Moonlighting enzymes: when cellular context defines specificity.

It is not often realized that the absolute protein specificity is an exception rather than a rule. Two major kinds of protein multi-specificities are promiscuity and moonlighting. This review discusses the idea of enzyme specificity and then focusses on moonlighting. Some important examples of protein moonlighting, such as crystallins, ceruloplasmin, metallothioniens, macrophage migration inhibitory factor, and enzymes of carbohydrate metabolism are discussed. How protein plasticity and intrinsic disorder enable the removing the distinction between enzymes and other biologically active proteins are outlined. Finally, information on important roles of moonlighting in human diseases is updated.

Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

Rethinking Opsins.

Opsins, the protein moieties of animal visual photo-pigments, have emerged as moonlighting proteins with diverse, light-dependent and -independent physiological functions. This raises the need to revise some basic assumptions concerning opsin expression, structure, classification, and evolution.

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors that are directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified which are mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Evolutionary conserved relocation of chromatin remodeling complexes to the mitotic apparatus.

BACKGROUND: ATP-dependent chromatin remodeling complexes are multi-protein machines highly conserved across eukaryotic genomes. They control sliding and displacing of the nucleosomes, modulating histone-DNA interactions and making nucleosomal DNA more accessible to specific binding proteins during replication, transcription, and DNA repair, which are processes involved in cell division. The SRCAP and p400/Tip60 chromatin remodeling complexes in humans and the related Drosophila Tip60 complex belong to the evolutionary conserved INO80 family, whose main function is promoting the exchange of canonical histone H2A with the histone variant H2A in different eukaryotic species. Some subunits of these complexes were additionally shown to relocate to the mitotic apparatus and proposed to play direct roles in cell division in human cells. However, whether this phenomenon reflects a more general function of remodeling complex components and its evolutionary conservation remains unexplored. RESULTS: We have combined cell biology, reverse genetics, and biochemical approaches to study the subcellular distribution of a number of subunits belonging to the SRCAP and p400/Tip60 complexes and assess their involvement during cell division progression in HeLa cells. Interestingly, beyond their canonical chromatin localization, the subunits under investigation accumulate at different sites of the mitotic apparatus (centrosomes, spindle, and midbody), with their depletion yielding an array of aberrant outcomes of mitosis and cytokinesis, thus causing genomic instability. Importantly, this behavior was conserved by the Drosophila melanogaster orthologs tested, despite the evolutionary divergence between fly and humans has been estimated at approximately 780 million years ago. CONCLUSIONS: Overall, our results support the existence of evolutionarily conserved diverse roles of chromatin remodeling complexes, whereby subunits of the SRCAP and p400/Tip60 complexes relocate from the interphase chromatin to the mitotic apparatus, playing moonlighting functions required for proper execution of cell division.

hu.MAP 2.0: integration of over 15,000 proteomic experiments builds a global compendium of human multiprotein assemblies.

A general principle of biology is the self-assembly of proteins into functional complexes. Characterizing their composition is, therefore, required for our understanding of cellular functions. Unfortunately, we lack knowledge of the comprehensive set of identities of protein complexes in human cells. To address this gap, we developed a machine learning framework to identify protein complexes in over 15,000 mass spectrometry experiments which resulted in the identification of nearly 7,000 physical assemblies. We show our resource, hu.MAP 2.0, is more accurate and comprehensive than previous state of the art high-throughput protein complex resources and gives rise to many new hypotheses, including for 274 completely uncharacterized proteins. Further, we identify 253 promiscuous proteins that participate in multiple complexes pointing to possible moonlighting roles. We have made hu.MAP 2.0 easily searchable in a web interface (http://humap2.proteincomplexes.org/), which will be a valuable resource for researchers across a broad range of interests including systems biology, structural biology, and molecular explanations of disease.

Als3-mediated attachment of enolase on the surface of Candida albicans cells regulates their interactions with host proteins.

The multifunctional protein enolase has repeatedly been identified on the surface of numerous cell types, including a variety of pathogenic microorganisms. In Candida albicans-one of the most common fungal pathogens in humans-a surface-exposed enolase form has been previously demonstrated to play an important role in candidal pathogenicity. In our current study, the presence of enolase at the fungal cell surface under different growth conditions was examined, and a higher abundance of enolase at the surface of C. albicans hyphal forms compared to yeast-like cells was found. Affinity chromatography and chemical cross-linking indicated a member of the agglutinin-like sequence protein family-Als3-as an important potential partner required for the surface display of enolase. Analysis of Saccharomyces cerevisiae cells overexpressing Als3 with site-specific deletions showed that the Ig-like N-terminal region of Als3 (aa 166-225; aa 218-285; aa 270-305; aa 277-286) and the central repeat domain (aa 434-830) are essential for the interaction of this adhesin with enolase. In addition, binding between enolase and Als3 influenced subsequent docking of host plasma proteins-high molecular mass kininogen and plasminogen-on the candidal cell surface, thus supporting the hypothesis that C. albicans can modulate plasma proteolytic cascades to affect homeostasis within the host and propagate inflammation during infection.

Proteomic Analysis Reveals Enzymes for ß-D-Glucan Formation and Degradation in Levilactobacillus brevis TMW 1.2112.

Bacterial exopolysaccharide (EPS) formation is crucial for biofilm formation, for protection against environmental factors, or as storage compounds. EPSs produced by lactic acid bacteria (LAB) are appropriate for applications in food fermentation or the pharmaceutical industry, yet the dynamics of formation and degradation thereof are poorly described. This study focuses on carbohydrate active enzymes, including glycosyl transferases (GT) and glycoside hydrolases (GH), and their roles in the formation and potential degradation of O2-substituted (1,3)-ß-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, ß-glucan, and D-glucose concentrations during the exponential, stationary, and early death phases. While the viscosity reached its maximum during the stationary phase and subsequently decreased, the ß-glucan concentration only increased to a plateau. Results were correlated with secretome and proteome data to identify involved enzymes and pathways. The suggested pathway for ß-glucan biosynthesis involved a ß-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the ß-glucan concentration did not decrease, most likely due to missing extracellular carbohydrate active enzymes. In addition, an operon was discovered containing known moonlighting genes, all of which were detected in both proteome and secretome samples.

Moonlighting matrix metalloproteinase substrates: Enhancement of proinflammatory functions of extracellular tyrosyl-tRNA synthetase upon cleavage.

Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/ß, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell-derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

The expanding world of metabolic enzymes moonlighting as RNA binding proteins.

RNA binding proteins play key roles in many aspects of RNA metabolism and function, including splicing, transport, translation, localization, stability and degradation. Within the past few years, proteomics studies have identified dozens of enzymes in intermediary metabolism that bind to RNA. The wide occurrence and conservation of RNA binding ability across distant branches of the evolutionary tree suggest that these moonlighting enzymes are involved in connections between intermediary metabolism and gene expression that comprise far more extensive regulatory networks than previously thought. There are many outstanding questions about the molecular structures and mechanisms involved, the effects of these interactions on enzyme and RNA functions, and the factors that regulate the interactions. The effects on RNA function are likely to be wider than regulation of translation, and some enzyme-RNA interactions have been found to regulate the enzyme's catalytic activity. Several enzyme-RNA interactions have been shown to be affected by cellular factors that change under different intracellular and environmental conditions, including concentrations of substrates and cofactors. Understanding the molecular mechanisms involved in the interactions between the enzymes and RNA, the factors involved in regulation, and the effects of the enzyme-RNA interactions on both the enzyme and RNA functions will lead to a better understanding of the role of the many newly identified enzyme-RNA interactions in connecting intermediary metabolism and gene expression.

Candida albicans and Candida glabrata triosephosphate isomerase - a moonlighting protein that can be exposed on the candidal cell surface and bind to human extracellular matrix proteins.

BACKGROUND: Triosephosphate isomerase (Tpi1) is a glycolytic enzyme that has recently been reported also to be an atypical proteinaceous component of the Candida yeast cell wall. Similar to other known candidal "moonlighting proteins", surface-exposed Tpi1 is likely to contribute to fungal adhesion during the colonization and infection of a human host. The aim of our present study was to directly prove the presence of Tpi1 on C. albicans and C. glabrata cells under various growth conditions and characterize the interactions of native Tpi1, isolated and purified from the candidal cell wall, with human extracellular matrix proteins. RESULTS: Surface plasmon resonance measurements were used to determine the dissociation constants for the complexes of Tpi1 with host proteins and these values were found to fall within a relatively narrow range of 10- 8-10- 7 M. Using a chemical cross-linking method, two motifs of the Tpi1 molecule (aa 4-17 and aa 224-247) were identified to be directly involved in the interaction with vitronectin. A proposed structural model for Tpi1 confirmed that these interaction sites were at a considerable distance from the catalytic active site. Synthetic peptides with these sequences significantly inhibited Tpi1 binding to several extracellular matrix proteins suggesting that a common region on the surface of Tpi1 molecule is involved in the interactions with the host proteins. CONCLUSIONS: The current study provided structural insights into the interactions of human extracellular matrix proteins with Tpi1 that can occur at the cell surface of Candida yeasts and contribute to the host infection by these fungal pathogens.

An overview of moonlighting proteins in Staphylococcus aureus infection.

Staphylococcus aureus is responsible for numerous instances of superficial, toxin-mediated, and invasive infections. The emergence of methicillin-resistant (MRSA), as well as vancomycin-resistant (VRSA) strains of S. aureus, poses a massive threat to human health. The tenacity of S. aureus to acquire resistance against numerous antibiotics in a very short duration makes the effort towards developing new antibiotics almost futile. S. aureus owes its destructive pathogenicity to the plethora of virulent factors it produces among which a majority of them are moonlighting proteins. Moonlighting proteins are the multifunctional proteins in which a single protein, with different oligomeric conformations, perform multiple independent functions in different cell compartments. Peculiarly, proteins involved in key ancestral functions and metabolic pathways typically exhibit moonlighting functions. Pathogens mainly employ those proteins as virulent factors which exhibit high structural conservation towards their host counterparts. Consequentially, the host immune system counteracts these invading bacterial virulent factors with minimal protective action. Additionally, many moonlighting proteins also play multiple roles in various stages of pathogenicity while augmenting the virulence of the bacterium. This has necessitated elaborative studies to be conducted on moonlighting proteins of S. aureus that can serve as drug targets. This review is a small effort towards understanding the role of various moonlighting proteins in the pathogenicity of S. aureus.

Virulence of Acinetobacter baumannii in proteins moonlighting.

The diverse function of the moonlighting proteins in Acinetobacter baumannii is highly associated with its virulence that had spurred renewed attention in recent years. The existing and newly formed hypothetical moonlighting proteins, evolve without jeopardizing the structural constraints of their original roles. It is yet uncertain and undefined to lucidly describe the functions of the moonlighting proteins in A. baumannii albeit its overwhelming evidences on few proteins. This commentary thus highlights the expression and occurrence of potent moonlighting proteins in A. baumannii, rendering virulence to the strains and the reasons to target the same portraying an active arena of research.

On the Need to Tell Apart Fraternal Twins eEF1A1 and eEF1A2, and Their Respective Outfits.

eEF1A1 and eEF1A2 are paralogous proteins whose presence in most normal eukaryotic cells is mutually exclusive and developmentally regulated. Often described in the scientific literature under the collective name eEF1A, which stands for eukaryotic elongation factor 1A, their best known activity (in a monomeric, GTP-bound conformation) is to bind aminoacyl-tRNAs and deliver them to the A-site of the 80S ribosome. However, both eEF1A1 and eEF1A2 are endowed with multitasking abilities (sometimes performed by homo- and heterodimers) and can be located in different subcellular compartments, from the plasma membrane to the nucleus. Given the high sequence identity of these two sister proteins and the large number of post-translational modifications they can undergo, we are often confronted with the dilemma of discerning which is the particular proteoform that is actually responsible for the ascribed biochemical or cellular effects. We argue in this review that acquiring this knowledge is essential to help clarify, in molecular and structural terms, the mechanistic involvement of these two ancestral and abundant G proteins in a variety of fundamental cellular processes other than translation elongation. Of particular importance for this special issue is the fact that several de novo heterozygous missense mutations in the human EEF1A2 gene are associated with a subset of rare but severe neurological syndromes and cardiomyopathies.

In Vitro Characterization of Guanylyl Cyclase BdPepR2 from Brachypodium distachyon Identified through a Motif-Based Approach.

In recent years, cyclic guanosine 3',5'-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules-BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP.

Moonlighting Proteins Shine New Light on Molecular Signaling Niches.

Plants as sessile organisms face daily environmental challenges and have developed highly nuanced signaling systems to enable suitable growth, development, defense, or stalling responses. Moonlighting proteins have multiple tasks and contribute to cellular signaling cascades where they produce additional variables adding to the complexity or fuzziness of biological systems. Here we examine roles of moonlighting kinases that also generate 3',5'-cyclic guanosine monophosphate (cGMP) in plants. These proteins include receptor like kinases and lipid kinases. Their guanylate cyclase activity potentiates the development of localized cGMP-enriched nanodomains or niches surrounding the kinase and its interactome. These nanodomains contribute to allosteric regulation of kinase and other molecules in the immediate complex directly or indirectly modulating signal cascades. Effects include downregulation of kinase activity, modulation of other members of the protein complexes such as cyclic nucleotide gated channels and potential triggering of cGMP-dependent degradation cascades terminating signaling. The additional layers of information provided by the moonlighting kinases are discussed in terms of how they may be used to provide a layer of fuzziness to effectively modulate cellular signaling cascades.

Glycogen synthase kinases: Moonlighting proteins with theranostic potential in cancer.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase is an archetypal multifunctional moonlighting protein involved in diverse cellular processes including metabolism, insulin signaling, proliferation, differentiation, apoptosis, neuronal function and embryonic development. The two known isoforms, GSK-3α and GSK-3ß that undergo activation/inactivation by post-translational, site-specific phosphorylation incorporate a vast number of substrates in their repertoire. Dysregulation of GSK-3 has been linked to diverse disease entities including cancer. The role of GSK-3 in cancer is paradoxical and enigmatic. The enzyme functions as a tumour promoter or suppressor based on the context, cell type and phosphorylation status. GSK-3 is the central hub that orchestrates signals from the Wnt/ß-catenin, PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK, hedgehog, Notch and TP53 pathways to elicit regulatory influences on cancer initiation, epithelial-mesenchymal transition, and resistance to therapy. As a direct target of several microRNAs, GSK-3 influences hallmark attributes of cancer, cancer stemness and treatment resistance. There is overwhelming evidence to indicate that GSK-3 is aberrantly regulated in different cancer types. Consequently, GSK-3 has emerged as a potential therapeutic target in cancer. A plethora of natural and synthetic GSK-3 modulators have been discovered and the number of patents published for GSK-3 inhibitors has also been steadily increasing in recent years. This review focuses on the intricate interactions between GSK-3 and oncogenic signalling circuits as well as the feasibility of targeting GSK-3 for the treatment of cancer.

Moonlighting proteins - an approach to systematize the concept.

Moonlighting refers to a protein with at least two unrelated, mechanistically different functions. As a concept, moonlighting describes a large and diverse group of proteins which have been discovered in a multitude of organisms. As of today, a systematized view on these proteins is missing. Here, we propose a classification of moonlighting proteins by two classifiers. We use the function of the protein as a first classifier: activating - activating (Type I), activating - inhibiting (Type II), inhibiting - activating (Type III) and inhibiting - inhibiting (Type IV). To further specify the type of moonlighting protein, we used a second classifier based on the character of the factor that switches the function of the protein: external factor affecting the protein (Type A), change in the first pathway (Type B), change in the second pathway (Type C), equal competition between both pathways (Type D). Using a small two-pathway model we simulated these types of moonlighting proteins to elucidate possible behaviors of the types of moonlighting proteins. We find that, using the results of our simulations, we can classify the behavior of the moonlighting types into Blinker, Splitter andSwitch.

On the move: redox-dependent protein relocation in plants.

Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterized. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalysing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual-targeted proteins in the classic sense of having targeting sequences that direct de novo synthesized proteins to specific cellular locations. We propose that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox-associated enzymes.

Understanding protein multifunctionality: from short linear motifs to cellular functions.

Moonlighting proteins perform multiple unrelated functions without any change in polypeptide sequence. They can coordinate cellular activities, serving as switches between pathways and helping to respond to changes in the cellular environment. Therefore, regulation of the multiple protein activities, in space and time, is likely to be important for the homeostasis of biological systems. Some moonlighting proteins may perform their multiple functions simultaneously while others alternate between functions due to certain triggers. The switch of the moonlighting protein's functions can be regulated by several distinct factors, including the binding of other molecules such as proteins. We here review the approaches used to identify moonlighting proteins and existing repositories. We particularly emphasise the role played by short linear motifs and PTMs as regulatory switches of moonlighting functions.