After you get home from work, perhaps after eating dinner, you may start working on other projects that you have, something that you might call a hobby. Humans aren’t the only ones that have a life after hours. Recently it’s been discovered that many proteins also have roles in the cell outside of their main function. This peculiar behavior led to the name ‘moonlighting,’ referencing a werewolf’s behavior under a full moon.
Keywords: Moonlighting, protein interactions, evolution, and disease therapy.
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Cryptic Sightings
Imagine you are studying sugar breakdown, or glycolysis, in yeast. The protein that you’re particularly interested in is 6-phosphofructokinase which is found in the nucleus. As you perform your cell-based experiments, you find it in a surprising location; all the way on the cell surface (Figure 1)! You wonder why this protein left the confines of the nucleus and what it’s doing on the surface membrane. Much to your surprise, it’s acting as a receptor to recognize other yeast.
Figure 1: Trying to study process #1 in the nucleus, you find that your protein is surprisingly on the surface, performing process #2.
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, it turns out that multifunctional proteins are not uncommon. Many moonlighting proteins have been discovered since then and it’s hypothesized that there could be countless more. The current research identifying secondary functions, understanding how these dual roles originate, and how they migrate within a cell to perform their different functions.
Biochemical Rules – Urban Myths and Legends
Moonlighting proteins was initially a shock because they go against so many established principles of proteins. Their function for the better half of the past century was thought to be unique with each protein having 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.
Though a simple model, scientists use the lock and key model because it is supported by another staple biochemical principle: form follows function. This convention prescribes that the chemical reaction a protein catalyzes is due to its physical shape.
The lock and key model does not take into account that proteins have a dynamic shape and can have perform the same reaction to different molecules or be a part of numerous chemical pathways within the cell. Since realizing moonlighting functions exist however, the lock and key model along with form follows function have come to mean different things. There are now at least two locks and two functions that have emerged dependent on the protein’s shape.
Evolutionary Perspective
One of the first rationales for the emergence of moonlighting functions was based in genetics. The theory was secondary functions evolved as a way to expand the functions of an organism without the expense of increasing the amount of genetic material. Transcription, going from DNA to protein, is a very intensive process for a cell, so getting a two-for-one deal on cellular functions would optimize the process. As such researchers who first noticed moonlighting initially called it gene sharing.
While this energy saving hypothesis makes a lot of sense, combined with the discovery that significant parts of the genome in most organisms have very little function at the protein level, the gene sharing model is unlikely. Instead of playing a big cellular role, large portions of DNA have products that regulate gene duplication instead of making a protein product. As such, there is less pressure for energy conservation from translation and therefore no drive for proteins to share genes.
It’s important to note that evolution has no real goal in mind — whatever happens to work is propagated but there’s no real sense of knowing 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 occur which enhance a protein’s capacity to accomplish a novel function are maintained and selected for over time.
Any external pressures or having functions evolve over time manifests itself in the protein as the alteration of its shape. This morphology could mean a change to better accommodate a substrate and keep other molecules out. Even small changes can destroy functionality rather than improve it. In fact, cells will often do something called gene duplication , where two copies of genes are stored in DNA. After the genes have been duplicated, the protein gene products can diverge but still remain essentially similar enough to perform the same role. This means that multiple functions could arise after gene duplication (Figure 2).
Figure 2: The initial gene products in a protein that changes a triangle into 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.
An alternative theory on the evolution of moonlighting functions is based on morphology (Figure 3). It hypothesizes that proteins either fold in a different shape or conformation, thus changing the form and therefore function, or can bind to other self-same proteins.
Figure 3: When the protein is on it’s own, it performs its normal reaction. However, if it forms a complex with another self-same protein, it has a different function as a dimer.
Rules of changing function, any patterns found
The pathway of a protein migrating, from the nucleus to the cell surface, for example, is not so straightforward. For many of the alterations of protein function and location, the rationale and methodology is not known: the only thing observed is that an already identified protein is seemingly not where it should be. Though the history and evolutionary perspective allow for understanding the basis of one protein evolving different jobs within the cell, it does not explain how the protein “knows” to clock in for it’s second shift.
One study looked specifically at moonlighting proteins that have different functions inside and outside of the cell, like 6-phosphofructokinase. Unfortunately, they did not find any features that are out of the ordinary — most of them had reasonable sizes and stability — and it turned out that many types of protein folding patterns can exhibit moonlighting. This ties in with the unfortunate case that most moonlighting functions are found through serendipity.
Significant amounts of effort has been poured into using computational means to predicting protein and enzymatic functions. Amino acid sequencing tools have so far been very useful in theorizing a protein’s molecular functions, but have thus far not been able to anticipate cellular functions. Some conserved, or ancestrally important, amino acids have been identified in specific cases but further studies need to confirm this.
The search continues
Even though origin story of moonlighting proteins, how they are able to travel long distances within the cell to perform their alternative function is still largely a mystery. Some research has shown that chemical modifications can signal it to move to a different location, but more work needs to be done to figure out how they are shuttled in a function-dependent way. Using bioinformatics, researchers hope to be able to find useful proteins without having to stumble around the forest. Once identifying and understanding them better, researchers want to call upon moonlighting protein in diseased states.
Sarah Kearns is a second-year PhD student at the University of Michigan studying Chemical Biology. She studies the recognition and mechanism of exchange of a CH3 group between enzyme and substrate in the Trievel Lab. Outside of the lab, she writes for MiSciWriters – for which she serves as a content editor and as the communications director – F1000, and her own blog, Annotated Science. You can find and connect with her on Twitter or in the comments below.
Images: Header; all other figures made by Sarah, with use of DNA stock photo.