 does not function correctly in many types of cancer. There is often too much of it, and it is too active, so the cells become “too efficient” to eject acid.
NHE1 may therefore become a possible target for cancer treatments when its function is dysregulated.
Finally, NHE1 is also being investigated as a target for treatments for the debilitating muscle disorder Duchenne’s muscular dystrophy and as a target for treating people with cardiovascular diseases.
“In both cases, the dysregulation of NHE1 is associated with calcium dysregulation, so the link between NHE1 and calcineurin could also become important,” explains Stine Falsig Pedersen.
”Molecular basis for the binding and selective dephosphorylation of Na+/H+ exchanger 1 by calcineurin” has been published in Nature Communications. In 2018, the Novo Nordisk Foundation awarded a grant to Stine Falsig Pedersen for the project Investigating the Role of NHE1-mediated Ca2+ dysregulation and Mitochondrial Damage in Nonalcoholic Steatohepatitis (NASH).