Author: Sarah Kearns

Editors: Zena Lapp, Jimmy Brancho, Noah Steinfeld

After you get home from work, perhaps after eating dinner, you may start working on other projects or hobbies. Humans aren’t the only ones that have a life after hours. Recently it’s been discovered that many proteins have roles in the cell outside of their main functions. This peculiar behavior led to the name ‘moonlighting,’ referencing individuals who have multiple jobs. A useful analogy might be a werewolf’s behavior under a full moon: being a person during the day, but a wolf at night.

Cryptic Sightings

Imagine you are studying sugar breakdown, known as glycolysis, in bacteria. One protein that you’re particularly interested in is 6-phosphofructokinase, which is found in the cytoplasm where it has a well-characterized role taking one substrate and converting it into another. However, to your great surprise, you find it in an unexpected location: all the way on the cell surface (Figure 1)! You wonder why this protein escaped the cytosol and what it’s doing on the surface membrane. Much to your surprise, it’s acting as a receptor that recognizes a host protein, helping the bacteria invade host organisms. This protein, as one of the first identified with two very different roles in the cell, highlights the functional and spacial variety that a protein can potentially have.

Figure 1: While studying process #1 in the cytoplasm, you find that your protein is surprisingly on the cell surface, performing process #2. Image Source: Sarah Kearns

Much like stumbling upon Wolfman while camping, researchers found ‘were-proteins’ purely by accident, noticing proteins in strange places in the cell. Since their first sighting in the late 1980’s, researchers have discovered many moonlighting proteins and hypothesize that there could be countless more. Current research aims to find moonlighting proteins, identify their secondary functions, understand how these dual roles originated, and determine how proteins migrate within cells to perform their different functions.

Biochemical Rules – Urban Myths and Legends

The discovery of moonlighting proteins was shocking because their existence challenged the established principles of how proteins function. For the better half of the past century each protein was thought have its own exclusive role in a complex biological system. One job per protein. This theory is referred to as the ‘lock and key model’ where the protein symbolizes a key that is able to unlock one door and one door only. This model goes hand in hand with another staple biochemical principle: form follows function. This convention prescribes that the physical shape of a protein determines the chemical reaction it performs.

Since the lock and key model was proposed, however, scientists have found that proteins have dynamic shapes, can perform the same reactions on different molecules, or be part of multiple chemical pathways within the cell. So instead of having static 3D structures like standard keys, proteins change their shapes as they move in solution or as they perform their reactions. With this new information, the lock and key model and form follows function have been updated: one key can open at least two locks, often by changing its shape.

How did proteins with multiple functions arise?

One of the first theories for the emergence of moonlighting functions suggested that secondary roles evolved as a way to allow an organism to expand its functions without the energy expense of making a second protein. Transcription and translation—going from DNA to RNA and then from RNA to protein—are expensive processes for a cell, so getting a two-for-one deal would save energy. As such, researchers who first noticed moonlighting initially called it gene sharing.

While this energy-saving hypothesis intuitively makes a lot of sense, the discovery that most genes do not encode proteins suggests the gene sharing model is unlikely, at least in terms of energy conservation at a transcriptional level. Many transcribed genes do not seem to have a role in the cell, at least at a protein level. Instead, large portions of DNA have products that regulate gene expression. Wasteful transcription in cells suggests that there is little pressure for energy conservation from transcription and therefore no drive for proteins to share genes themselves.

It’s important to note that evolution has no real goal in mind — whatever happens to work is propagated but there’s no way to know what will or will not be more efficient. This is sometimes referred to as the ‘tinkerer’s way’ of evolution. Any mutations, then, that randomly or spontaneously enhance a protein’s capacity to accomplish a novel function are maintained and selected for over time. These mutations manifest as alterations of a protein’s shape. Such changes could better accommodate substrates to keep other molecules out or could even introduce entirely new functions. However, not all mutations are beneficial: even small changes can destroy functionality rather than improve it. Because of this, a duplicate gene can be beneficial to an species, and may arise through an error itself. After a gene has been duplicated, because these two copies function redundantly, the protein gene products can diverge but often remain similar enough to perform the same role. This can lead to the rise of multiple functions and preservation of the original role of the gene (Figure 2).

Figure 2: The initial gene products result in a yellow protein that changes a triangle to a square. But when the gene becomes duplicated, while it maintains its original function, it can also become a receptor for a blue signal molecule over time. Image Source: Sarah Kearns

An alternative theory on the evolution of moonlighting functions postulates that a single protein can fold into multiple shapes, changing its form and therefore function. This theory is suggested by certain disease states where a protein changes from its normal shape to a misfolded shape that gives it an aberrant function. This finding suggests that morphing into other shapes to perform different reactions could be possible in moonlighting proteins. Similarly, proteins can bind to other self-same proteins, allowing different forms and functions (Figure 3). Tau proteins combine both of these concepts, both functioning with other self-same proteins and misfolding, typically serving as cellular scaffolding but forming plaques in neurodegenerative disease states.

Figure 3: When the protein is on its own, it performs a reaction. However, if it forms a complex with another self-same protein, it has a different function as a dimer. Image Source: Sarah Kearns

The search continues

Even though we have hypotheses about the origin of moonlighting proteins, how they are able to act at different locations within the cell is still largely a mystery. Some research has shown that chemical modifications can signal certain proteins to move to different locations, but it is still unclear how they are shuttled in a function-dependent manner. Researchers also hope to predict which proteins have moonlighting functions without having to simply observe proteins to see which exhibit this behavior. Once we understand them better, researchers want to call on moonlighting proteins and use their alternative functions to combat diseases. If researchers could induce a cellular signal that called moonlighting proteins away from their werewolf role in disease, perhaps they could coerce them into becoming human again!

About the author:


Sarah Kearns is a second-year PhD student at the University of Michigan studying Chemical Biology. She’s interested in using structural biology to probe substrate recognition in proteins to design better drug therapies. Outside of the lab, she writes for MiSciWriters for which she serves as a content editor and as the communications director. When not researching or writing about science, she is an open access advocate, an avid baker, and always has at least three books on her current reading list. Sarah has her own blog, Annotated Science, and can be found on Twitter.

Read all posts by Sarah here.

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