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

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.

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.

Investigating and understanding the response of microbes to various surfaces requires a versatile parametrisation of the surface, and multiple assays that captures the complexity of the biofilm structures. Here, Staphylococcus aureus biofilm viability, polysaccharide poly-N-acetylglucosamine, and proteins on the cell surface were analysed with agar plate- and well plate-based biofilm formation assays. Biofilms were grown on a set of nanostructured polymeric surfaces, which were thoroughly characterised for their surface chemistry and topography. Surface hydrophobicity, summit density as well as peak and valley structure were found to influence the microbial viability and exopolysaccharide abundance level in the agar plate assay. In the well plate assay, surface chemical parameters had a lesser influence on the viability, but roughness caused by valley structures increased the viability and decreased the exopolysaccharide expression. Surface proteins relating to pathogenicity were affected by the biofilm formation assay. The abundance profile of these proteins correlated clearly with several roughness parameters, especially fine structure parameters in the agar plate assay and lateral roughness in the well plate assay. These results highlight the necessity of describing the surfaces with a versatile set of different roughness parameters to completely understand what specific features of a surface drive a certain bacterial response.

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.

Graphical abstract, Highlights • 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.

Bacterial biofilms are an important underlying cause for chronic infections. By switching into the biofilm state, bacteria can evade host defenses and withstand antibiotic chemotherapy. Despite the fact that biofilms at clinical and environmental settings are mostly composed of multiple microbial species, biofilm research has largely been focused on single-species biofilms. In this study, we investigated the interaction between two clinically relevant bacterial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) by label-free quantitative proteomics focusing on proteins associated with the bacterial cell surfaces (surfaceome) and proteins exported/released to the extracellular space (exoproteome). The changes observed in the surfaceome and exoproteome of P. aeruginosa pointed toward higher motility and lower pigment production when co-cultured with S. aureus. In S. aureus, lower abundances of proteins related to cell wall biosynthesis and cell division, suggesting increased persistence, were observed in the dual-species biofilm. Complementary phenotypic analyses confirmed the higher motility and the lower pigment production in P. aeruginosa when co-cultured with S. aureus. Higher antimicrobial tolerance associated with the co-culture setting was additionally observed in both species. To the best of our knowledge, this study is among the first systematic explorations providing insights into the dynamics of both the surfaceome and exoproteome of S. aureus and P. aeruginosa dual-species biofilms.

Les proteïnes "moonlighting" són aquelles que duen a terme més d'una funció bioquímica. En microorganismes patògens, s'ha vist que moltes d'aquestes proteïnes tenen funcions secundàries relacionades amb l'adhesió a l'hoste i la capacitat d'infecció. Conèixer quines regions de la proteïna tenen a veure amb la virulència pot ajudar a entendre millor els mecanismes que hi ha implicats. Addicionalment, s'ha vist que moltes d'aquestes proteïnes relacionades amb la virulència tenen originalment funcions molt bàsiques per a la cèl·lula. Això ha suggerit la hipòtesi que aquestes proteïnes, estant evolutivament molt conservades en els hostes, podrien ser utilitzades pels patògens com una forma de camuflatge, exposant-les a la superfície cel·lular perquè l'hoste les identifiqui com a pròpies. En aquest treball s'han buscat, en un conjunt de deu proteïnes moonlighting de patògens, elements estructurals, glicosilacions i motius de seqüència possiblement relacionats amb la virulència. Per fer-ho, s'han dut a terme, per a cada una de les proteïnes estudiades, comparacions amb diverses proteïnes homòlogues, tant d'altres patògens com de no patògens. La selecció d'elements sospitosos de tenir relació amb la virulència s'ha fet escollint aquells que es troben conservats exclusivament en els patògens. De forma semblant, en el cas de les glicosilacions, s'han seleccionat també aquelles conservades exclusivament en no patògens, com a sospitoses de tenir relació amb l'absència de virulència. Las proteínas "moonlighting" son aquellas que llevan a cabo más de una función bioquímica. En microorganismos patógenos, se ha visto que muchas de estas proteínas tienen funciones secundarias relacionadas con la adhesión al huésped y la capacidad de infección. Conocer qué regiones de la proteína tienen que ver con la virulencia puede ayudar a entender mejor los mecanismos que hay implicados. Adicionalmente, se ha visto que muchas de estas proteínas relacionadas con la virulencia tienen originalmente funciones muy básicas para la célula. Esto ha sugerido la hipótesis que estas proteínas, estando evolutivamente muy conservadas en los huéspedes, podrían ser utilizadas por los patógenos como una forma de camuflaje, exponiéndolas en la superficie celular porque el huésped las identifique como propias. En este trabajo se han buscado, en un conjunto de diez proteínas moonlighting de patógenos, elementos estructurales, glicosilacions y motivos de secuencia posiblemente relacionados con la virulencia. Para hacerlo, se han llevado a cabo, para cada una de las proteínas estudiadas, comparaciones con varias proteínas homólogas, tanto otros patógenos como de no patógenos. La selección de elementos sospechosos de tener relación con la virulencia se ha hecho escogiendo aquellos que se encuentran conservados exclusivamente en los patógenos. De forma parecida, en el caso de las glicosilacions, se han seleccionado también aquellas conservadas exclusivamente en no patógenos, como sospechosas de tener relación con la ausencia de virulencia. "Moonlighting" proteins are those that carry out more than one biochemical function. In pathogen microorganisms, it has been observed that many of such proteins have secondary functions related to host adhesion and to the ability to infect. Knowing which regions of the protein are related to virulence can help better understand the mechanisms involved. In addition, it has been observed that many of such proteins related to virulence originally have very essential cellular functions. This fact has suggested the hypothesis that those proteins, being evolutionarily very preserved in hosts, may be used by pathogens as a form of camouflage, exposing them to the cell surface so the host identifies them as its own. With this project it has been sought, in a set of ten moonlighting proteins of pathogens, structural elements, glycosylations and sequence motifs possibly related to virulence. To do so, it has been carried out, for each of the studied proteins, a series of comparisons with several homologue proteins, both from other pathogens and from nonpathogens. The selection of elements suspicious of being related to virulence has been done choosing those preserved exclusively in pathogens. Similarly, in the case of glycosylations, have also been selected those preserved exclusively in non-pathogens, as suspicious of being related to the absence of virulence. A total of 7 structural regions, 40 predicted glycosylations and 31 sequence motifs have been obtained. Those elements may help direct future research efforts, starting by its experimental validation.

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

The swine pathogen Streptococcus suis is a Gram-positive bacterium which causes infections in pigs, with an impact in animal health and in the livestock industry, and it is also an important zoonotic agent. During the infection process, surface and secreted proteins are essential in the interaction between microorganisms and their hosts. Here, we report a comparative proteomic analysis of the proteins released to the extracellular milieu in six human clinical isolates belonging to the highly prevalent and virulent serotype 2. The total secreted content was precipitated and analyzed by GeLC-MS/MS. In the six strains, 144 proteins assigned to each of the categories of extracellular or surface proteins were identified, as well as 680 predicted cytoplasmic proteins, many of which are putative moonlighting proteins. Of the nine predicted signal peptide-I secreted proteins, seven had relevant antigenic potential when they were analyzed through bioinformatic analysis. This is the first work comparing the exoproteome fraction of several human isolates of this important pathogen.

The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM and envelope proteins of Mycobacterium marinum ATCC 927, a nontuberculous mycobacterial model, were monitored over 3 months by label-free proteomics and compared with cell surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC 927 forms pellicle-type and submerged-type biofilms (PBFs and SBFs, respectively) after 2 weeks and 2 days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggest that specific changes in mycolic acid synthesis (cord factor), Esx1 secretion, and cell wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting 64× MIC rifampicin (persisters) was detected in both biofilm subtypes and already in 1-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of various ambient oxygen tensions on each cell type and nonclassical protein secretion are likely factors explaining the majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in planktonic (PL) and PBF cells, while SBF may prefer both the Esx5 and nonclassical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports the first proteomic insight into aging mycobacterial biofilm ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence of nontuberculous mycobacteria., mSystems, 6 (3), ISSN:2379-5077

Moonlighting and multitasking proteins refer to proteins with two or more functions performed by a single polypeptide chain. An amazing example of the Gain of Function (GoF) phenomenon of these proteins is that 25% of the moonlighting functions of our Multitasking Proteins Database (MultitaskProtDB-II) are related to pathogen virulence activity. Moreover, they usually have a canonical function belonging to highly conserved ancestral key functions, and their moonlighting functions are often involved in inducing extracellular matrix (ECM) protein remodeling. There are three main questions in the context of moonlighting proteins in pathogen virulence: (A) Why are a high percentage of pathogen moonlighting proteins involved in virulence? (B) Why do most of the canonical functions of these moonlighting proteins belong to primary metabolism? Moreover, why are they common in many pathogen species? (C) How are these different protein sequences and structures able to bind the same set of host ECM protein targets, mainly plasminogen (PLG), and colonize host tissues? By means of an extensive bioinformatics analysis, we suggest answers and approaches to these questions. There are three main ideas derived from the work: first, moonlighting proteins are not good candidates for vaccines. Second, several motifs that might be important in the adhesion to the ECM were identified. Third, an overrepresentation of GO codes related with virulence in moonlighting proteins were seen.

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.

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.

eEF1A1 and eEF1A2 are paralogous proteins whose presence in most normal eukaryotic cells is mutually exclusive and developmentally regulated. Often described in the scientific litera-ture under the collective name eEF1A, which stands for eukaryotic elongation factor 1A, their best known activity (in a monomeric, GTP-bound conformation) is to bind aminoacyl-tRNAs and deliver them to the A-site of the 80S ribosome. However, both eEF1A1 and eEF1A2 are endowed with mul-titasking abilities (sometimes performed by homo-and heterodimers) and can be located in different subcellular compartments, from the plasma membrane to the nucleus. Given the high sequence identity of these two sister proteins and the large number of post-translational modifications they can undergo, we are often confronted with the dilemma of discerning which is the particular pro-teoform that is actually responsible for the ascribed biochemical or cellular effects. We argue in this review that acquiring this knowledge is essential to help clarify, in molecular and structural terms, the mechanistic involvement of these two ancestral and abundant G proteins in a variety of funda-mental cellular processes other than translation elongation. Of particular importance for this special issue is the fact that several de novo heterozygous missense mutations in the human EEF1A2 gene are associated with a subset of rare but severe neurological syndromes and cardiomyopathies., This research was funded by the Spanish MICINN (Project PID2019-104070RB-C22 to F.G.) and PharmaMar S.A.U. (Colmenar Viejo, Madrid, Spain). The APC was defrayed by University of Alcalá.

La multifuncionalidad de proteínas se refiere a su habilidad para llevar a cabo un cabo dos o más funciones biológicas. El objetivo de este trabajo fue examinar con herramientas bioinformáticas la relación entre proteínas multifuncionales y enfermedades humanas, tomando en cuenta los mecanismos moleculares de la enfermedad y su interacción con dianas farmacológicas. La aproximación tomada involucraba evaluar estadísticamente la asociación entre enfermedades humanas y proteínas multifuncionales, y revisar bases de datos de interacción de proteínas para analizar la interacción local entre ellas y un análisis funcional de las proteínas de estas redes. No se observaron suficientes evidencias para afirmar que el número de clases funcionales de proteínas moonlighting en humanos es distinto al resto de los organismos. Sí se obtuvo suficiente información para sugerir que las proteínas moonlighting son propensas a estar involucradas en enfermedades humanas. Además, se hicieron análisis de redes de interacción de proteínas moonlighting involucradas en enfermedades humanas, y se encontraron 7 grupos de interacción. También se hizo un análisis de términos GO de cada grupo asociado a enfermedades, para una caracterización de los procesos biológicos, funcione moleculares y compartimientos celulares donde se encontraban. Se encontraron redes de interacción proteína-proteína para los grupos de interacción asociados a enfermedades, y se hizo un acoplamiento molecular entre proteínas pertenecientes a uno de los grupos. Se puede concluir que en las proteínas moonlighting humanas asociadas a enfermedades existen redes de interacción de tamaño considerable que tienen conexiones entre sí. Estudios de Máster Protein multifunctionality refers to their ability to carry out two or more biological functions. The objective of this work was to examine with bioinformatics tools the relationship between multifunctional proteins and human diseases, considering the molecular mechanisms of the disease and its interaction with drug targets. The approach taken involved statistically evaluating the association between human diseases and multifunctional proteins and reviewing protein interaction databases to analyze the local interaction between them and a functional analysis of the proteins in these networks. Not enough evidence was observed to affirm that the number of functional classes of moonlighting proteins in humans is different from other organisms. Enough information was btained to suggest that moonlighting proteins are likely to be involved in human disease. In addition, analysis of interaction networks of moonlighting proteins involved in human diseases were made, and 7 interaction groups were found. An analysis of GO terms of each group associated with diseases was also done, for a characterization of the biological processes, molecular functions, and cellular compartments where they were found. Protein-protein interaction networks were found for the disease-associated interaction groups, and a molecular coupling was made between proteins belonging to one of the groups. It can be concluded that in human moonlighting proteins associated with diseases there are interaction networks of considerable size that have connections between them. La multifuncionalitat de proteïnes es refereix a la seva habilitat per dur a terme un terme dues o més funcions biològiques. L'objectiu d'aquest treball va ser examinar amb eines bioinformàtiques la relació entre proteïnes multifuncionals i malalties humanes, tenint en compte els mecanismes moleculars de la malaltia i la seva interacció amb dianes farmacològiques. L'aproximació presa involucrava avaluar estadísticament l'associació entre malalties humanes i proteïnes multifuncionals, i revisar bases de dades d'interacció de proteïnes per analitzar la interacció local entre elles i una anàlisi funcional de les proteïnes d'aquestes xarxes. No es van observar suficients evidències per afirmar que el nombre de classes funcionals de proteïnes moonlighting en humans és diferent a la resta dels organismes. Sí que es va obtenir suficient informació per suggerir que les proteïnes moonlighting són propenses a estar involucrades en malalties humanes. A més, es van fer anàlisis de xarxes d'interacció de proteïnes moonlighting involucrades en malalties humanes, i es van trobar 7 grups d'interacció. També es va fer una anàlisi de termes GO de cada grup associat a malalties, per a una caracterització dels processos biològics, funcioni moleculars i compartiments cel·lulars on es trobaven. Es van trobar xarxes d'interacció proteïna-proteïna per als grups d'interacció associats a malalties, i es va fer un acoblament molecular entre proteïnes pertanyents a un dels grups. Es pot concloure que en les proteïnes moonlighting humanes associades a malalties existeixen xarxes d'interacció de mida considerable que tenen connexions entre si.

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.

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

Protein science has moved from a focus on individual molecules to an integrated perspective in which proteins emerge as dynamic players with multiple functions, rather than monofunctional specialists. Annotation of the full functional repertoire of proteins has impacted the fields of biochemistry and genetics, and will continue to influence basic and applied science questions - from the genotype-to-phenotype problem, to our understanding of human pathologies and drug design. In this review, we address the phenomena of pleiotropy, multidomain proteins, promiscuity, and protein moonlighting, providing examples of multitasking biomolecules that underlie specific mechanisms of human disease. In doing so, we place in context different types of multifunctional proteins, highlighting useful attributes for their systematic definition and classification in future research directions.

Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge.

International audience; We analyzed the adhesion capacity to mucus of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives using an immobilized mucin model. On the basis of their adhesive capacity to mucin, three phenotypes were selected for cell-wall protein proteomic analysis to pinpoint proteins involved in the adhesion process: the highly adhesive L. pentosus CF1-43 N (73.49% of adhesion ability), the moderately adhesive L. pentosus CF1-37 N (49.56% of adhesion ability) and the poorly adhesive L. pentosus CF2-20P (32.79% of adhesion ability). The results revealed four moonlighting proteins over-produced in the highly adhesive L. pentosus CF1-43 N, which were under/not produced in the other two L. pentosus strains (CF1-37 N and CF2-20P). These proteins were involved in glycolytic pathway (phosphoglycerate mutase and glucosamine-6-phosphate deaminase), stress response (small heat shock protein) and transcription (transcription elongation factor GreA). Furthermore, the relative fold change in gene expression analysis showed significant up-regulation of the genes coding for these four moonlighting proteins in the highly adhesive L. pentosus CF1-43 N versus the poorly adhesive L. pentosus CF2-20P and also in response to mucin for 20 h which clearly indicate the significant role of these genes in the adhesion capacity of L. pentosus. Thus, these proteins could be used as biomarkers for mucus adhesion in L. pentosus. On the other hand, mucin exposure induced other probiotic effects in L. pentosus strains, enhancing their co-aggregation ability with pathogens and possible inactivation.

Clostridium difficile (CD) infections are a growing threat due to the strain resistance to antibiotic treatment and the emergence of hypervirulent strains. One solution to this problem is the search for new vaccine antigens, preferably surface-localized that will be recognized by antibodies at an early stage of colonization. The purpose of the study was to assess the usefulness of novel immunoreactive surface proteins (epitopes) as potential vaccine antigens. Such approach might be tough to pursue since pathogens have acquired strategies to subvert adaptive immune response to produce humoral response against non-essential proteins for their survival. In this study CD surface proteins were isolated, immunoreactive proteins identified and mapped to select potential epitopes. The results of the study exclude the use of CD glyceraldehyde 3-phosphate dehydrogenase as a vaccine antigen, especially as a whole protein. Sequences P9 (201AAGNIVPNTTGAAKAI218) and P10 (224KGKLDGAAQRVPVVTG241) recognized by patients sera are conserved and widespread among CD strains. They show cross-reactivity with sera of people suffering from other bacterial infections and are recognized by sera of autoimmune disease patients. Our study documents that special care in analyzing the sequence of new epitope should be taken to avoid side effects prior to consider it as a vaccine antigen.

This study compared the secretomes (proteins exported out of the cell) of Propionibacterium freudenreichii of different origin to identify plausible adaptation factors. Phylosecretomics indicated strain-specific variation in secretion of adhesins/invasins (SlpA, InlA), cell-wall hydrolysing (NlpC60 peptidase, transglycosylase), protective (RpfB) and moonlighting (DnaK, GroEL, GaPDH, IDH, ENO, ClpB) enzymes and/or proteins. Detailed secretome comparison suggested that one of the cereal strains (JS14) released a tip fimbrillin (FimB) in to the extracellular milieu, which was in line with the electron microscopy and genomic analyses, indicating the lack of surface-associated fimbrial-like structures, predicting a mutated type-2 fimbrial gene cluster (fimB-fimA-srtC2) and production of anchorless FimB. Instead, the cereal strain produced high amounts of SlpB that tentatively mediated adherent growth on hydrophilic surface and adherence to hydrophobic material. One of the dairy strains (JS22), producing non-covalently bound surface-proteins (LspA, ClpB, AraI) and releasing SlpA and InlA into the culture medium, was found to form clumps under physiological conditions. The JS22 strain lacked SlpB and displayed a non-clumping and biofilm-forming phenotype only under conditions of increased ionic strength (300mM NaCl). However, this strain cultured under the same conditions was not adherent to hydrophobic support, which supports the contributory role of SlpB in mediating hydrophobic interactions. Thus, this study reports significant secretome variation in P.freudenreichii and suggests that strain-specific differences in protein export, modification and protein-protein interactions have been the driving forces behind the adaptation of this bacterial species.