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Moonlighting proteins (MPs), characterized by their ability to perform multiple physiologically unrelated functions without alterations to their primary structures, represent a fascinating class of biomolecules with significant implications for host-pathogen interactions. This Review highlights the emerging importance of metabolic moonlighting proteins (MetMPs) in bacterial pathogenesis, focusing on their non-canonical secretion and unconventional surface anchoring mechanisms. Despite lacking typical signal peptides and anchoring motifs, MetMPs such as acetaldehyde alcohol dehydrogenase (AdhE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are secreted and localized to the bacterial surface under stress conditions, facilitating host colonization and immune evasion. The secretion of MetMPs, often observed during conditions such as resource scarcity or infection, suggests a complex regulation akin to the overexpression of heat shock proteins in response to environmental stresses. This Review proposes two potential pathways for MetMP secretion: membrane damage-induced permeability and co-transportation with traditionally secreted proteins, highlighting a remarkable bacterial adaptability. Biophysically, surface anchoring of MetMPs is driven by electrostatic interactions, bypassing the need for conventional anchoring sequences. This mechanism is exemplified by the interaction between the bifunctional enzyme AdhE (known as Listeria adhesion protein, LAP) and the internalin B (InlB) in Listeria monocytogenes, which is mediated by charged residues facilitating adhesion to host tissues. Furthermore, MetMPs play critical roles in iron homeostasis, immune modulation, and evasion, underscoring their multifaceted roles in bacterial pathogenicity. The intricate dynamics of MetMP secretion and anchoring underline the need for further research to unravel the molecular mechanisms underpinning these processes, offering potential new targets for therapeutic intervention against bacterial infections.

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INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

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The rationale for replacing the old binary of structure-function with the trinity of structure, disorder, and function has gained considerable ground in recent years. A continuum model based on the expanded form of the existing paradigm can now subsume importance of both conformational flexibility and intrinsic disorder in protein function. The disorder is actually critical for understanding the protein-protein interactions in many regulatory processes, formation of membrane-less organelles, and our revised notions of specificity as amply illustrated by moonlighting proteins. While its importance in formation of amyloids and function of prions is often discussed, the roles of intrinsic disorder in infectious diseases and protein function under extreme conditions are also becoming clear. This review is an attempt to discuss how our current understanding of protein function, specificity, and evolution fit better with the continuum model. This integration of structure and disorder under a single model may bring greater clarity in our continuing quest for understanding proteins and molecular mechanisms of their functionality.

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Cyclic nucleotides 3',5'-cAMP and 3',5'-cGMP are now established signaling components of the plant cell while their 2',3' positional isomers are increasingly recognized as such. 3',5'-cAMP/cGMP is generated by adenylate cyclases (ACs) or guanylate cyclases (GCs) from ATP or GTP, respectively, whereas 2',3'-cAMP/cGMP is produced through the hydrolysis of double-stranded DNA or RNA by synthetases. Recent evidence suggests that the cyclic nucleotide generating and inactivating enzymes moonlight in proteins with diverse domain architecture operating as molecular tuners to enable dynamic and compartmentalized regulation of cellular signals. Further characterization of such moonlighting enzymes and extending the studies to noncanonical cyclic nucleotides promises new insights into the complex regulatory networks that underlie plant development and responses, thus offering exciting opportunities for crop improvement.

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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.

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Moonlighting proteins, known for their ability to perform multiple, often unrelated functions within a single polypeptide chain, challenge the traditional "one gene, one protein, one function" paradigm. As organisms evolved, their genomes remained relatively stable in size, but the introduction of post-translational modifications and sub-strategies like protein promiscuity and intrinsic disorder enabled multifunctionality. Enzymes, in particular, exemplify this phenomenon, engaging in unrelated processes alongside their primary catalytic roles. This study employs a systematic, quantitative informatics approach to shed light on human moonlighting protein sequences. Phylogenetic analyses of human moonlighting proteins are presented, elucidating the distal-proximal relationships among these proteins based on sequence-derived quantitative features. The findings unveil the captivating world of human moonlighting proteins, urging further investigations in the emerging field of moonlighting proteomics, with the potential for significant contributions to our understanding of multifunctional proteins and their roles in diverse cellular processes and diseases.

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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.

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Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins' enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 °C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 °C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment.

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Leptospirosis is a neglected worldwide zoonosis involving farm animals and domestic pets caused by the Gram-negative spirochete Leptospira interrogans. This bacterium deploys a variety of immune evasive mechanisms, some of them targeted at the complement system of the host's innate immunity. In this work, we have solved the X-ray crystallographic structure of L. interrogans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to 2.37-Å resolution, a glycolytic enzyme that has been shown to exhibit moonlighting functions that potentiate infectivity and immune evasion in various pathogenic organisms. Besides, we have characterized the enzyme's kinetic parameters toward the cognate substrates and have proven that the two natural products anacardic acid and curcumin are able to inhibit L. interrogans GAPDH at micromolar concentration through a noncompetitive inhibition modality. Furthermore, we have established that L. interrogans GAPDH can interact with the anaphylatoxin C5a of human innate immunity in vitro using bio-layer interferometry and a short-range cross-linking reagent that tethers free thiol groups in protein complexes. To shed light into the interaction between L. interrogans GAPDH and C5a, we have also carried out cross-link guided protein-protein docking. These results suggest that L. interrogans could be placed in the growing list of bacterial pathogens that exploit glycolytic enzymes as extracellular immune evasive factors. Analysis of the docking results indicates a low affinity interaction that is consistent with previous evidence, including known binding modes of other α-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system.

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Bacterial membrane vesicles (MVs) are abundant in the oceans, but their potential functional roles remain unclear. In this study we characterized MV production and protein content of six strains of Alteromonas macleodii, a cosmopolitan marine bacterium. Alteromonas macleodii strains varied in their MV production rates, with some releasing up to 30 MVs per cell per generation. Microscopy imaging revealed heterogenous MV morphologies, including some MVs aggregated within larger membrane structures. Proteomic characterization revealed that A. macleodii MVs are rich in membrane proteins related to iron and phosphate uptake, as well as proteins with potential functions in biofilm formation. Furthermore, MVs harbored ectoenzymes, such as aminopeptidases and alkaline phosphatases, which comprised up to 20% of the total extracellular enzymatic activity. Our results suggest that A. macleodii MVs may support its growth through generation of extracellular 'hotspots' that facilitate access to essential substrates. This study provides an important basis to decipher the ecological relevance of MVs in heterotrophic marine bacteria.

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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.

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The term moonlighting proteins refers to those proteins that present alternative functions performed by a single polypeptide chain acquired throughout evolution (called canonical and moonlighting, respectively). Over 78% of moonlighting proteins are involved in human diseases, 48% are targeted by current drugs, and over 25% of them are involved in the virulence of pathogenic microorganisms. These facts encouraged us to study the link between the functions of moonlighting proteins and disease. We found a large number of moonlighting functions activated by pathological conditions that are highly involved in disease development and progression. The factors that activate some moonlighting functions take place only in pathological conditions, such as specific cellular translocations or changes in protein structure. Some moonlighting functions are involved in disease promotion while others are involved in curbing it. The disease-impairing moonlighting functions attempt to restore the homeostasis, or to reduce the damage linked to the imbalance caused by the disease. The disease-promoting moonlighting functions primarily involve the immune system, mesenchyme cross-talk, or excessive tissue proliferation. We often find moonlighting functions linked to the canonical function in a pathological context. Moonlighting functions are especially coordinated in inflammation and cancer. Wound healing and epithelial to mesenchymal transition are very representative. They involve multiple moonlighting proteins with a different role in each phase of the process, contributing to the current-phase phenotype or promoting a phase switch, mitigating the damage or intensifying the remodeling. All of this implies a new level of complexity in the study of pathology genesis, progression, and treatment. The specific protein function involved in a patient's progress or that is affected by a drug must be elucidated for the correct treatment of diseases.

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Estrogen accounts for several biological processes in the body; embryo implantation and pregnancy being one of the vital events. This manuscript aims to unearth the nuclear role of Son of sevenless1 (SOS1), its interaction with estrogen receptor alpha (ERα), and signal transducer and activator of transcription 3 (STAT3) in the uterine nucleus during embryo implantation. SOS1, a critical cytoplasmic linker between receptor tyrosine kinase and rat sarcoma virus signaling, translocates into the nucleus via its bipartite nuclear localization signal (NLS) during the 'window of implantation' in pregnant mice. SOS1 associates with chromatin, interacts with histones, and shows intrinsic histone acetyltransferase (HAT) activity specifically acetylating lysine 16 (K16) residue of histone H4. SOS1 is a coactivator of STAT3 and a co-repressor of ERα. SOS1 creates a partial mesenchymal-epithelial transition by acting as a transcriptional modulator. Finally, our phylogenetic tree reveals that the two bipartite NLS surface in reptiles and the second acetyl coenzymeA (CoA) (RDNGPG) important for HAT activity emerges in mammals. Thus, SOS1 has evolved into a moonlighting protein, the special class of multi-tasking proteins, by virtue of its newly identified nuclear functions in addition to its previously known cytoplasmic function.

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Biological systems consist of multiple components of different physical and chemical properties that require complex and dynamic regulatory loops to function efficiently. The discovery of ever more novel interacting sites in complex proteins suggests that we are only beginning to understand how cellular and biological functions are integrated and tuned at the molecular and systems levels. Here we review recently discovered interacting sites which have been identified through rationally designed amino acid motifs diagnostic for specific molecular functions, including enzymatic activities and ligand-binding properties. We specifically discuss the nature of the latter using as examples, novel hormone recognition and gas sensing sites that occur in moonlighting protein complexes. Drawing evidence from the current literature, we discuss the potential implications at the cellular, tissue, and/or organismal levels of such non-catalytic interacting sites and provide several promising avenues for the expansion of amino acid motif searches to discover hitherto unknown protein interactants and interaction networks. We believe this knowledge will unearth unexpected functions in both new and well-characterized proteins, thus filling existing conceptual gaps or opening new avenues for applications either as drug targets or tools in pharmacology, cell biology and bio-catalysis. Beyond this, motif searches may also support the design of novel, effective and sustainable approaches to crop improvements and the development of new therapeutics.

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Aim: Pathogenic invasion of Staphylococcus aureus is critically dependent on host plasminogen activation. Materials & methods: The pathophysiological implications of the interactions between S. aureus recombinant enolase and host plasminogen were investigated. The effects of mutation and small synthetic peptide inhibitors on interactions were assessed. Results: In vitro, the S. aureus recombinant enolase exists as a catalytically active fragile octamer and a robust dimer. The dimer interacts with the host plasminogen on the S. aureus surface. Conclusion: The interaction of host plasminogen and S. aureus enolase might mediate bacterial adherence to the host, activate the plasminogen with the help of plasminogen activators and prevent α2-antiplasmin-mediated inhibition of plasmin. Incorporating mutant and synthetic peptides inhibited the interactions and their associated pathophysiological consequences.

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Moonlighting proteins have at least two independent functions and are widely found in animals, plants and microorganisms. Moonlighting proteins play important roles in signal transduction, cell growth and movement, tumor inhibition, DNA synthesis and repair, and metabolism of biological macromolecules. Moonlighting proteins are difficult to find through biological experiments, so many researchers identify moonlighting proteins through bioinformatics methods, but their accuracies are relatively low. Therefore, we propose a new method. In this study, we select SVMProt-188D as the feature input, and apply a model combining linear discriminant analysis and basic classifiers in machine learning to study moonlighting proteins, and perform bagging ensemble on the best-performing support vector machine. They are identified accurately and efficiently. The model achieves an accuracy of 93.26% and an F-sorce of 0.946 on the MPFit dataset, which is better than the existing MEL-MP model. Meanwhile, it also achieves good results on the other two moonlighting protein datasets.

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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.

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Plant secretomics has been especially important in understanding the molecular basis of plant development, stress resistance and biomarker discovery. In addition to sharing a similar role in maintaining cell metabolism and biogenesis with the animal secretome, plant-secreted proteins actively participate in signaling events crucial for cellular homeostasis during stress adaptation. However, investigation of the plant secretome remains largely overlooked, particularly in pulse crops, demanding urgent attention. To better understand the complexity of the secretome, we developed a reference map of a stress-resilient orphan legume, Lathyrus sativus (grasspea), which can be utilized as a potential proteomic resource. Secretome analysis of L. sativus led to the identification of 741 nonredundant proteins belonging to a myriad of functional classes, including antimicrobial, antioxidative and redox potential. Computational prediction of the secretome revealed that ∼29% of constituents are predicted to follow unconventional protein secretion (UPS) routes. We conducted additional in planta analysis to determine the localization of two secreted proteins, recognized as cell surface residents. Sequence-based homology comparison revealed that L. sativus shares ∼40% of the constituents reported thus far from in vitro and in planta secretome analysis in model and crop species. Significantly, we identified 571 unique proteins secreted from L. sativus involved in cell-to-cell communication, organ development, kinase-mediated signaling, and stress perception, among other critical roles. Conclusively, the grasspea secretome participates in putative crosstalk between genetic circuits that regulate developmental processes and stress resilience.

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The proteolytic activity of human plasmin (hPm) is utilized by various cells to provide a surface protease that increases the potential of cells to migrate and disseminate. Skin-trophic Pattern D strains of Streptococcus pyogenes (GAS), e.g., GAS isolate AP53, contain a surface M-protein (PAM) that directly and strongly interacts (Kd ~ 1 nM) with human host plasminogen (hPg), after which it is activated to hPm by a specific coinherited bacterial activator, streptokinase (SK2b), or by host activators. Another ubiquitous class of hPg binding proteins on GAS cells includes "moonlighting" proteins, such as the glycolytic enzyme, enolase (Sen). However, the importance of Sen in hPg acquisition, especially when PAM is present, has not been fully developed. Sen forms a complex with hPg on different surfaces, but not in solution. Isogenic AP53 cells with a targeted deletion of PAM do not bind hPg, but the surface expression of Sen is also greatly diminished upon deletion of the PAM gene, thus confounding this approach for defining the role of Sen. However, cells with point deletions in PAM that negate hPg binding, but fully express PAM and Sen, show that hPg binds weakly to Sen on GAS cells. Despite this, Sen does not stimulate hPg activation by SK2b, but does stimulate tissue-type plasminogen activator-catalyzed activation of hPg. These data demonstrate that PAM plays the dominant role as a functional hPg receptor in GAS cells that also contain surface enolase.