Moonlighting Proteins: Diverse Functions Found in Fungi.

Moonlighting proteins combine multiple functions in one polypeptide chain. An increasing number of moonlighting proteins are being found in diverse fungal taxa that vary in morphology, life cycle, and ecological niche. In this mini-review we discuss examples of moonlighting proteins in fungi that illustrate their roles in transcription and DNA metabolism, translation and RNA metabolism, protein folding, and regulation of protein function, and their interaction with other cell types and host proteins.

Moonlighting Proteins in the Fuzzy Logic of Cellular Metabolism.

The numerous interconnected biochemical pathways that make up the metabolism of a living cell comprise a fuzzy logic system because of its high level of complexity and our inability to fully understand, predict, and model the many activities, how they interact, and their regulation. Each cell contains thousands of proteins with changing levels of expression, levels of activity, and patterns of interactions. Adding more layers of complexity is the number of proteins that have multiple functions. Moonlighting proteins include a wide variety of proteins where two or more functions are performed by one polypeptide chain. In this article, we discuss examples of proteins with variable functions that contribute to the fuzziness of cellular metabolism.

Multitalented actors inside and outside the cell: recent discoveries add to the number of moonlighting proteins.

During the past few decades, it's become clear that many enzymes evolved not only to act as specific, finely tuned and carefully regulated catalysts, but also to perform a second, completely different function in the cell. In general, these moonlighting proteins have a single polypeptide chain that performs two or more distinct and physiologically relevant biochemical or biophysical functions. This mini-review describes examples of moonlighting proteins that have been found within the past few years, including some that play key roles in human and animal diseases and in the regulation of biochemical pathways in food crops. Several belong to two of the most common subclasses of moonlighting proteins: trigger enzymes and intracellular/surface moonlighting proteins, but a few represent less often observed combinations of functions. These examples also help illustrate some of the current methods used for identifying proteins with multiple functions. In general, a greater understanding about the functions and molecular mechanisms of moonlighting proteins, their roles in the regulation of cellular processes, and their involvement in health and disease could aid in many areas including developing new antibiotics, predicting the functions of the millions of proteins being identified through genome sequencing projects, designing novel proteins, using biological circuitry analysis to construct bacterial strains that are better producers of materials for industrial use, and developing methods to tweak biochemical pathways for increasing yields of food crops.

Protein moonlighting: what is it, and why is it important?

Members of the GroEL/HSP60 protein family have been studied for many years because of their critical roles as ATP-dependent molecular chaperones, so it might come as a surprise that some have important functions in ATP-poor conditions, for example, when secreted outside the cell. At least some members of each of the HSP10, HSP70, HSP90, HSP100 and HSP110 heat shock protein families are also 'moonlighting proteins'. Moonlighting proteins exhibit more than one physiologically relevant biochemical or biophysical function within one polypeptide chain. In this class of multifunctional proteins, the multiple functions are not due to gene fusions or multiple proteolytic fragments. Several hundred moonlighting proteins have been identified, and they include a diverse set of proteins with a large variety of functions. Some participate in multiple biochemical processes by using an active site pocket for catalysis and a different part of the protein's surface to interact with other proteins. Moonlighting proteins play a central role in many diseases, and the development of novel treatments would be aided by more information addressing current questions, for example, how some are targeted to multiple cellular locations and how a single function can be targeted by therapeutics without targeting a function not involved in disease.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.

Moonlighting proteins - nature's Swiss army knives.

The human body is a complex biological machine with billions of cells and vast numbers of biochemical processes - but our genome only contains 22,000 protein-encoding genes. Moonlighting proteins provide one way to increase the number of cellular activities. Moonlighting proteins exhibit more than one physiologically relevant biochemical or biophysical function within one polypeptide chain. Already more than 300 moonlighting proteins have been identified, and they include a diverse set of proteins with a large variety of functions. This article discusses examples of moonlighting proteins, how one protein structure can perform two different functions, and how the multiple functions can be regulated. In addition to learning more about what our proteins do and how they work together in complex multilayered interaction networks and processes in our bodies, the study of moonlighting proteins can inform future synthetic biology projects in making proteins that perform new functions and new combinations of functions, for example, for synthesising new materials, delivering drugs into cells, and in bioremediation.

Disruption of the monocarboxylate transporter-4-basigin interaction inhibits the hypoxic response, proliferation, and tumor progression.

We have previously shown that glioblastoma stem cells (GSCs) are enriched in the hypoxic tumor microenvironment, and that monocarboxylate transporter-4 (MCT4) is critical for mediating GSC signaling in hypoxia. Basigin is involved in many physiological functions during early stages of development and in cancer and is required for functional plasma membrane expression of MCT4. We sought to determine if disruption of the MCT-Basigin interaction may be achieved with a small molecule. Using a cell-based drug-screening assay, we identified Acriflavine (ACF), a small molecule that inhibits the binding between Basigin and MCT4. Surface plasmon resonance and cellular thermal-shift-assays confirmed ACF binding to basigin in vitro and in live glioblastoma cells, respectively. ACF significantly inhibited growth and self-renewal potential of several glioblastoma neurosphere lines in vitro, and this activity was further augmented by hypoxia. Finally, treatment of mice bearing GSC-derived xenografts resulted in significant inhibition of tumor progression in early and late-stage disease. ACF treatment inhibited intratumoral expression of VEGF and tumor vascularization. Our work serves as a proof-of-concept as it shows, for the first time, that disruption of MCT binding to their chaperon, Basigin, may be an effective approach to target GSC and to inhibit angiogenesis and tumor progression.

An analysis of surface proteomics results reveals novel candidates for intracellular/surface moonlighting proteins in bacteria.

Proteins expressed on the bacterial cell surface play important roles in infection and virulence and can be targets for vaccine development or used as biomarkers. Surprisingly, an increasing number of surface proteins are being found to be identical to intracellular enzymes and chaperones, and a few dozen intracellular/surface moonlighting proteins have been found that have different functions inside the cell and on the cell surface. The results of twenty-two published bacterial surface proteomics studies were analyzed using bioinformatics tools to consider how many additional intracellular proteins are also found on the cell surface. More than 1000 out of the 3619 proteins observed on the cell surface lack the transmembrane alpha-helices or transmembrane beta-barrels found in integral membrane proteins and also lack the signal peptides found in proteins secreted through the Sec pathway. Many of the proteins found on the cell surface are intracellular chaperones or enzymes involved in central metabolic pathways, including some that have previously been shown to have a moonlighting function on the cell surface in at least one species, such as Hsp60/GroEL, DnaK, glyceraldehyde 3-phosphate dehydrogenase, enolase, and fructose 1,6-bisphosphate aldolase. The results of the proteomics studies suggest they could also be moonlighting on the surface of many other species. Hundreds of other intracellular proteins are also found on the cell surface, although a second function on the surface has not yet been demonstrated, for example, glutamine synthetase, gamma-glutamyl phosphate reductase, and cysteine desulfurase. The presence of intracellular proteins on the cell surface is more common than previously expected and suggests that many additional proteins might be candidates for being intracellular/surface moonlighting proteins.

MoonProt: a database for proteins that are known to moonlight.

Moonlighting proteins comprise a class of multifunctional proteins in which a single polypeptide chain performs multiple biochemical functions that are not due to gene fusions, multiple RNA splice variants or pleiotropic effects. The known moonlighting proteins perform a variety of diverse functions in many different cell types and species, and information about their structures and functions is scattered in many publications. We have constructed the manually curated, searchable, internet-based MoonProt Database (http://www.moonlightingproteins.org) with information about the over 200 proteins that have been experimentally verified to be moonlighting proteins. The availability of this organized information provides a more complete picture of what is currently known about moonlighting proteins. The database will also aid researchers in other fields, including determining the functions of genes identified in genome sequencing projects, interpreting data from proteomics projects and annotating protein sequence and structural databases. In addition, information about the structures and functions of moonlighting proteins can be helpful in understanding how novel protein functional sites evolved on an ancient protein scaffold, which can also help in the design of proteins with novel functions.

An introduction to protein moonlighting.

Moonlighting proteins comprise a class of multifunctional proteins in which a single polypeptide chain performs multiple physiologically relevant biochemical or biophysical functions. Almost 300 proteins have been found to moonlight. The known examples of moonlighting proteins include diverse types of proteins, including receptors, enzymes, transcription factors, adhesins and scaffolds, and different combinations of functions are observed. Moonlighting proteins are expressed throughout the evolutionary tree and function in many different biochemical pathways. Some moonlighting proteins can perform both functions simultaneously, but for others, the protein's function changes in response to changes in the environment. The diverse examples of moonlighting proteins already identified, and the potential benefits moonlighting proteins might provide to the organism, such as through coordinating cellular activities, suggest that many more moonlighting proteins are likely to be found. Continuing studies of the structures and functions of moonlighting proteins will aid in predicting the functions of proteins identified through genome sequencing projects, in interpreting results from proteomics experiments, in understanding how different biochemical pathways interact in systems biology, in annotating protein sequence and structure databases, in studies of protein evolution and in the design of proteins with novel functions.

Utilizing the folate receptor for active targeting of cancer nanotherapeutics.

The development of specialized nanoparticles for use in the detection and treatment of cancer is increasing. Methods are being proposed and tested that could target treatments more directly to cancer cells, which could lead to higher efficacy and reduced toxicity, possibly even eliminating the adverse effects of damage to the immune system and the loss of quick replicating cells. In this mini-review we focus on recent studies that employ folate nanoconjugates to target the folate receptor. Folate receptors are highly overexpressed on the surface of many tumor types. This expression can be exploited to target imaging molecules and therapeutic compounds directly to cancerous tissues.

Proteins with neomorphic moonlighting functions in disease.

One gene can encode multiple protein functions because of RNA splice variants, gene fusions during evolution, promiscuous enzyme activities, and moonlighting protein functions. In addition to these types of multifunctional proteins, in which both functions are considered "normal" functions of a protein, some proteins have been described in which a mutation or conformational change imparts a second function on a protein that is not a "normal" function of the protein. We propose to call these new functions "neomorphic moonlighting functions". The most common examples of neomorphic moonlighting functions are due to conformational changes that impart novel protein-protein interactions resulting in the formation of protein aggregates in Alzheimers, Parkinsons disease, and the systemic amyloidoses. Other changes that can result in a neomorphic moonlighting function include a mutation in SMAD4 that causes the protein to bind to new promoters and thereby alter gene transcription patterns, mutations in two isocitrate dehydrogenase isoforms that impart a new catalytic activity, and mutations in dihydrolipoamide dehydrogenase that activate a hidden protease activity. These neomorphic moonlighting functions were identified because of their connection to disease. In the cases described herein, the new functions cause cancers or severe neurological impairment, although in most cases the mechanism by which the new function leads to disease is unknown.

Recombinant expression screening of P. aeruginosa bacterial inner membrane proteins.

BACKGROUND: Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. RESULTS: Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. CONCLUSIONS: In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this one could aid in overcoming the technical bottlenecks in working with TM proteins and could potentially aid in increasing the rate of structure determination.

Expression, detergent solubilization, and purification of a membrane transporter, the MexB multidrug resistance protein.

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP). The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC). In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

Moonlighting proteins--an update.

A growing number of diverse proteins are being identified that moonlight. Moonlighting proteins comprise an interesting subset of multifunctional proteins in which the two functions are found in a single polypeptide chain. They do not include proteins that are multifunctional due to gene fusions, families of homologous proteins, splice variants, or promiscuous enzyme activities. This review summarizes recent discoveries that add to the list of known moonlighting proteins. They include several different kinds of proteins and combinations of functions. In one case, a novel DNA binding function was found for a biosynthetic enzyme through a proteomics microarray project. The review also summarizes recent X-ray crystal structures that provide clues to the molecular mechanisms of one or both functions, and in some cases how a protein can switch between functions. In addition, the possibility that many proteins with intrinsically unstructured regions might also moonlight is discussed.

Crystal structure of phosphoglucose isomerase from Trypanosoma brucei complexed with glucose-6-phosphate at 1.6 A resolution.

Enzymes of glycolysis in Trypanosoma brucei have been identified as potential drug targets for African sleeping sickness because glycolysis is the only source of ATP for the bloodstream form of this parasite. Several inhibitors were previously reported to bind preferentially to trypanosomal phosphoglucose isomerase (PGI, the second enzyme in glycolysis) than to mammalian PGIs, which suggests that PGI might make a good target for species-specific drug design. Herein, we report recombinant expression, purification, crystallization and X-ray crystal structure determination of T. brucei PGI. One structure solved at 1.6 A resolution contains a substrate, D-glucose-6-phosphate, in an extended conformation in the active site. A second structure solved at 1.9 A resolution contains a citrate molecule in the active site. The structures are compared with the crystal structures of PGI from humans and from Leishmania mexicana. The availability of recombinant tPGI and its first high-resolution crystal structures are initial steps in considering this enzyme as a potential drug target.

The crystal structure of rabbit phosphoglucose isomerase complexed with D-sorbitol-6-phosphate, an analog of the open chain form of D-glucose-6-phosphate.

Phosphoglucose isomerase (PGI) catalyzes the isomerization of D-glucose-6-phosphate (G6P) and D-fructose-6-phosphate (F6P) in glycolysis and gluconeogenesis. Analysis of previously reported X-ray crystal structures of PGI without ligand, with the cyclic form of F6P, or with inhibitors that mimic the cis-enediol intermediate led to proposed mechanisms for the ring opening and isomerization steps in the multistep catalytic mechanism. To help complete our model of the overall mechanism, information is needed about the state of PGI between the ring opening and isomerization steps, in other words, a structure of the enzyme complexed with the open form of a substrate or an analog. Here, we report the crystal structure of rabbit PGI complexed with D-sorbitol-6-phosphate (S6P), an analog of the open chain form of G6P, at 2.0 A resolution. As was seen in the PGI/F6P structure, a helix containing amino acid residues 512-520 is found in the "out" position, which provides sufficient space in the active site for a substrate in its cyclic form and which is probably the location of that helix just after ring opening (or just before ring closure). However, the S6P ligand is in an extended conformation, as was seen previously with ligands that mimic the cis-enediol intermediate. The extended conformation enables the ligand to interact with Glu357, which transfers a proton during the isomerization step. The PGI/S6P structure represents the conformation of the enzyme and substrate between the ring opening (or ring closing) step and the isomerization step and helps to complete the model for PGI's catalytic mechanism.

Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor.

The phosphomannose isomerases (PMI) comprise three families of proteins: type I, type II, and type III PMIs. Members of all three families catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) but share little or no sequence identity. Because (1) PMIs are essential for the survival of several microorganisms, including yeasts and bacteria, and (2) the PMI enzymes from several pathogens do not share significant sequence identity to the human protein, PMIs have been considered as potential therapeutic targets. Elucidation of the catalytic and regulatory mechanisms of the different types of PMIs is strongly needed for rational species-specific drug design. To date, inhibition and crystallographic studies of all PMIs are still largely unexplored. As part of our research program on aldose-ketose isomerases, we report in this paper the evaluation of two new inhibitors of type I and type II PMIs from baker's yeast and Pseudomonas aeruginosa, respectively. We found that 5-phospho-D-arabinonohydroxamic acid (5PAH), which is the most potent inhibitor of phosphoglucose isomerase (PGI), is by far the best inhibitor ever reported of both type I and type II PMI-catalyzed isomerization of M6P to F6P. 5PAH, which has an inhibition constant at least 3 orders of magnitude smaller than that of previously reported PMI inhibitors, may be the first high-energy intermediate analogue inhibitor of the enzymes. We also tested the related molecule 5-phospho-D-arabinonate (5PAA), which is a strong competitive inhibitor of PGI, and found that it does not inhibit either PMI. All together, our results are consistent with a catalytic role for the metal cofactor in PMI activity.

Moonlighting proteins: old proteins learning new tricks.

Recently, several laboratories identifying proteins involved in the complex processes of replication, transcription and tumor suppression found that the 'new' protein they discovered had another, previously identified, function. A single protein with multiple functions might seem surprising, but there are actually many cases of proteins that 'moonlight', or have more than one role in an organism. As well as adding to the number and types of proteins that are known to moonlight, these new examples add to our understanding of the potential importance of moonlighting proteins.

Multifunctional proteins: examples of gene sharing.

Adding to the difficulty of interpreting the human genome sequence and annotating protein sequence databases is the observation that a single protein can 'moonlight' or perform multiple, apparently unrelated, functions. This review summarizes examples of moonlighting proteins in cellular activities and biochemical pathways important in cancer and other diseases. The proteins include a variety of combinations of functions and mechanisms to switch between functions. Moonlighting proteins can be beneficial to the organism, such as by coordinating cellular activities. However, moonlighting proteins can potentially make more difficult the determination of the molecular mechanisms of disease and the process of rational drug design.

Conformational changes in phosphoglucose isomerase induced by ligand binding.

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is the second enzyme in glycolysis, where it catalyzes the isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate. It is the same protein as autocrine motility factor, differentiation and maturation mediator, and neuroleukin. Here, we report a new X-ray crystal structure of rabbit PGI (rPGI) without ligands bound in its active site. The structure was solved at 1.8A resolution by isomorphous phasing with a previously solved X-ray crystal structure of the rPGI dimer containing 6-phosphogluconate in its active site. Comparison of the new structure to previously reported structures enables identification of conformational changes that occur during binding of substrate or inhibitor molecules. Ligand binding causes an induced fit of regions containing amino acid residues 209-215, 245-259 and 385-389. This conformational change differs from the change previously reported to occur between the ring-opening and isomerization steps, in which the helix containing residues 513-521 moves toward the bound substrate. Differences between the liganded and unliganded structures are limited to the region within and close to the active-site pocket.