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Background: Diseases of veterinary importance, such as bovine Anaplasmosis, cause significant economic losses. Due to this, the study of various proteins of the causal agent Anaplasma marginale has focused on surface proteins. However, a vaccine for this disease is not yet available. To this end, in this work, moonlighting proteins (MLPs) are presented as an alternative approach for the design of immunogens against A. marginale. Methods: The proteins of the strain MEX-15-099-01 were analyzed, and its MLPs were identified. Subsequently, four virulence-associated MLP genes were selected and identified using PCR. The proteins were analyzed using a structural homology approach and the collection of B-cell epitopes was predicted for each MLP. Finally, a pair of AmEno peptides were synthesized and the antigenic potential was tested using an iELISA. Results: Our bioinformatics analysis revealed the potential of AmEno, AmGroEl, AmEF-Tu, and AmDnaK proteins as promising candidates for designing immunogens. The PCR allowed the gene sequence identification in the genome of the strain MEX-15-099-01. Notably, AmEno-derived synthetic peptides showed antigenicity in an ELISA. Conclusions: Our study has shed light on the potential use of MLPs for immunogen design, demonstrating the antigenic potential of AmEno. [ABSTRACT FROM AUTHOR]

The cyclic nucleotides 3′,5′-cAMP/cGMP are now established as essential components of cellular processes in plants. The discovery of 2′,3′-cAMP/cGMP synthetases in Toll/interleukin-1 receptor (TIR) proteins supports an increasing recognition of both isomers as signaling molecules. Amino acid motifs diagnostic for adenylate cyclases (ACs) and guanylate cyclases (GCs) have enabled the discovery of moonlighting catalytic activities in both well-characterized and new proteins. 3′,5′-cAMP/cGMP operate as intramolecular tuners in microenvironments, while the 2′,3′ isoforms operate at the systems level in defense responses. Certain phosphodiesterases (PDEs) that hydrolyze 3′,5′-cAMP/cGMP function in the same protein as the enzymes that generate it. The twin AC-PDE domain architecture enables the tuning of cyclic nucleotide activities in a transient, dynamic, and compartmentalized manner. Research on cyclic nucleotide moonlighting enzymes is expected to drive biotechnological innovations and crop improvements. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Downstream signalling involving adenylyl cyclases (ACs) and kinases is a key component of several processes in plants including cell division, growth, and response to stress. ACs are enzymes that generate the second messenger molecule, 3′,5′-cyclic adenosine monophosphate (cAMP) from 5′-adenosine triphosphate (ATP) while kinases are enzymes that catalyze the addition of a phosphate group to other molecules (trans-phosphorylation) or themselves (auto-phosphorylation). Apparently, while there has been an expanded record of various ACs and kinases identified in plants, no plant molecule to date has been shown to possess both the AC and kinase activities/functions and with such activities/functions having the characteristic of cross-talking interactions. Therefore, in an endeavor to find such a molecule, we searched the amino acid sequence of a known Arabidopsis AC, pentatricopeptide repeat (AtPPR) protein, and found a kinase-specific sequence signature (KSSS), which we speculated to be working in synergy with the AC center in this protein during downstream signalling. So, in order to test if this additional center is catalytically active and perhaps also having some cross-talking interactions with the AC center, we cloned, expressed, and affinity purified a truncated version of AtPPR, harboring both the AC and KSSS centers (AtPPR-AC/K). When tested in vitro, the recombinant AtPPR-AC/K showed a Mn2+-dependent AC activity that is positively enhanced by Ca2+ and HCO3− and a trans-/auto-phosphorylation kinase activity capable of utilizing both ATP and GTP as substrates and specific to the serine, threonine, and tyrosine amino acids as target residues. In addition, the kinase activity of AtPPR-AC/K was found to be reduced by cAMP while at the same time, it was totally shut down by Ca2+. This thus qualified both cAMP and Ca2+ as molecular switches or modulators, capable of regulating AtPPR functions through cross-talking interactions between the activities of its two domains. Our work, therefore, has essentially established AtPPR as the first member of a new class of moonlighting proteins with AC and kinase activities that have cross-talking interactions between themselves, conceivably presenting this protein as an ideal candidate for further explorations to improve plants, particularly agricultural crops. [ABSTRACT FROM AUTHOR]

Background: Diseases of veterinary importance, such as bovine Anaplasmosis, cause significant economic losses. Due to this, the study of various proteins of the causal agent Anaplasma marginale has focused on surface proteins. However, a vaccine for this disease is not yet available. To this end, in this work, moonlighting proteins (MLPs) are presented as an alternative approach for the design of immunogens against A. marginale. Methods: The proteins of the strain MEX-15-099-01 were analyzed, and its MLPs were identified. Subsequently, four virulence-associated MLP genes were selected and identified using PCR. The proteins were analyzed using a structural homology approach and the collection of B-cell epitopes was predicted for each MLP. Finally, a pair of AmEno peptides were synthesized and the antigenic potential was tested using an iELISA. Results: Our bioinformatics analysis revealed the potential of AmEno, AmGroEl, AmEF-Tu, and AmDnaK proteins as promising candidates for designing immunogens. The PCR allowed the gene sequence identification in the genome of the strain MEX-15-099-01. Notably, AmEno-derived synthetic peptides showed antigenicity in an ELISA. Conclusions: Our study has shed light on the potential use of MLPs for immunogen design, demonstrating the antigenic potential of AmEno.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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/β, 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.

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.

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 a-helical proteins with GAPDH. These findings allow us to propose L. interrogans GAPDH as a potential immune evasive factor targeting the complement system. [ABSTRACT FROM AUTHOR]

The function of a newly discovered protein is often assessed by matching its new sequence to sequences of proteins with known functions. However, protein superfamilies can contain homologous elements that catalyze different reactions. Some homologous proteins differ in that they perform a second or even a third function and are called moonlighting proteins, which can be translated as compatible proteins or part-time proteins. Also, such proteins are called multifunctional. In addition to these, the superfamilies of proteins with multiple functions also include pseudoenzymes that have a common catalytically active domain but no catalytic activity, as well as metamorphs and morpheins. This review discusses examples of such proteins, their diversity of functions, and their importance in the life of the cell. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The second messenger cyclic diguanylate (c-di-GMP, cdG) is a bacterial lifestyle-switch molecule, well known for its role in biofilm formation. Extracellular biofilm matrix components include diverse biopolymers such as polysaccharides, nucleic acids, proteins and lipids. The production and/or secretion of many these matrix biopolymers can be directly or indirectly regulated by cdG. We studied the extracellular proteome of a Rhizobium etli strain expressing artificially high levels of intracellular cdG. We found that, in addition to promoting the secretion of various extracellular proteins likely involved in adhesion and biofilm formation, high cdG levels also promote the export of cytoplasmic proteins (ECP) to the cell exterior. Intriguingly, most these cytoplasmic proteins have been previously described as moonlighting or multifunctional proteins in other organisms, often found extracellularly or at the bacterial cell surface. We obtained evidence that this ECP may involve an active process that would be enhanced by cdG. For a typical cytoplasmic protein, glyceraldehyde 3-phosphate dehydrogenase (Gap), we also observed that cdG increases the number of extracellular Gap proteoforms that can be separated by two-dimensional gel electrophoresis. The results suggest that cdG promotes the active exportation of cytoplasmic proteins through yet unknown mechanisms involving the post-translational modification of proteins. Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second messenger cyclic diguanylate (cdG). We have performed quantitative proteomics to determine the extracellular protein contents of a Rhizobium etli strain expressing high cdG intracellular levels. cdG promoted the exportation of proteins that likely participate in adhesion and biofilm formation: the rhizobial adhesion protein RapA and two previously undescribed likely adhesins, along with flagellins. Unexpectedly, cdG also promoted the selective exportation of cytoplasmic proteins. Nearly 50% of these cytoplasmic proteins have been previously described as moonlighting or candidate moonlighting proteins in other organisms, often found extracellularly. Western blot assays confirmed cdG-promoted export of two of these cytoplasmic proteins, the translation elongation factor (EF-Tu) and glyceraldehyde 3-phosphate dehydrogenase (Gap). Transmission Electron Microscopy immunolabeling located the Gap protein in the cytoplasm but was also associated with cell membranes and extracellularly, indicative of an active process of exportation that would be enhanced by cdG. We also obtained evidence that cdG increases the number of extracellular Gap proteoforms, suggesting a link between cdG, the post-translational modification and the export of cytoplasmic proteins. [ABSTRACT FROM AUTHOR]

Estrogen accounts for several biological processes in the body; embryo implantation and pregnancy being one of the vital events. This manuscript aims t o 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 a cetyl 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. [ABSTRACT FROM AUTHOR]

To overcome their limited genetic capacity, numerous viruses encode multifunctional proteins. The birnavirus VP3 protein plays key roles during infection, including scaffolding of the viral capsid during morphogenesis, recruitment, and regulation of the viral RNA polymerase, shielding of the double-stranded RNA genome and targeting of host endosomes for genome replication, and immune evasion. The dimeric form of VP3 is critical for these functions. In previous work, we determined the X-ray structure of the central domains (D2-D3) of VP3 from the infectious bursal disease virus (IBDV). However, the structure and function of the IBDV VP3 N-terminal domain (D1) could not be determined at that time. Using integrated structural biology approaches and functional cell assays, here we characterize the IBDV VP3 D1 domain, unveiling its unexplored roles in virion stability and infection. The X-ray structure of D1 shows that this domain folds in four α-helices arranged in parallel dimers, which are essential for maintaining the dimeric arrangement of the full-length protein. Combining small-angle X-ray scattering analyses with molecular dynamics simulations allowed us to build a structural model for the D1-D3 domains. This model consists of an elongated structure with high flexibility in the D2-D3 connection, keeping D1 as the only driver of VP3 dimerization. Using reverse genetics tools, we show that the obliteration of D1 domain prevents the VP3 scaffold function during capsid assembly and severely impacts IBDV infection. Altogether, our study elucidates the structure of the VP3 D1 domain and reveals its role in VP3 protein dimerization and IBDV infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

The plant proteins called ERECTA family play important role in inflorescence architecture, stomatal patterning and phloem-xylem organization. ERECTA proteins belong to the moonlighting proteins family containing the guanylyl cyclase (GC) catalytic center embedded within the intracellular kinase domain. This characteristic architecture of ERECTA proteins prompted us to experimentally confirm of enzymatic activity of one of these, BdERL1 (ERECTA-like1 from Brachypodium distachyon). We have shown that BdERL1 is dual-function protein with both kinase and GC activity. Moreover, our mutagenesis studies also revealed the catalytic roles of key conserved amino acid residues at the GC center and importantly, probing of the kinase and GC with Ca2+ and/or cGMP, shed light on the intramolecular regulations of BdERL1.

The fractions of 26S and 20S proteasomes have been isolated from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to the proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes involved in various metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract 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.

In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. 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. 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. Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Lipocalins for Hungry Animals in a Chilly World As for other lipid-binding proteins, metabolism regulation has been assigned to practically all Lipocalins, and correlations of metabolic diseases with Lipocalin expression are abundant. Keywords: immunomodulation; antioxidant; cellular barriers; metabolism; reproduction; development; social behaviour; moonlighting proteins EN immunomodulation antioxidant cellular barriers metabolism reproduction development social behaviour moonlighting proteins 1 4 4 05/02/22 20220428 NES 220428 Introduction Lipocalins form an ancestral protein family found in all kingdoms of life, except for Archaea. Lipocalins can synthesize immune system mediators [PGD SB 2 sb (Urade)] or control the availability of small immunomodulating molecules [like LPC by ApoD, or LPC and PAF by AGP (Ruiz; Sanchez and Ganfornina)]. Lipocalins as Optimizers A major conclusion we extract from the wide array of physiological processes reviewed in this Research Topic is that Lipocalins are key to organismal fitness, despite not being indispensable genes. [Extracted from the article]

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)-β-D-glucan of Levilactobacillus (L.) brevis TMW 1.2112. The fermentation broth of L. brevis TMW 1.2112 was analyzed for changes in viscosity, β-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 β-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 β-glucan biosynthesis involved a β-1,3 glucan synthase (GT2) and enzymes from maltose phosphorylase (MP) operons. The decreased viscosity appeared to be associated with cell lysis as the β-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. [ABSTRACT FROM AUTHOR]

The coagulase-negative staphylococcal (CoNS) species Staphylococcus lugdunensis is unique in causing serious infections in humans that resemble those of Staphylococcus aureus rather than those of other CoNS species. The colonization and invasion of host tissue presupposes the presence of adherence factors, but only a few proteins mediating adhesion of S. lugdunensis to biotic surfaces are known yet. Here, we report on the functionality of the S. lugdunensis enolase (SlEno), which performs two distinct roles, first, as the metabolic enzyme of the glycolysis, and second, as an adherence factor to the extracellular matrix (ECM) of cells. Phylogenetic analyses of the SlEno confirmed their high conservation to enolases of other species and revealed a closer relationship to Staphylococcus epidermidis than to S. aureus. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot experiments, we identified SlEno to be located in the cytoplasm as well as on the cell surface of S. lugdunensis. Recombinantly generated and surface-associated SlEno showed the usual enolase activity by catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate but, in addition, also displayed strong binding to immobilized laminin, fibronectin, fibrinogen, and collagen type IV in a dose-dependent manner. We also showed a strong binding of SlEno to plasminogen (Plg) and observed a tissue plasminogen activator (tPA)-dependent conversion of Plg to plasmin (Pln) whereby the Plg activation significantly increased in the presence of SlEno. This interaction might be dependent on lysines of the SlEno protein as binding to Plg was inhibited by ε-aminocaproic acid. Furthermore, the enhanced activation of the Plg/Pln system by SlEno enabled S. lugdunensis to migrate through a fibrin matrix. This migration was about 10-fold higher than without exogenously added SlEno. Finally, we observed a significantly higher clearance of S. lugdunensis by freshly prepared granulocytes and in the presence of anti-SlEno antibodies. In conclusion, these data demonstrate for the first time a moonlighting function of the S. lugdunensis enolase, which is an underrated virulence factor for colonization and invasion of tissues. Hence, SlEno might be a potential vaccine candidate to prevent severe infections caused by this pathogen. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The coagulase-negative staphylococcal (CoNS) species Staphylococcus lugdunensis is unique in causing serious infections in humans that resemble those of Staphylococcus aureus rather than those of other CoNS species. The colonization and invasion of host tissue presupposes the presence of adherence factors, but only a few proteins mediating adhesion of S. lugdunensis to biotic surfaces are known yet. Here, we report on the functionality of the S. lugdunensis enolase (SlEno), which performs two distinct roles, first, as the metabolic enzyme of the glycolysis, and second, as an adherence factor to the extracellular matrix (ECM) of cells. Phylogenetic analyses of the SlEno confirmed their high conservation to enolases of other species and revealed a closer relationship to Staphylococcus epidermidis than to S. aureus. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot experiments, we identified SlEno to be located in the cytoplasm as well as on the cell surface of S. lugdunensis. Recombinantly generated and surface-associated SlEno showed the usual enolase activity by catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate but, in addition, also displayed strong binding to immobilized laminin, fibronectin, fibrinogen, and collagen type IV in a dose-dependent manner. We also showed a strong binding of SlEno to plasminogen (Plg) and observed a tissue plasminogen activator (tPA)-dependent conversion of Plg to plasmin (Pln) whereby the Plg activation significantly increased in the presence of SlEno. This interaction might be dependent on lysines of the SlEno protein as binding to Plg was inhibited by ε-aminocaproic acid. Furthermore, the enhanced activation of the Plg/Pln system by SlEno enabled S. lugdunensis to migrate through a fibrin matrix. This migration was about 10-fold higher than without exogenously added SlEno. Finally, we observed a significantly higher clearance of S. lugdunensis by freshly prepared granulocytes and in the presence of anti-SlEno antibodies. In conclusion, these data demonstrate for the first time a moonlighting function of the S. lugdunensis enolase, which is an underrated virulence factor for colonization and invasion of tissues. Hence, SlEno might be a potential vaccine candidate to prevent severe infections caused by this pathogen.

Despite significant and coordinated efforts to combat schistosomiasis, such as providing clean water, sanitation, hygiene, and snail control, these strategies still fall short, as regions previously thought to be disease-free have shown active schistosomiasis transmission. Therefore, it is necessary to implement integrated control methods, emphasizing vaccine development for sustainable control of schistosomiasis. Vaccination has significantly contributed to global healthcare and has been the most economically friendly method for avoiding pathogenic infections. Over the years, different vaccine candidates for schistosomiasis have been investigated with varying degrees of success in clinical trials with many not proceeding past the early clinical phase. Recently, proteins have been mentioned as targets for drug discovery and vaccine development, especially those with multiple functions in schistosomes. Moonlighting proteins are a class of proteins that can perform several functions besides their known functions. This multifunctional property is believed to have been expressed through evolution, where the polypeptide chain gained the ability to perform other tasks without undergoing any structural changes. Since proteins have gained more traction as drug targets, multifunctional proteins have thus become attractive for discovering and developing novel drugs since the drug can target more than one function. Moonlighting proteins are promising drug and vaccine candidates for diseases such as schistosomiasis, since they aid in disease promotion in the human host. This manuscript elucidates vital moonlighting proteins used by schistosomes to drive their life cycle and to ensure their survival in the human host, which can be used to develop anti-schistosomal therapeutics and vaccinomics., Competing Interests: None., (AJTR Copyright © 2024.)

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.

Periodontal disease depends on the presence of different microorganisms in the oral cavity that during the colonization of periodontal tissues form a multispecies biofilm community, thus allowing them to survive under adverse conditions or facilitate further colonization of host tissues. Not only numerous bacterial species participate in the development of biofilm complex structure but also fungi, especially Candida albicans, that often commensally inhabits the oral cavity. C. albicans employs an extensive armory of various virulence factors supporting its coexistence with bacteria resulting in successful host colonization and propagation of infection. In this article, we highlight various aspects of individual fungal virulence factors that may facilitate the collaboration with the associated bacterial representatives of the early colonizers of the oral cavity, the bridging species, and the late colonizers directly involved in the development of periodontitis, including the “red complex” species. In particular, we discuss the involvement of candidal cell surface proteins—typical fungal adhesins as well as originally cytosolic “moonlighting” proteins that perform a new function on the cell surface and are also present within the biofilm structures. Another group of virulence factors considered includes secreted aspartic proteases (Sap) and other secreted hydrolytic enzymes. The specific structure of the candidal cell wall, dynamically changing during morphological transitions of the fungus that favor the biofilm formation, is equally important and discussed. The non-protein biofilm-composing factors also show dynamic variability upon the contact with bacteria, and their biosynthesis processes could be involved in the stability of mixed biofilms. Biofilm-associated changes in the microbe communication system using different quorum sensing molecules of both fungal and bacterial cells are also emphasized in this review. All discussed virulence factors involved in the formation of mixed biofilm pose new challenges and influence the successful design of new diagnostic methods and the application of appropriate therapies in periodontal diseases.

Periodontal disease depends on the presence of different microorganisms in the oral cavity that during the colonization of periodontal tissues form a multispecies biofilm community, thus allowing them to survive under adverse conditions or facilitate further colonization of host tissues. Not only numerous bacterial species participate in the development of biofilm complex structure but also fungi, especially Candida albicans , that often commensally inhabits the oral cavity. C. albicans employs an extensive armory of various virulence factors supporting its coexistence with bacteria resulting in successful host colonization and propagation of infection. In this article, we highlight various aspects of individual fungal virulence factors that may facilitate the collaboration with the associated bacterial representatives of the early colonizers of the oral cavity, the bridging species, and the late colonizers directly involved in the development of periodontitis, including the "red complex" species. In particular, we discuss the involvement of candidal cell surface proteins—typical fungal adhesins as well as originally cytosolic "moonlighting" proteins that perform a new function on the cell surface and are also present within the biofilm structures. Another group of virulence factors considered includes secreted aspartic proteases (Sap) and other secreted hydrolytic enzymes. The specific structure of the candidal cell wall, dynamically changing during morphological transitions of the fungus that favor the biofilm formation, is equally important and discussed. The non-protein biofilm-composing factors also show dynamic variability upon the contact with bacteria, and their biosynthesis processes could be involved in the stability of mixed biofilms. Biofilm-associated changes in the microbe communication system using different quorum sensing molecules of both fungal and bacterial cells are also emphasized in this review. All discussed virulence factors involved in the formation of mixed biofilm pose new challenges and influence the successful design of new diagnostic methods and the application of appropriate therapies in periodontal diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Proteus mirabilis oligomeric urease is a moonlighting virulence factor. • Holo-urease promoted platelet aggregation and toxicity in fungal and insect models. • Subunit Ureβ has the highest toxicity while also activating platelets. • Bioinformatics analyses revealed gene/segment duplication. • Ureβ and its counterparts in other ureases carry most of the non-enzymatic activities. Ureases are moonlighting proteins displaying non-catalytic properties, including platelet activation, antifungal and entomotoxic effects. The structure-activity mapping of these properties is poorly developed. Proteus mirabilis urease (PMU) consists of three subunits, PmUreα, PmUreβ and PmUreγ, in an (αβγ) 3 organization. In order to study the structure-activity relationships of PMU we obtained the recombinant subunits of this urease and evaluated their biological activities. The holo-urease promoted platelet aggregation, and toxicity in fungal and insect models. Similar to Jaburetox, a plant urease-derived polypeptide, PmUreβ showed the highest toxicity against yeasts and insects, and activated human platelets. PmUreγ and PmUreα presented insecticidal action upon injection. In addition, only PmUreγ and PmUreβ promote hemocytes aggregation. Bioinformatics analyses revealed gene/segment duplication and evolutionary divergence among ureases. Our findings show that PmUreβ (and probably its counterparts in other ureases) carries most of the non-enzymatic activities of these proteins. [ABSTRACT FROM AUTHOR]

The gut microbiota use proteins on their surface to form and maintain interactions with host cells and tissues. In recent years, many of these cell surface proteins have been found to be identical to intracellular enzymes and chaperones. When displayed on the cell surface these moonlighting proteins help the microbe attach to the host by interacting with receptors on the surface of host cells, components of the extracellular matrix, and mucin in the mucosal lining of the digestive tract. Binding of these proteins to the soluble host protein plasminogen promotes the conversion of plasminogen to an active protease, plasmin, which activates other host proteins that aid in infection and virulence. In this mini-review, we discuss intracellular/surface moonlighting proteins of pathogenic and probiotic bacteria and eukaryotic gut microbiota.

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. [ABSTRACT FROM AUTHOR]

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.proteincomple xes.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. [ABSTRACT FROM AUTHOR]

Dengue is emerging as one of the most prevalent mosquito-borne viral diseases of humans. The 11kb RNA genome of the dengue virus encodes three structural proteins (envelope, pre-membrane, capsid) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), all of which are translated as a single polyprotein that is subsequently cleaved by viral and host cellular proteases at specific sites. Non-structural protein 5 (NS5) is the largest of the non-structural proteins, functioning as both an RNA-dependent RNA polymerase (RdRp) that replicates the viral RNA and an RNA methyltransferase enzyme (MTase) that protects the viral genome by RNA capping, facilitating polyprotein translation. Within the human host, NS5 interacts with several proteins such as those in the JAK-STAT pathway, thereby interfering with anti-viral interferon signalling. This mini-review presents annotated, consolidated lists of known and potential NS5 interactors in the human host as determined by experimental and computational approaches respectively. The most significant protein interactors and the biological pathways they participate in are also highlighted and their implications discussed, along with the specific serotype of dengue virus as appropriate. This information can potentially stimulate and inform further research efforts towards providing an integrative understanding of the mechanisms by which NS5 manipulates the human-virus interface in general and the innate and adaptive immune responses in particular.

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. [ABSTRACT FROM AUTHOR]

Dengue is emerging as one of the most prevalent mosquito-borne viral diseases of humans. The 11kb RNA genome of the dengue virus encodes three structural proteins (envelope, pre-membrane, capsid) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), all of which are translated as a single polyprotein that is subsequently cleaved by viral and host cellular proteases at specific sites. Non-structural protein 5 (NS5) is the largest of the non-structural proteins, functioning as both an RNA-dependent RNA polymerase (RdRp) that replicates the viral RNA and an RNA methyltransferase enzyme (MTase) that protects the viral genome by RNA capping, facilitating polyprotein translation. Within the human host, NS5 interacts with several proteins such as those in the JAK-STAT pathway, thereby interfering with anti-viral interferon signalling. This mini-review presents annotated, consolidated lists of known and potential NS5 interactors in the human host as determined by experimental and computational approaches respectively. The most significant protein interactors and the biological pathways they participate in are also highlighted and their implications discussed, along with the specific serotype of dengue virus as appropriate. This information can potentially stimulate and inform further research efforts towards providing an integrative understanding of the mechanisms by which NS5 manipulates the human-virus interface in general and the innate and adaptive immune responses in particular. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plasticity and adaptation to environmental stress are the main features that tumor and immune system share. Except for intrinsic and high-defined properties, cancer and immune cells need to overcome the opponent’s defenses by activating more effective signaling networks, based on common elements such as transcriptional factors, protein-based complexes and receptors. Interestingly, growing evidence point to an increasing number of proteins capable of performing diverse and unpredictable functions. These multifunctional proteins are defined as moonlighting proteins. During cancer progression, several moonlighting proteins are involved in promoting an immunosuppressive microenvironment by reprogramming immune cells to support tumor growth and metastatic spread. Conversely, other moonlighting proteins support tumor antigen presentation and lymphocytes activation, leading to several anti-cancer immunological responses. In this light, moonlighting proteins could be used as promising new potential targets for improving current cancer therapies. In this review, we describe in details 12 unprecedented moonlighting proteins that during cancer progression play a decisive role in guiding cancer-associated immunomodulation by shaping innate or adaptive immune response.