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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]

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]

[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]

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. [ABSTRACT FROM AUTHOR]

• Surface shaving was established in B. pertussis and identified 126 proteins. • Key surfaceome differences provides further evidence of proteomic adaptation • 22 common proteins consistently identified in every sub-proteome. • 11 were essential immunogenic housekeeping proteins, potential as vaccine targets. Since acellular vaccines (ACV) were introduced in Australia, epidemic Bordetella pertussis strains changed from single nucleotide polymorphism (SNP) cluster II to SNP cluster I. Our previous proteomic analysis identified potential proteomic adaptations in the whole cell and secretome of SNP cluster I. Additionally, current ACVs were shown to be less efficacious against cluster I in mice models and there is a pressing need to discover new antigens to improve the ACV. One important source of novel antigens is the surfaceome. Therefore, in this study we established surface shaving in B. pertussis to compare the surfaceome of SNP cluster I (L1423) and II (L1191), and identify novel surface antigens for vaccine development. Surface shaving using 1 μg of trypsin for 5 min identified 126 proteins with the most abundant being virulence-associated and known outer membrane proteins. Cell viability counts showed minimal lysis from shaving. The proportion of immunogenic proteins was higher in the surfaceome than in the whole cell and secretome. Key differences in the surfaceome were identified between SNP cluster I and II, consistent with those identified in the whole cell proteome and secretome. These differences include unique transport proteins and decreased immunogenic proteins in L1423, and provides further evidence of proteomic adaptation in SNP cluster I. Finally, a comparison of proteins in each sub-proteome identified 22 common proteins. These included 11 virulence proteins (Prn, PtxA, FhaB, CyaA, TcfA, SphB1, Vag8, BrkA, BopD, Bsp22 and BipA) and 11 housekeeping proteins (TuF, CtpA, TsF, OmpH, GltA, SucC, SucD, FusA, GroEL, BP3330 and BP3561) which were immunogenic, essential and consistently expressed thus demonstrating their potential as future targets. This study established surface shaving in B. pertussis, confirmed key expression differences and identified unknown surface proteins which may be potential vaccine antigens. [ABSTRACT FROM AUTHOR]

• ADH of Candida albicans was cloned and purified for the first time. • ADH triggers THP-1 cells to differentiate into M1 macrophages. • ERK pathway plays an important role for macrophage differentiation. • ADH induces the productions of IL-1β, IL-6, and TNF-α by THP-1 cells. • As an antigen protein, ADH may induce the host innate immune system. Candida albicans proteins located on the cell wall and in the cytoplasm have gained great attention because they are not only involved in cellular metabolism and the maintenance of integrity but also interact with host immune systems. Previous research has reported that enolase from C. albicans exhibits high immunogenicity and effectively protects mice against disseminated candidiasis. In this study, alcohol dehydrogenase (ADH) of C. albicans was cloned and purified for the first time, and this study focused on evaluating its effects on the differentiation of the human monocytic cell line THP-1. The morphological features of THP-1 cells exposed to ADH were similar to those of phorbol-12-myristate acetate-differentiated (PMA-differentiated) macrophages. Functionally, ADH enhanced the adhesion, phagocytosis, and killing capacities of THP-1 cells. A flow cytometric assay demonstrated that ADH-induced THP-1 cells significantly increased CD86 and CD11b expression. The production of IL-1β, IL-6, and TNF-α by cells increased in the presence of ADH. As expected, after pretreatment with a MEK inhibitor (U0126), ADH-induced THP-1 cells exhibited unaltered morphological features, eliminated ERK1/2 phosphorylation, prevented CD86/CD11b upregulation and inhibited pro-inflammatory cytokine increase. Collectively, these results suggest that ADH enables THP-1 cells to differentiate into macrophages via the ERK pathway, and it may play an important role in the immune response against fungal invasion. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH or Gap) is a ubiquitously distributed enzyme that plays an essential role in the glycolytic and gluconeogenic pathways. However, additional roles have been described unrelated to its enzymatic function in diverse organisms, often linked to its presence in the cell surface or as a secreted protein. Despite being a paradigm among multifunctional/moonlighting proteins, little is known about its possible roles in phytopathogenic bacteria. In the present work we have studied three putative gap paralogous genes identified in the genome of Pseudomonas syringae pv. tomato (Pto) DC3000, an important model in molecular plant pathology, with the aim of determining their physiological and possible non-canonical roles in this bacterium and in the plant infection process. We have established that the Gap1 protein has a predominantly glycolytic activity, whereas the NADPH-dependent Gap2 main activity is gluconeogenic. The third paralogue lacks GAPDH activity in Pto but is indispensable for vitamin B6 metabolism and displays erythrose-4-phosphate dehydrogenase activity, thus referred as epd. Both Gap enzymes exhibit distinct functional characteristics depending on the bacterium physiological state, with Gap1 presenting a substantial role in motility, biosurfactant production and biofilm formation. On the other hand, solely Gap2 appears to be essential for growth on tomato plant. Furthermore, Gap1 and Gap2 present a distinctive transcriptional regulation and both have been identified exported outside the cells with different definite media compositions. This serves as compelling evidence of additional roles beyond their central metabolic functions. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Mycoplasma pneumoniae is a frequent cause of community-acquired infections of the human respiratory tract. During the evolutionary adaptation of the bacteria to the host, the genome of the pathogen is strongly reduced resulting in the loss of cell wall, limited metabolic pathways and a relatively small number of virulence factors. As interacting with host proteins, surface-exposed proteins with a primary function in cytosol-located processes of metabolism and regulation such as glycolytic enzymes, heat-shock proteins and chaperones have been considered as contributing to pathogenesis. Among these moonlighting proteins, some members are confirmed as binding to several host components. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of M. pneumoniae is a typical example of such multi-binding proteins. To investigate the organization of these interactions, GAPDH was divided into four parts. Recombinant proteins were successfully expressed in Escherichia coli and polyclonal antisera were produced. Binding of full length and parts of GAPDH to human A549 cells was proven. Furthermore, interactions with human plasminogen, vitronectin, fibronectin and fibrinogen were demonstrated for nearly all recombinant GAPDH proteins. In the presence of these proteins, plasminogen can be activated to the protease plasmin. In contrast, the localization on the surface of bacterial cell was confirmed for the C-terminal part of GAPDH only. By using overlapping peptides covering this region, binding of the investigated host components to the sequence 326QLVRVVNYCAKL337 was found. The results of the study suggest a prominent role of the surface-localized C-terminal part of GAPDH in associations with different human proteins indicating its importance for host-pathogen-interactions. [ABSTRACT FROM AUTHOR]

Dysfunctions in mitochondria – the powerhouses of the cell – lead to several human pathologies. Because mitochondria integrate nuclear and mitochondrial genetic systems, they are richly intertwined with cellular activities. The nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key components of the mitochondrial translation apparatus. Mutations in these enzymes predominantly affect the central nervous system (CNS) but also target other organs. Comparable mutations in mt-aaRSs can lead to vastly diverse diseases, occurring at different stages in life, and within different tissues; this represents a confounding issue. With newer information available, we propose that the pleiotropy and tissue-specificity of mt-aaRS-associated diseases result from the molecular integration of mitochondrial translation events within the cell; namely, through specific crosstalk between the cellular program and the energy demands of the cell. We place particular focus on neuronal cells. [ABSTRACT FROM AUTHOR]

Fluctuations in nutrient availability are a fact of life for bacterial cells in the ‘wild’. To survive and compete, bacteria must rapidly modulate cell-cycle processes to accommodate changing nutritional conditions and concomitant changes in cell growth. Our understanding of how this is achieved has been transformed in recent years, with cellular metabolism emerging as a central player. Several metabolic enzymes, in addition to their normal catalytic functions, have been shown to directly modulate cell-cycle processes in response to changing nutrient levels. Here we focus on cell division, the final event in the bacterial cell cycle, and discuss recent compelling evidence connecting division regulation to nutritional status and metabolic activity. [ABSTRACT FROM AUTHOR]

In the past few decades, hundreds of moonlighting proteins have been identified that perform two or more distinct and physiologically relevant biochemical or biophysical functions that are not due to gene fusions, multiple RNA splice variants, or pleiotropic effects. For this special issue on protein species, this article discusses three topics related to moonlighting proteins that illustrate how small changes or differences in protein covalent structures can result in different functions. Examples are given of moonlighting proteins that switch between functions after undergoing post-translational modifications (PTMs), proteins that share high levels of amino acid sequence identity to a moonlighting protein but share only one of its functions, and several “neomorphic moonlighting proteins” in which a single amino acid mutation results in the addition of a new function. Biological significance For this special issue on protein species, this article discusses three topics related to moonlighting proteins : Post-translational modifications (PTMs) that can cause a switch between functions, homologs that share only one of multiple functions, and proteins in which a single amino acid mutation results in the creation of a new function. The examples included illustrate that even in an average protein of hundreds of amino acids, a relatively small difference in sequence or PTMs can result in a large difference in function, which can be important in predicting protein functions, regulation of protein functions, and in the evolution of new functions. [ABSTRACT FROM AUTHOR]

River blindness, caused by the filarial parasite Onchocerca volvulus , is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase ( Ov -GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov -GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis . Mice were either vaccinated with Ov -GAPDH.DNA plasmid ( Ov - p GAPDH.DNA) alone or in combination with recombinantly expressed Ov -GAPDH protein ( Ov - r GAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis , those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov -GAPDH peptides ( Ov -GAPDH pept ), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov -GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host–parasite relationship. [ABSTRACT FROM AUTHOR]

The definition of safety and efficacy of food-employed bacteria as well as probiotic strains is a continuous, often unattended, challenge. Proteomic techniques such as 2DE, DIGE and LC/LC-MS/MS are suitable and powerful tools to reveal new aspects (positive and negative) of “known” and “unknown” strains that can be employed in food making and as nutraceutical supplements for human health. Unfortunately, these techniques are not used as extensively as it should be wise. The present report describes the most significant results obtained by our research group in 10 years of study on subproteomes in bacteria, chiefly lactic acid bacteria. Production of desired and undesired metabolites, differences between strains belonging to same species but isolated from different ecological niches, the effect of cryoprotectants on survival to lyophilization as well as the adhesive capability of strains, were elucidated by analysis of cytosolic, membrane-enriched, surface and extracellular proteomes. The present review opens a window on a yet largely underexplored field and highlights the huge potential of subproteome investigations for more rational choice of microbial strains as food starters, probiotics and for production of nutraceuticals. These analyses will hopefully contribute to manufacturing safer and healthier food and food supplements in the near future. This article is part of a Special Issue entitled: HUPO 2014. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Lathyrus sativus shares ∼40% of the secretome constituents reported thus far. • We identified 571 unique secretory proteins, involved in critical functions. • One-third of L. sativus proteins is predicted to follow unconventional secretion. • The secretome recognized biological processes underlying stress resilience. [ABSTRACT FROM AUTHOR]

Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a multifunctional protein with defined functions in numerous mammalian cellular processes. GAPDH functional diversity depends on various factors such as covalent modifications, subcellular localization, oligomeric state and intracellular concentration of substrates or ligands, as well as protein–protein interactions. In bacteria, alternative GAPDH functions have been associated with its extracellular location in pathogens or probiotics. In this study, new intracellular functions of Escherichia coli GAPDH were investigated following a proteomic approach aimed at identifying interacting partners using in vivo formaldehyde cross-linking followed by mass spectrometry. The identified proteins were involved in metabolic processes, protein synthesis and folding or DNA repair. Some interacting proteins were also identified in immunopurification experiments in the absence of cross-linking. Pull-down experiments and overlay immunoblotting were performed to further characterize the interaction with phosphoglycolate phosphatase (Gph). This enzyme is involved in the metabolism of 2-phosphoglycolate formed in the DNA repair of 3′-phosphoglycolate ends generated by bleomycin damage. We show that interaction between Gph and GAPDH increases in cells challenged with bleomycin, suggesting involvement of GAPDH in cellular processes linked to DNA repair mechanisms. [Copyright &y& Elsevier]

Proteomics projects involving biochemical, genetic and computational methods are being used to elucidate the functions of thousands of proteins in healthy and disease states. The results provide valuable information both for understanding basic physiological processes and for the development of novel therapeutics. However, the ability of a protein to moonlight, or to have more than one function, can complicate the interpretation and application of the results from these proteomics studies. This review provides examples of how moonlighting proteins can affect the results of proteomics projects being used to determine protein function based on patterns of protein expression, protein–protein interactions, analysis of protein sequences and structures, and other methods. [Copyright &y& Elsevier]