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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.

Methicillin-resistant Staphylococcus aureus (MRSA) is the common name for a heterogeneous group of highly drug-resistant staphylococci. Two major MRSA classes are distinguished based on epidemiology, namely community-associated (CA) and hospital-associated (HA) MRSA. Notably, the distinction of CA- and HA-MRSA based on molecular traits remains difficult due to the high genomic plasticity of S. aureus. Here we sought to pinpoint global distinguishing features of CA- and HA-MRSA through a comparative genome and proteome analysis of the notorious MRSA lineage USA300. We show for the first time that CA- and HA-MRSA isolates can be distinguished by 2 distinct extracellular protein abundance clusters that are predictive not only for epidemiologic behavior, but also for their growth and survival within epithelial cells. This ‘exoproteome profiling’ also groups more distantly related HA-MRSA isolates into the HA exoproteome cluster. Comparative genome analysis suggests that these distinctive features of CA- and HA-MRSA isolates relate predominantly to the accessory genome. Intriguingly, the identified exoproteome clusters differ in the relative abundance of typical cytoplasmic proteins, suggesting that signatures of cytoplasmic proteins in the exoproteome represent a new distinguishing feature of CA- and HA-MRSA. Our comparative genome and proteome analysis focuses attention on potentially distinctive roles of ‘liberated’ cytoplasmic proteins in the epidemiology and intracellular survival of CA- and HA-MRSA isolates. Such extracellular cytoplasmic proteins were recently invoked in staphylococcal virulence, but their implication in the epidemiology of MRSA is unprecedented.

Las proteínas moonlighting son aquellas que realizan más de una función utilizando la misma cadena polipeptídica. Identificar estas proteínas es necesario tanto para su anotación funcional como por su relevancia en múltiples aspectos bioquímicos y fisiopatológicos, estando implicadas en virulencia de microorganismos y patologías humanas. Es un fenómeno de gran complejidad, pues comprender cómo surgen y coexisten diferentes funciones sin interferir entre ellas supone un reto. Aún quedan muchas cuestiones por resolver como cuál es su origen evolutivo o cuáles son los mecanismos por los que se adquieren estas funciones adicionales. En este trabajo se ha estudiado la relación entre las proteínas multifuncionales y el Non Orthologous Gene Displacement (NOGD), un fenómeno por el que un componente esperado de una vía enzimática está ausente y su función es realizada por otra proteína. Se han encontrado 124 proteínas moonlighting que realizan funciones de proteínas NOGD e incluso hay varios ejemplos de proteínas NOGD que son multifuncionales. Se analizó la relación filogenética entre las proteínas multifuncionales y NOGD que realizan la misma función y se ha visto que las dos proteínas NOGD tienen líneas evolutivas diferentes y que una de ellas es o se relaciona con una proteína multifuncional. Este hecho sugiere que el NOGD puede ser un mecanismo por el cual una proteína podría adquirir una nueva función para sustituir al componente faltante de la vía y se convertiría, por tanto, en una proteína multifuncional si conserva su función original. Moonlighting proteins performs more than one function using the same polypeptide chain. Identifying these proteins is necessary for their functional annotation and for their relevance in multiple biochemical and pathophysiological aspects, being implicated in the virulence of microorganisms and human diseases. It is a highly complex phenomenon, since understanding how different functions arise and coexist without interfering with each other is a challenge. Many questions remain to be resolved, such as their evolutionary origin or the mechanisms by which these additional functions are acquired. In this work, the relationship between multifunctional proteins and Non Orthologous Gene Displacement (NOGD) has been studied. NOGD is a phenomenon in which an expected component of an enzyme pathway is absent and its function is performed by another protein. We have found 124 moonlighting proteins with functions of NOGD proteins and even some examples in which NOGD proteins are multifunctional itselves. We have analyzed the phylogenetic relationship between multifunctional proteins and NOGD performing the same function. We obtained that NOGD proteins have different evolutionary lines and almost one of them is a multifunctional protein or is related to that. This fact suggests that NOGD may be a mechanism by which a protein could acquire a new function to replace the missing component of the pathway and would therefore become a multifunctional protein if it retains its original function. Les proteïnes moonlighting són aquelles que realitzen més d'una funció utilitzant la mateixa cadena polipeptídica. Identificar aquestes proteïnes és necessari tant per a la seva anotació funcional com per la seva rellevància en múltiples aspectes bioquímics i fisiopatològics, estant implicades en virulència de microorganismes i patologies humanes. És un fenomen de gran complexitat, ja que comprendre com sorgeixen i coexisteixen diferents funcions sense interferir entre elles suposa un repte. Encara queden moltes qüestions per resoldre com quin és el seu origen evolutiu o quins són els mecanismes pels quals s'adquireixen aquestes funcions addicionals. En aquest treball s'ha estudiat la relació entre les proteïnes multifuncionals i el Non Orthologous Gene Displacement (NOGD), un fenomen pel qual un component esperat d'una via enzimàtica està absent i la seva funció és realitzada per una altra proteïna. S'han trobat 124 proteïnes moonlighting que realitzen funcions de proteïnes NOGD i fins i tot hi ha diversos exemples de proteïnes NOGD que són multifuncionals. Es va analitzar la relació filogenètica entre les proteïnes multifuncionals i NOGD que realitzen la mateixa funció i s'ha vist que les dues proteïnes NOGD tenen línies evolutives diferents i que una d'elles és o es relaciona amb una proteïna multifuncional. Aquest fet suggereix que el NOGD pot ser un mecanisme pel qual una proteïna podria adquirir una nova funció per substituir el component que falta de la via i es convertiria, per tant, en una proteïna multifuncional si conserva la seva funció original.

Pathogenic bacteria and metastasic tumor cells try to spread to new parts of the human body and survive to the immune response. Since both need to overcome the same obstacles, the aim of the study is to find common characteristics between both processes, starting from the so called moonlighting proteins. Moonlighting proteins are multifunctional proteins and in bacteria are usually virulence factors. Therefore, functions associated with them are related to the infection and survival of the pathogen. It is wanted to identify the functions that are mostly shared between virulence moonlighting proteins and metastasic proteins. In order to perform this comparison, metastasic proteins were divided according to the processes in which they were involved: glucose metabolism, remodeling/regeneration of tissues, interaction with plasminogen and interaction with extracellular matrix. Subsequently, their GO terms were compared to those of the moonlighting proteins, differentiating which of them were virulence factors. The results obtained showed that GO terms related to the binding to extracelullar matrix, such as collagen, fibronectin or laminin, are widely represented among virulence moonlighting proteins and metastasic proteins. Further investigation is needed to prove if the binding sites used by both types of proteins are the same, if they share binding sequences or to discover new blocker drugs specific of these binding sites. Tanto las bacterias patógenas como las células tumorales metastásicas luchan por diseminarse por el cuerpo humano y sobrevivir a la respuesta del Sistema Inmunitario. Al tener que superar los mismos obstáculos, el objetivo del estudio es encontrar los puntos comunes entre ambos procesos, partiendo de las proteínas moonlighting. Las proteínas moonlighting son proteínas multifuncionales que en bacterias suelen actuar como factores de virulencia, por lo que las funciones asociadas a ellas están relacionadas con la infección y la supervivencia del patógeno. Se quiere comprobar qué funciones están compartidas en mayor medida entre proteínas moonlighting de virulencia y proteínas relacionadas con la metástasis. Para realizar la comparación, las proteínas metastásicas se dividieron por procesos en las que estaban involucradas: metabolismo de glucosa, remodelación/regeneración de tejidos, interacción con plasminógeno e interacción con la matriz extracelular. Posteriormente, se compararon sus códigos GO con los de las proteínas moonlighting, diferenciando las que son factores de virulencia de las que no. Los resultados obtenidos indican que los códigos GO relacionados con la unión a proteínas de la matriz extracelular, como el colágeno, fibronectina o laminina, están ampliamente representados entre las proteínas moonlighting de virulencia y proteínas metastásicas. Son necesarios más estudios para comprobar si los sitios de unión que utilizan ambos tipos de proteínas son los mismos, si comparten secuencias de unión, descubrir nuevos fármacos bloqueadores de sitios de unión etc. Tant els bacteris patògens com les cèl·lules tumorals metastàsiques lluiten per disseminar-se pel cos humà i sobreviure a la resposta del Sistema Immunitari. En haver de superar els mateixos obstacles, l'objectiu de l'estudi és trobar els punts comuns entre tots dos processos, partint de les proteïnes moonlighting. Les proteïnes moonlighting són proteïnes multifuncionals que en bacteris solen actuar com a factors de virulència, per la qual cosa les funcions associades a elles estan relacionades amb la infecció i la supervivència del patogen. Es vol comprovar quines funcions estan compartides en major mesura entre proteïnes moonlighting de virulència i proteïnes relacionades amb la metàstasi. Per a realitzar la comparació, les proteïnes metastàsiques es van dividir per processos en les quals estaven involucrades: metabolisme de glucosa, remodelació/regeneració de teixits, interacció amb plasminogen i interacció amb la matriu extracelul·lar. Posteriorment, es van comparar els seus codis GO amb els de les proteïnes moonlighting, diferenciant les que són factors de virulència de les quals no. Els resultats obtinguts indiquen que els codis GO relacionats amb la unió a proteïnes de la matriu extracelul·lar, com el col·lagen, fibronectina o laminina, estan àmpliament representats entre les proteïnes moonlighting de virulència i proteïnes metastásicas. Són necessaris més estudis per a comprovar si els llocs d'unió que utilitzen tots dos tipus de proteïnes són els mateixos, si comparteixen seqüències d'unió, descobrir nous fàrmacs blocadors de llocs d'unió etc.

Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials&mdash, borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene&mdash, at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-&beta, (1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points&mdash, from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points.

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Plants as sessile organisms face daily environmental challenges and have developed highly nuanced signaling systems to enable suitable growth, development, defense, or stalling re-sponses. Moonlighting proteins have multiple tasks and contribute to cellular signaling cascades where they produce additional variables adding to the complexity or fuzziness of biological sys-tems. Here we examine roles of moonlighting kinases that also generate 3’,5’-cyclic guanosine monophosphate (cGMP) in plants. These proteins include receptor like kinases and lipid kinases. Their guanylate cyclase activity potentiates the development of localized cGMP-enriched nanodomains or niches surrounding the kinase and its interactome. These nanodomains contribute to allosteric regulation of kinase and other molecules in the immediate complex directly or indirectly modulating signal cascades. Effects include downregulation of kinase activity, modulation of other members of the protein complexes such as cyclic nucleotide gated channels and potential triggering of cGMP-dependent degradation cascades terminating signaling. The additional layers of information provided by the moonlighting kinases are discussed in terms of how they may be used to provide a layer of fuzziness to effectively modulate cellular signaling cascades.

Highlight This article comments on: Bross CD, Howes TR, Abolhassani Rad S, Kljakic O, Kohalmi SE. 2017. Subcellular localizations of Arabidopsis arogenate dehydratases suggest novel and non-enzymatic roles. Journal of Experimental Botany 68, 1425–1440.

Surface-associated proteins from Mycobacterium bovis BCG Moreau RDJ are important components of the live Brazilian vaccine against tuberculosis. They are important targets during initial BCG vaccine stimulation and modulation of the host's immune response, especially in the bacterial-host interaction. These proteins might also be involved in cellular communication, chemical response to the environment, pathogenesis processes through mobility, colonization, and adherence to the host cell, therefore performing multiple functions. In this study, the proteomic profile of the surface-associated proteins from M. bovis BCG Moreau was compared to the BCG Pasteur reference strain. The methodology used was 2DE gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF), leading to the identification of 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits. Moonlighting proteins are multifunctional proteins in which two or more biological functions are fulfilled by a single polypeptide chain. Therefore, the identification of such proteins with moonlighting predicted functions can contribute to a better understanding of the molecular mechanisms unleashed by live BCG Moreau RDJ vaccine components.

In cestodiasis, mechanical and molecular contact between the parasite and the host activates the immune response of the host and may result in inflammatory processes, leading to ulceration and intestinal dysfunctions. The aim of the present study was to identify antigenic proteins of the adult cestode Hymenolepis diminuta by subjecting the total protein extracts from adult tapeworms to 2DE immunoblotting (two-dimensional electrophoresis combined with immunoblotting) using sera collected from experimentally infected rats. A total of 36 protein spots cross-reacting with the rat sera were identified using LC-MS/MS. As a result, 68 proteins, including certain structural muscle proteins (actin, myosin, and paramyosin) and moonlighters (heat shock proteins, kinases, phosphatases, and glycolytic enzymes) were identified; most of these were predicted to possess binding and/or catalytic activity required in various metabolic and cellular processes, and reported here as potential antigens of the adult cestode for the first time. As several of these antigens can also be found at the cell surface, the surface-associated proteins were extracted and subjected to in-solution digestion for LC-MS/MS identification (surfaceomics). As a result, a total of 76 proteins were identified, from which 31 proteins, based on 2DE immunoblotting, were predicted to be immunogenic. These included structural proteins actin, myosin and tubulin as well as certain moonlighting proteins (heat-shock chaperones) while enzymes with diverse catalytic activities were found as the most dominating group of proteins. In conclusion, the present study shed new light into the complexity of the enteric cestodiasis by showing that the H. diminuta somatic proteins exposed to the host possess immunomodulatory functions, and that the immune response of the host could be stimulated by diverse mechanisms, involving also those triggering protein export via yet unknown pathways.

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein–protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin. Electronic supplementary material The online version of this article (10.1186/s13567-018-0610-2) contains supplementary material, which is available to authorized users.

It is indeed remarkable how many developments in biological sciences have their origin in earlier ideas which were nearly forgotten and were buried. This review illustrates this with a few examples. Free solution capillary electrophoresis is a refined version of moving boundary electrophoresis. Immobilised metal affinity chromatography rests on the early work on protein precipitation with metal ions. The concept of proteins as templates was rejected in early times but has resurfaced in the context of prions and intrinsically disordered protein regions. The approach of using enzymes in organic solvents is credited to papers published in late 1980s; the idea though is as old at least as the beginning of the 20th century.

Moonlighting (multitasking, multifunctional) es la capacidad de algunas proteínas de ejecutar dos o más funciones bioquímicas. Normalmente, las proteínas moonlighting son descubiertas por "serendipia". La multifuncionalidad de las proteínas adquiere una mayor importancia a partir de los recientes descubrimientos acerca del proteoma humano (y de animales modelo como el ratón). El mecanismo de “splicing” no da lugar a un gran número de proteínas como se pensaba hasta ahora, sinó que los genes humanos mayoritariamente expresan la denominada isoforma principal, a nivel de proteína. Por ello el moonlighting puede contribuir, o ser la clave, a la complejidad de la célula. Por esta razón, sería de utilidad que la bioinformática pudiera predecir la multifuncionalidad, especialmente debido a la gran cantidad de nuevas secuencias provenientes de los proyectos genómicos. Durante el presente trabajo se han analizado y descrito diferentes aproximaciones que utilizan secuencias, estructuras, interactómica, algoritmos bioinformáticos e información contenida en las bases de datos como UniProt, OMIM, etc, para tratar de contribuir a la identificación de las proteínas multifuncionales. Un primer objetivo de este trabajo ha sido la actualización de la base de datos de proteínas moonlighting, MultitaskProtDB-II (http://wallace.uab.es/multitaskII), que ha servido como banco de trabajo para todos los análisis realizados. Por ejemplo, hemos intentado mapar los dominios funcionales de cada función dentro de la estructura de algunas proteínas importantes de nuestra base de datos. Esto ha permitido, en un cierto número de casos, identificar los dominios relacionados con una o más enfermedades humanas basadas en la proteína multifuncional analizada. De MultitaskProtDB-II, se ha encontrado que un porcentaje significativo de proteínas moonlighting (78%) están relacionadas con enfermedades humanas y que un 48% de ellas son dianas para fármacos actuales. Además, un 25% de proteínas moonlighting de la base de datos actualizada están implicadas en la virulencia de microorganismos patógenos, y hemos propuesto una explicación basada en el hecho de que las proteínas moonlighting, al ser evolutivamente muy conservadas, el huésped evitaría desarrollar una respuesta inmune que podría desencadenar una enfermedad autoinmune. Esto tiene gran importancia en la selección de proteínas candidatas a ser inmunogénicas protectivas en estudios de vacunología reversa. Desde el punto de vista de la evolución de las proteínas moonlighting y a partir del análisis de identificadores GO y de la ortología de interactomas, se sugiere que la segunda función estaría conservada filogenéticamente y que habría muchas más proteínas multifuncionales de lo que se pensaba anteriormente. Finalmente se ha analizado la posible relación evolutiva entre la multifuncionalidad y el desplazamiento de gen no ortólogo. Moonlighting (multitasking, multifunctional) is the ability of some proteins to perform two or more biochemical functions. Normally, moonlighting proteins are discovered by "serendipity". The multifunctionality of proteins acquires greater importance from recent discoveries about the human proteome (and animal models such as mice). The splicing mechanism does not give rise to a large number of proteins as was thought until now, but human genes mainly express the so-called main isoform, at the protein level. Therefore, moonlighting can contribute, or be the key, to the complexity of the cell. For this reason, it would be useful to predict multifunctionality from a bioinformatics point of view, especially due to the large number of new sequences coming from the genomic projects. During this work we have analyzed and described different approaches that use sequences, structures, interactomics, bioinformatic algorithms and the information contained in databases such as UniProt, OMIM, etc., to try to contribute to the identification of multifunctional proteins. A first objective of this work was to update the moonlighting protein database, MultitaskProtDB-II (http://wallace.uab.es/multitaskII), which has been used as a work platform for all the analyses. For example, we mapped the functional domains of each function within the structure of the protein and this allowed, in a certain number of cases, the identification of the domains related to one or more human diseases caused by this multifunctional protein. From MultitaskProtDB-II, it has been found that a significant percentage of moonlighting proteins (78%) are related to human diseases and that 48% of them are targets for current drugs. In addition, 25% of moonlighting proteins from the updated database are involved in the virulence of pathogenic microorganisms, and we have proposed an explanation based on the fact that the moonlighting proteins, being evolutionarily very conserved, the host would avoid developing a response immune that could trigger an autoimmune disease. This is of great importance in the selection of candidate proteins in order to be immunogenic and protective in reverse vaccinology studies. From the point of view of the evolution of moonlighting proteins and from the analysis of GO identifiers and the orthology of interactomes, it has been suggested that the second function would be phylogenetically conserved and that there would be many more multifunctional proteins than previously thought. Finally, the possible evolutionary relationship between multifunctionality and non-orthologous gene displacement has been analysed.

Moonlighting or multitasking proteins refer to those proteins with two or more functions performed by a single polypeptide chain. Proteins that belong to key ancestral functions and metabolic pathways such as primary metabolism typically exhibit moonlighting phenomenon. We have collected 698 moonlighting proteins in MultitaskProtDB-II database. A survey shows that 25% of the proteins of the database correspond to moonlighting functions related to pathogens virulence activity. Why is the canonical function of these virulence proteins mainly from ancestral key biological functions (especially of primary metabolism)? Our hypothesis is that these proteins present a high conservation between the pathogen protein and the host counterparts. Therefore, the host immune system will not elicit protective antibodies against pathogen proteins. The fact of sharing epitopes with host proteins (known as epitope mimicry) might be the cause of autoimmune diseases. Although many pathogen proteins can be antigenic, only a few of them would elicit a protective immune response. This would also explain the lack of successful vaccines based in these conserved moonlighting proteins., This review looks at why so many pathogen virulence proteins are from the primary metabolism and are conserved between pathogen and host.

Arogenate dehydratases (ADTs) have been identified to catalyze the last step of phenylalanine (Phe) biosynthesis in plants. All ADTs have a transit peptide sequence that targets them into the chloroplasts where the biosynthesis of Phe happens. Subcellular localization studies using fluorescently tagged Arabidopsis thaliana ADTs demonstrated that all six ADTs localize to chloroplast stromules (stroma filled tubules). However, one member of this family, ADT5, was also detected in the nucleus. As dual targeting of proteins to different cell compartments is an indication of multifunctionality, ADT5 nuclear localization suggests that this member of the ADT protein family is a moonlighting protein with a non-enzymatic role in the nucleus. In this study, first the nuclear localization of the ADT5 was confirmed by expression of ADT5-CFP under the regulation of ADT5 native promoter. Using confocal microscopy and Western blot analysis it was shown that ADT5 localizes into the nucleus. Next, different possible mechanisms that could result to the nuclear localization of ADT5 were studied. It was tested if ADT5 can move directly from chloroplast stroma to the nucleus through stromules or if ADT5 enter the nucleus from cytoplasm using the nuclear import system. Data presented are consistent with a translocation from cytoplasm. A combination of an AQEH motif and a single amino acid Asn28 present in the N-terminus of ADT5 ACT domain were identified as potential protein interaction sites required for nuclear targeting of ADT5. Y2H screenings and protein-protein interaction analyses suggest that nuclear localization of ADT5 occurs through the interaction of the cytosolic portion of an ER membrane bound protein, PHOSPHOLIPID DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1). A nuclear targeting sequence was identified in the N-terminal cytosolic portion of PDAT1. Hence, it is possible that PDAT1 piggybacks ADT5 into the nucleus through the nuclear import system. This study introduces ADT5 as a moonlighting protein and identifies a possible mechanism for ADT5 nuclear translocation.

Moonlighting proteini predstavljaju zanimljivu skupinu proteina koji zaobilaze klasičnu formulaciju „jedan gen – jedan protein – jedna funkcija“ izvodeći dvije ili više biokemijski nepovezanih zadaća. Ne uključuju proteine nastale genskim fuzijama, alternativnim izrezivanjem, proteolizom ili onima koji posjeduju višestruke funkcije unutar različitih domena ili pak uzrokuju pleiotropni učinak na fenotip. Često je riječ o proteinima koji su uključeni u esencijalne metaboličke procese poput glikolize, Krebsova ciklusa, smatanja i stabilnosti dugih proteina, transkripciju i translaciju, itd. Postoje različiti mehanizmi na koje može doći do ispoljavanja i promjene između kanonske i moonlighting funkcije, od kojih je u ovom seminaru najznačajniji onaj koji uključuje unutarstanične proteine s alternativnom funkcijom na staničnoj površini. Biofizička osnova proteinske multifunkcionalnosti leži u posttranslacijskim modifikacijama, točkastim mutacijama ili duplikaciji gena koji prolazi niz mutacija i poprima drugu funkciju. Bakterije koriste površinski asocirane moonlighting proteine kako bi pospješili svoju adhezivnost i kolonizaciju tkiva, bilo vezanjem za komponente izvanstaničnog matriksa, vezujući plazminogen ili pak koju od komponenata imunosnog sustava, čime ga mogu i utišati. U bakterija je do dan danas ostalo otvoreno pitanje njihova transporta budući da ne posjeduju karakteristične signalne sekvence za translokaciju. Važnost istraživanja dotičnih proteina je višestruka – za mnoge se pokazalo kako mogu biti glavni uzročnici patogenosti i katkad poticati razvoj tumora, dok s druge strane, mnogi umrežuju metaboličke puteve na nepredvidive načine koji su od velikog značaja u bilo kakvom istraživanju sistematske biologije na razini proteomike i metabolomike. Moonlighting proteins represent an interesting group of proteins which do not follow the classical formulation „one gene – one protein – one function“ by simultaneously performing two or more biochemically unrelated tasks. They do not include those proteins that have been genetically fused, alternatively spliced or proteolytically cut, or those that contain multiple functions within different domains or exert pleiotropic effect on phenotype. It's common for moonlighting proteins to be part of an essential metabolic pathway such as glycolysis, Krebs cycle, folding and stabilising of other proteins, transcription and translation, etc. There are plenty of mechanisms which moonlighting proteins use to switch between their canonical and alternative functions, of which the most important for this seminary is the execution of alternative function when outside of the cell, commonly attached to the plasma membrane. Biophysical fundaments on which moonlighting effect occurs include post-translational modifications, point mutations or gene duplications which make an alternate version of an original protein with an added moonlighting function. Bacteria use surface-associated moonlighting proteins to adhere to extracellular matrix of host epithelial cell, to activate plasminogen or even to evade host immune response by binding certain proteins of the immune system. Even to this day there are still no clues to how bacteria translocate their intracellular proteins outside of the cell without any signal sequence. The importance of research in the field of moonlighting is multifaceted – some have been observed to cause disease and cancer, others on the contrary make the web of metabolism more complex and intertwined by connecting distant pathways. The latter is very important to have in mind before attempting any systems biology approach.

Las proteínas moonlighting son proteínas con funciones múltiples totalmente independientes y ocultas, es decir, se desconocen muchas de las funciones que tienen las proteínas moonlighting. Además, las proteínas moonlitghting están estrechamente ligadas a enfermedades genéticas. En este articulo se han descubierto nuevas funciones desconocidas en las proteínas de una forma inédita, ya que, hasta el momento, las que se han descubierto han sido, principalmente, en laboratorio. Y, en este trabajo, se ha realizado el proceso de detección de nuevas funciones desconocidas desde la bioinformática. Para ello, se recoge información en lenguaje natural de las bases de datos Gene Ontology[1], funciones asociadas a los genes, y UniProt[2], funciones asociadas a las proteínas. La base de datos de Gene Ontology fue creada para tener toda la información de todos los genes y la base de datos de Uniprot para tener toda la información sobre las proteínas. En este trabajo utilizamos los datos que están relacionados con el Homo sapiens y usamos las bases de datos para descubrir funciones desconocidas en las proteínas mediante un algoritmo de árbol de decisiones basado en reglas biológicas. El descubrimiento de las nuevas funciones desconocidas puede permitir saber las aflicciones que sufren las proteínas, conocer reacciones desconocidas al entrar en contacto con algún medicamento, o la relación con algunas patologías. Moonlighting proteins are proteins with multiple totally independent and hidden function sand, that is to say, that many of the functions that have the moonlighting proteins are unknown. They are also closely linked to genetic diseases. This Article pretends to discover new hidden functions in proteins in an unpublished way, since those that have been discovered have been by laboratory and now, the process of detecting new unknown functions has been performed from bioinformatics. For it collects information in natural language from database, Gene Ontology[1], functions associated to genes, and the database Uni-prot[2], functions associated to protein. The Gene Ontology database was created to have all the information of all the genes and the Uniprot database to have all the information about the proteins. In this article, we use data that are related to Homo sapiens and we use databases to discover unknown functions in proteins using a decision tree algorithm based on biological rules. The discovery of the new unknown functions can allow to know the afflictions that suffer the proteins when coming in contact with some medicine or the relation with some pathologies. Les proteïnes moonlighting són proteïnes amb funcions múltiples totalment independents i ocultes, és a dir, es desconeixen moltes de les funcions que tenen les proteïnes moonlighting. A més, les proteïnes moonlitghting estan estretament lligades a malalties genètiques. En aquest article s'han descobert noves funcions desconegudes en les proteïnes d'una forma inèdita, ja que, fins al moment, les que s'han descobert han estat, principalment, en laboratori. I, en aquest treball, s'ha realitzat el procés de detecció de noves funcions desconegudes des de la bioinformàtica. Per a això, es recull informació en llenguatge natural de les bases de dades Gene Ontology [1], funcions associades als gens, i UniProt [2], funcions associades a les proteïnes. La base de dades de Gene Ontology va ser creada per tenir tota la informació de tots els gens i la base de dades de UniProt per tenir tota la informació sobre les proteïnes. En aquest treball utilitzem les dades que estan relacionats amb l'Homo sapiens i fem servir les bases de dades per descobrir funcions desconegudes en les proteïnes mitjançant un algoritme d'arbre de decisions basat en regles biològiques. El descobriment de les noves funcions desconegudes pot permetre saber les afliccions que pateixen les proteïnes, conèixer reaccions desconegudes en entrar en contacte amb algun medicament, o la relació amb algunes patologies.

International audience; Neurohormones diffuse in sweat and epidermis leading skin bacterial microflora to be largely exposed to these host factors. Bacteria can sense a multitude of neurohormones, but their role in skin homeostasis was only investigated recently. The first study focused on substance P (SP), a neuropeptide produced in abundance by skin nerve terminals. SP is without effect on the growth of Gram-positive (Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermidis) and Gram-negative (Pseudomonas fluorescens) bacteria. However, SP is stimulating the virulence of Bacillus and Staphylococci. The action of SP is highly specific with a threshold below the nanomolar level. Mechanisms involved in the response to SP are different between bacteria although they are all leading to increased adhesion and/or virulence. The moonlighting protein EfTu was identified as the SP-binding site in B. cereus and Staphylococci. In skin nerve terminals, SP is co-secreted with the calcitonin gene-related peptide (CGRP), which was shown to modulate the virulence of S. epidermidis. This effect is antagonized by SP. Identification of the CGRP sensor, DnaK, allowed understanding this phenomenon as EfTu and DnaK are apparently exported from the bacterium through a common system before acting as SP and CGRP sensors. Many other neuropeptides are expressed in skin, and their potential effects on skin bacteria remain to be investigated. Integration of these host signals by the cutaneous microbiota now appears as a key parameter in skin homeostasis.

In a recent work we have identified, from a bigger gene regulatory network, a seven-node module involved in the control of the life cycle of Trypanosoma cruzi (T. cruzi) (Carrea and Diambra, 2016). To that end, we have analyzed microarray gene-expression data of the four different T. cruzi’s life cycle stages, by means of a systems biology approach., Artículo original disponible en http://sedici.unlp.edu.ar/handle/10915/86040, Facultad de Ciencias Exactas

Lactobacillus acidophilus NCFM is a well-known probiotic bacterium extensively studied for its beneficial health effects. Exoproteome (proteins exported into culture medium) and surface proteome (proteins attached to S-layer) of this probiotic were identified by using 2DE followed by MALDI TOF MS to find proteins potentially involved in bacteria-host interactions. The exo- and surface proteomes included 43 and 39 different proteins from 72 and 49 successfully identified spots, respectively. Twenty-two proteins were shared between the two proteomes; both contained the major surface layer protein that participates in host interaction as well as several well-known and putative moonlighting proteins. The exoproteome contained nine classically-secreted (containing a signal sequence) and ten nonclassically-secreted proteins, while the surface proteome contained four classically-secreted and eight nonclassically secreted proteins. Identification of exo- and surface proteomes contributes describing potential protein-mediated probiotic-host interactions.