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What is Protein Moonlighting and Why is it Important?

Constance J. Jeffery

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

1.1 What is Protein Moonlighting?

Moonlighting proteins exhibit more than one physiologically relevant biochemical or biophysical function within one polypeptide chain (Jeffery 1999). In this class of multifunctional proteins, the multiple functions are not due to gene fusions, multiple RNA splice variants or multiple proteolytic fragments. The moonlighting proteins do not include pleiotropic proteins, where a protein has multiple downstream cellular roles in different pathways or physiological processes that result from a single biochemical or biophysical function of a protein. Moonlighting proteins also do not include families of homologous proteins if the different functions are performed by different members of the protein family.

Some of the first moonlighting proteins to be identified were taxon-specific crystallins in the lens of the eye. These proteins, including the delta 2 crystallin/arginosuccinate lyase in the duck (Wistow and Piatigorsky 1987), upsilon crystallin/lactate dehydrogenase A in the duckbill platypus (van Rheede et al. 2003), eta-crystallin/cytosolic aldehyde dehydrogenase (ALDH class 1) in the elephant shrew (Bateman et al. 2003), and several others, are ubiquitous soluble enzymes that were adopted as structural proteins in the lens. Other well-known moonlighting proteins include soluble enzymes in biochemical pathways that also bind to DNA or RNA to regulate transcription or translation. Human thymidylate synthase (TS), a cytosolic enzyme in the de novo synthesis of the DNA precursor thymidylate, also binds to mRNA encoding TS to inhibit translation (Chu et al. 1991). The Salmonella typhimurium PutA protein is an enzyme with proline dehydrogenase and proline oxidase pyrroline-5-carboxylic acid dehydrogenase activity when it is bound to the inner side of the plasma membrane (Menzel and Roth 1981a, b), but it also binds to DNA and moonlights as a transcriptional repressor of the put operon (Ostrovsky de Spicer et al. 1991; Ostrovsky de Spicer and Maloy 1993). The E. coli BirA biotin synthase is an enzyme in the biotin biosynthetic pathway that is also a bio operon suppressor (Barker and Campbell 1981). Saccharomyces cerevisiae N-acetylglutamate kinase/N-acetylglutamyl-phosphate reductase (Arg5,6p) is an enzyme in the arginine biosynthetic pathway (Boonchird et al. 1991; Abadjieva et al. 2001) and also binds to mitochondrial and nuclear DNA to regulate expression of several genes (Hall et al. 2004). Kluyveromyces lactis galactokinase (GAL1) phosphorylates galactose and is also a transcriptional activator of genes in the GAL operon (Meyer et al. 1991).

Perhaps even more surprising than the fact that some proteins can perform such different functions is that such a large variety of proteins moonlight. Over the past few decades, hundreds of proteins have been shown to moonlight (Mani et al. 2015; moonlightingproteins.org). They include many types of proteins: enzymes, scaffolds, receptors, adhesins, channels, transcription and translation regulators, extracellular matrix proteins, growth factors, and many others. They are active in a variety of physiological processes and biochemical pathways, are found in the cytoplasm, nucleus, mitochondria, on cell surface, and other cellular compartments, and some are secreted. They are also expressed in many different cell types within a species. They are found in a variety of species from throughout the evolutionary tree. They are common in eukaryotes in humans and other placental and monotreme (i.e., platypus) mammals, reptiles, birds, amphibians, fish, worms, insects, plants, fungi, and protozoans. A few are found in archea and many more have been identified in eubacteria, including pathogenic species (Clostridium difficile, Helicobacter pylori, Pseudomonas aeruginosa, Staphylococcus, etc.) as well as nonpathogenic, commensal bacteria, including health-promoting or "pro-biotic" species (Bifidobacterium). A few moonlighting proteins have even been found in viruses.

The variety also extends to the combinations of functions that are observed. Many of the known moonlighting proteins are cytosolic enzymes, chaperones, or other proteins that exhibit a second function in other cellular locations, for example as a receptor on the cell surface. Several proteins described in more detail in other chapters are cytosolic enzymes or chaperones that are secreted to serve as growth hormones or cytokines. For example, an enzymatic function and an extracellular cytokine function are found in phosphoglucose isomerase/autocrine motility factor (Gurney et al. 1986a, b; Chaput et al. 1988; Faik et al. 1988; Watanabe et al. 1996; Xu et al. 1996). Many of the moonlighting proteins have surprisingly unrelated functions, such as the PHGPx (glutathione peroxidase), a soluble enzyme that is also a sperm structural protein (Scheerer et al. 2007), adopted during evolution for its structural characteristics in the same way as taxon-specific crystallins above. Other proteins can exhibit two functions even within the same cellular compartment and may change function as cellular conditions change, for example changes in pH, or the concentration of a ligand, substrate, cofactor, or product. Still other moonlighting proteins have one function as a monomer or homo-multimer but interact with other proteins in a multiprotein complex, such as the proteasome or ribosome. Some proteins even have more than two functions, for example glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase.

1.2 Why is Moonlighting Important?

1.2.1 Many More Proteins Might Moonlight

The diverse examples of moonlighting proteins already identified suggest that many more moonlighting proteins are likely to found. The ability of one protein to perform multiple functions greatly expands the possible number of functions that can be performed by the proteome. In addition, the study of the molecular mechanisms and regulation of moonlighting functions helps broaden our understanding of protein biochemistry and suggests additional activities that might be encoded by genomes.

1.2.2 Protein Structure/Evolution

X-ray crystal structures and other biochemical and biophysical studies of some of the moonlighting proteins have added to our understanding of how one protein can perform two different functions and, in some cases, provided information about the triggers and molecular mechanisms involved in switching between two activities (several examples reviewed in Jeffery 2004, 2009).

In cases where the function of the protein changes in response to changes in the environment, moonlighting proteins provide examples of how a protein can sense and respond to these changes, and thereby provide interesting examples of regulation of protein function. The hemagglutinin-neuraminidase of paramyxovirus, which causes mumps, has different conformations at high- and low-pH conditions. The protein first enables binding of the virus to the surface of host cells. A change in pH promotes the movement of several amino acid side-chains and a loop in the active site to switch between the protein's sialic acid binding and hydrolysis functions so that it can cleave the glycosidic linkages of neuraminic acids (Crennell et al. 2000). The E. coli periplasmic serine endoprotease/heat-shock protein DegP (Protease Do) switches from a peptidase at high temperatures to a protein-folding chaperone at lower temperatures (Krojer et al. 2002).

In some moonlighting proteins the two functional sites are located distant from each other on the protein surface and the protein can perform both functions simultaneously, but in other proteins the functional sites are close to each other or even overlapping. Streptomyces coelicolor albaflavenone monooxygenase/synthase has a heme-dependent monooxygenase activity to catalyze the reaction (+)-epi-isozizaene?+?2 NADPH?+?2 O(2)?<?=?>?albaflavenone?+?2 NADP(+)?+?3?H(2)O and has a typical cytochrome P450 fold. However, the protein was also found to exhibit terpene synthase activity. After solution of its X-ray crystal structure a second active site pocket, for terpene synthase activity, was identified in an alpha-helical barrel near the monooxygenase active site (Zhao et al. 2008, 2009). It was recently found that the fructose-1,6-bisphosphate aldolase/phosphatase enzymes from the hyperthermophiles Thermoproteus neutrophilususes and Sulfolobus tokodaii utilize a single active site pocket to catalyze two reactions in the same biochemical pathway (Du et al. 2011; Fushinobu et al. 2011). After completion of the first catalytic function, several loops undergo conformational changes in order to bind the second substrate and perform the second catalysis.

A much larger conformational change occurs in cytosolic aconitase that renders the protein unable to perform one function but able to perform a second function. Aconitase is an enzyme...