Search

A major feature of eukaryotic cells is the separation between nucleus and cytoplasm. Although physically separated, these two compartments are in permanent communication through a transport system that must be precisely regulated to maintain cell homeostasis and avoid serious diseases, such as cancer. A crucial element in this transport system is the exportin XPO1, which exports many proteins (so-called cargoes) from the nucleus to cytoplasm. XPO1 alteration has been frequently associated with cancer and XPO1 is an important therapeutic target. The cellular effect of XPO1 inhibition is expected to be mediated by changes in the subcellular distribution of its cargoes. However, while many of XPO1 cargoes have been already identified, the complete set remains uncharacterized, making XPO1 a very interesting candidate for proteomic studies. Thus, we have designed a novel proteomics strategy, based on compartment-specific proximity biotinylation using the APEX2 peroxidase, to search for XPO1 cargos. To this end, we have targeted APEX2 to cytoplasm and nucleus, isolated the biotinylated proteins by affinity purification, and identified them by mass spectrometry. The results of a proof-of concept experiment reported here show that this strategy, combined with specific XPO1 inhibition, can lead to the identification of XPO1 cargoes. This novel approach, therefore, may advance our understanding of XPO1-dependent nuclear export by facilitating the identification of novel cargoes, and may also contribute to better characterize the cellular effect of therapeutically used XPO1 inhibitors.; Nukleo eta zitoplasmaren arteko banaketa da zelula eukariotoen ezaugarririk bereizgarriena. Konpartimentuok fisikoki banaturik egon arren, elkarren arteko komunikazioa etengabea da, eta horretarako estuki erregulaturiko garraio-sistema dago zeina zelularen homeostasia mantendu eta minbizia bezalako gaixotasunak ekiditeko ezinbestekoa den. Garraio-sistema honetako pieza gakoa XPO1 esportina da zeinak kargo deritzen proteina askoren nukleotik zitoplasmaranzko esportazioa egikaritzen duen. Esportina honen eta minbiziaren arteko lotura maiz aipatu izan da, eta badira XPO1 inhibitzen duten agente terapeutikoak. XPO1en inhibizioak bere kargoen banaketa azpizelularrean aldaketak eragingo dituela espero daiteke. Alabaina, XPO1en kargo asko ezagunak badira ere, bere kargo bilduma osoa ez da zehaztu oraindik, eta honek XPO1 ikerketa proteomikoetarako kandidatu oso interesgarri bilakatzen du. Hau honela, estrategia proteomiko berri bat diseinatu dugu, zeinetan APEX2 peroxidasa erabiliz konpartimentu-espezifikoko gertuko biotinilazioa burutu dugun XPO1en kargo berriak bilatzeko. Horretarako, APEX2 zitoplasma eta nukleora ituratu dugu eta, biotinilatutako proteinak afinitate-purifikazioz arrantzatu ostean, masa-espektrometriaz identifikatu ditugu. Lan honetan azaldutako kontzeptu-froga esperimentuaren emaitzek erakusten dute estrategia hau, XPO1en inhibizio espezifikoarekin konbinatuz, kargo berriak identifikatzeko baliagarria izan daitekeela. Hurbilketa berri honek beraz, kargo gehiagoren identifikazioa erraztearekin batera, XPO1en mendeko garraioan sakondu eta terapeutikoki erabiltzen diren XPO1en inhibitzaileek zelula mailan duten eragina argitzeko balio dezake ere.

Seven members of the Coronaviridae family infect humans, but only three (SARS-CoV, SARS-CoV-2 and MERS-CoV) cause severe disease with a high case fatality rate. Using in silico analyses (machine learning techniques and comparative genomics), several features associated to coronavirus pathogenicity have been recently proposed, including a potential increase in the strength of a predicted novel nuclear export signal (NES) motif in the nucleocapsid protein.Here, we have used a well-established nuclear export assay to experimentally establish whether the recently proposed nucleocapsid NESs are capable of mediating nuclear export, and to evaluate if their activity correlates with coronavirus pathogenicity.The six NES motifs tested were functional in our assay, but displayed wide differences in export activity. Importantly, these differences in NES strength were not related to strain pathogenicity. Rather, we found that the NESs of the strains belonging to the genus Alphacoronavirus were markedly stronger than the NESs of the strains belonging to the genus Betacoronavirus.We conclude that, while some of the genomic features recently identified in silico could be crucial contributors to coronavirus pathogenicity, this does not appear to be the case of nucleocapsid NES activity, as it is more closely related to coronavirus genus than to pathogenic capacity.

The nuclear export receptor CRM1 (XPO1) recognizes and binds specific sequence motifs termed nuclear export signals (NESs) in cargo proteins. About 200 NES motifs have been identified, but over a thousand human proteins are potential CRM1 cargos, and most of their NESs remain to be identified. On the other hand, the interaction of NES peptides with the &ldquo, NES-binding groove&rdquo, of CRM1 was studied in detail using structural and biochemical analyses, but a better understanding of CRM1 function requires further investigation of how the results from these in vitro studies translate into actual NES export in a cellular context. Here we show that a simple cellular assay, based on a recently described reporter (SRVB/A), can be applied to identify novel potential NESs motifs, and to obtain relevant information on different aspects of CRM1-mediated NES export. Using cellular assays, we first map 19 new sequence motifs with nuclear export activity in 14 cancer-related proteins that are potential CRM1 cargos. Next, we investigate the effect of mutations in individual NES-binding groove residues, providing further insight into CRM1-mediated NES export. Finally, we extend the search for CRM1-dependent NESs to a recently uncovered, but potentially vast, set of small proteins called micropeptides. By doing so, we report the first NES-harboring human micropeptides.

The nuclear envelope, a double membrane that encloses the nucleus of eukaryotic cells, establishes a physical separation between the two main cellular compartments: the nucleus and the cytoplasm. Continuous communication between these compartments is crucial for the maintenance of cellular homeostasis. This communication relies on the bidirectional transport of macromolecules across the nuclear envelope. We focus here on a protein, called XPO1, which plays a key role in nucleocytoplasmic transport. XPO1 is the main receptor that mediates the export of hundreds of proteins and several RNA molecules from the nucleus to the cytoplasm. In this article we first review the molecular mechanisms that underlie nucleocytoplasmic transport. Next, we focus on XPO1 to describe some of the experimental approaches that are frequently applied to investigate its function. Finally, we illustrate the use of these approaches using the recently described case of the USP12/WDR20 complex [1] as an example.; Zelula eukariotoen nukleoa inguratzen duen mintzak zelularen bi konpartimentu nagusien, nukleoa eta zitoplasmaren, arteko banaketa fisikoa ezartzen du. Bi konpartimentu hauen arteko komunikazioa etengabea izateaz gain, ezinbestekoa ere bada zelularen homeostasia mantentzeko. Komunikazio hori, nukleoaren mintzean zehar noranzko bietan ematen den molekulen mugikortasunak egikaritzen du. Komunikazio horretan funtsezkoa den proteina bat dugu mintzagai honakoan, XPO1 izeneko proteina alegia. XPO1 nukleotik zitoplasmarako garraioan jarduten duen esportazio-hartzaile nagusia da, ehundaka proteina eta baita zenbait RNA molekula esportatzen dituelarik. Artikulu honetan garraio nukleozitoplasmikoaren mekanismo molekularrak deskribatzeaz gain, XPO1 esportazio-hartzailean zentratu gara eta bere funtzioa aztertzeko erabili ohi diren hurbilketa esperimentalak jaso ditugu. Azken horien adibidetzat berriki deskribatutako USP12/WDR20 konplexuaren kasua [1] aztertu dugu.

Following the rapid expansion of the proteomics field, the investigation of post translational modifications (PTM) has become extremely popular changing our perspective of how proteins constantly fine tune cellular functions. Reversible protein phosphorylation plays a pivotal role in virtually all biological processes in the cell and it is one the most characterized PTM up to date. During the last decade, the development of phosphoprotein/phosphopeptide enrichment strategies and mass spectrometry (MS) technology has revolutionized the field of phosphoproteomics discovering thousands of new site-specific phosphorylations and unveiling unprecedented evidence about their modulation under distinct cellular conditions. The field has expanded so rapidly that the use of traditional methods to validate and characterize the biological role of the phosphosites is not feasible any longer. Targeted MS holds great promise for becoming the method of choice to study with high precision and sensitivity already known site-specific phosphorylation events. This review summarizes the contribution of large-scale unbiased MS analyses and highlights the need of targeted MS-based approaches for follow-up investigation. Additionally, the article illustrates the biological relevance of protein phosphorylation by providing examples of disease-related phosphorylation events and emphasizes the benefits of applying targeted MS in clinics for disease diagnosis, prognosis and drug-response evaluation.

2-interleukina (IL-2) eta IL-15 zitokinek berebiziko garrantzia daukate sistema immunologikoaren erregulazioan, besteak beste T linfozitoen proliferazioa sustatzen dutelako. Zitokina biek bi hartzaile-azpiunitate partekatzen dituztenez (IL-2Rβ eta IL-2Rγ) ez da harritzekoa teilakatutako hainbat funtzio izatea, baina bai da harrigarria, ordea, bakoitzak erantzun immunologiko espezikoak eta maiz kontrajarriak eragitea. Nahiz eta mekanismo asko proposatu diren IL-2 eta IL-15 arteko desberdintasunak azaltzeko, ez dago argi nola den posible biek, hartzaile berdinak erabiltzen dituztelarik, zeluletan erantzun desberdinak sustatzea. Horren gakoa aurkitzeko asmoz, IL-2 eta IL-15ez kitzikatutako T-zeluletan piztutako seinalizazio-bidezidorrak aztertu eta elkarren artean konparatu ditugu, masa-espektrometria erabilita. Gure lanak erakutsi du bi zitokinek kitzikatutako T linfozitoen seinalizazio-sareak oso parekoak izan arren, badirela ñabardura batzuk bi zitokinen arteko alde funtzionala azaltzen lagun dezaketenak.; L-2 and IL-15 are key cytokines regulating the immune system i n part by promoting T-cell proliferation. Both cytokines signal through the same receptor subunits (IL-2Rβ and IL-2Rγ) and not surprisingly they exert some overlapping functions. However they also have specific and even sometimes opposing roles raising the paradigm of how they mediate distinct immune reponses by using the same receptors. Although several options have been suggested, it remains controversial the molecular mechanism underlying the functional dichotomy of IL-2 and IL-15. Aiming to find the key to answering such enigma, we assessed and compared by mass spectrometry the signaling networks activated by both cytokines. Our study revealed that the transduction pathways initiated by IL-2 and IL-15 are highly similar but not identical since we detected faint differences that may account for the functional discrepancy observed between both cytokines.

Interleukin-2 (IL-2) is major cytokine involved in T cell proliferation, differentiation and apoptosis. Association between IL-2 and its receptor (IL-2R), triggers activation of complex signaling cascade governed by tyrosine phosphorylation that culminates in transcription of genes involved in modulation of the immune response. The complete characterization of the IL-2 pathway is essential to understand how aberrant IL-2 signaling results in several diseases such as cancer or autoimmunity and also how IL-2 treatments affect cancer patients. To gain insights into the downstream machinery activated by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5min of IL-2 stimulation resulted in identification of 172 proteins among which 79 were found with increased abundance in the tyrosine-phosphorylated complexes, including several previously not reported IL-2 downstream effectors. Combinatorial site-specific phosphoproteomic analysis resulted in identification of 99 phosphorylated sites mapping to the identified proteins with increased abundance in the tyrosine-phosphorylated complexes, of which 34 were not previously described. In addition, chemical inhibition of the identified IL-2-mediated JAK, PI3K and MAPK signaling pathways, resulted in distinct alteration on the IL-2 dependent proliferation.

Udgivelsesdato: 2007-Jul-20 Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified a number of phosphorylated residues by mass spectrometry and generated mutants in which these amino acids are replaced by Asp to mimic the effect of phosphorylation. Our results show that, among the eight phosphoryl groups experimentally detected, four are located at the flexible N terminus, and the rest are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin.

Genes that encode glycosylphosphatidylinositol anchored proteins (GPI-APs) constitute an estimated 1-2% of eukaryote genomes. Current computational methods for the prediction of GPI-APs are sensitive and specific; however, the analysis of the processing site (omega- or omega-site) of GPI-APs is still challenging. Only 10% of the proteins that are annotated as GPI-APs have the omega-site experimentally verified. We describe an integrated computational and experimental proteomics approach for the identification and characterization of GPI-APs that provides the means to identify GPI-APs and the derived GPI-anchored peptides in LC-MS/MS data sets. The method takes advantage of sequence features of GPI-APs and the known core structure of the GPI-anchor. The first stage of the analysis encompasses LC-MS/MS based protein identification. The second stage involves prediction of the processing sites of the identified GPI-APs and prediction of the corresponding terminal tryptic peptides. The third stage calculates possible GPI structures on the peptides from stage two. The fourth stage calculates the scores by comparing the theoretical spectra of the predicted GPI-peptides against the observed MS/MS spectra. Automated identification of C-terminal GPI-peptides from porcine membrane dipeptidase, folate receptor and CD59 in complex LC-MS/MS data sets demonstrates the sensitivity and specificity of this integrated computational and experimental approach.

Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti- C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans , which exerts three anti- C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae , Cryptococcus neoformans , Aspergillus fumigatus , and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans , and exhibited a potent fungicidal effect against C. albicans , C. lusitaniae , Cryptococcus neoformans , A. fumigatus , and S. prolificans , showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala , since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used alone or in combination with current antimicrobial agents.

The cell wall is the major fungal structure involved in the interaction with the host and most of the immunological effects observed with intact fungal cells have been reproduced with cell-wall components. As a result of the exposure to fungal antigens, most individuals develop both cellular and antibody responses intended to limit the invasiveness or to eradicate the fungus from the infected tissues. However, a number of fungi including Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Trichophyton spp. and Histoplasma capsulatum can also induce T- and B-suppressive activities. A wide diversity of immunodominant cell-wall antigens for both cell-mediated and humoral responses have been identified in the most important fungal pathogens, although considerable differences exist in the information available at the molecular level among the different mycoses. Cellular responses require macrophage and Th1 activation, whereas humoral responses comprise the activation of the complement system and the induction of antibodies. The ability of fungal cell-wall components to elicit cellular or humoral immune responses has been traditionally used in the serodiagnosis of mycoses, the identification of fungal organisms and the development of vaccines for the prevention of mycoses. In the future, the analysis of such molecules will provide critical information in understanding the nature of host-fungus interactions.

Monoclonal antibody (MAb) C7 reacted with a >200-kDa component from the Candida albicans cell wall identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry as Als3. It also bound the recombinant N terminus of Als3. Binding of MAb C7 to Als3 may explain the biological activities exerted by the MAb on C. albicans .

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are posttranslationally processed proteins that become tethered to the extracellular leaflet of the plasma membrane via a C-terminal glycan-like moiety. Since the first GPI-AP was described in the 1970s, more than 500 GPI-APs have been reported in a range of species, including plants, microbes, and mammals. GPI-APs are probably involved in cell signaling, cell recognition, and cell remodeling processes, and they may potentially serve as cell surface antigens or vaccine targets in pathogenic microorganisms or transformed mammalian cells. Due to the structural complexity and physicochemical properties of GPI-APs, their identification and structural characterization is a demanding analytical task. Here, we report a simple, fast and sensitive method for isolation and structural analysis of GPI-anchors using a combination of hydrophilic interaction liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry. This method allowed analysis of GPI peptides derived from low picomole levels of the porcine kidney membrane dipeptidase. Furthermore, it allowed unambiguous assignment of the omega site via amino acid sequencing of the modified peptides. GPI-anchor-specific diagnostic ions were observed by MALDI-MS/MS at m/z 162, 286, 422, and 447, corresponding to glucosamine, mannose ethanolamine phosphate, glucosamine inositol phosphate, and mannose ethanolamine phosphate glucosamine, respectively. Thus, the methodology described herein may enable sensitive and specific detection of GPI-anchored peptides in large-scale proteomic studies of plasma membrane proteins.

Immunization of mice with a stress mannoprotein of > 200 kDa from the cell wall of Candida albicans led to the production of monoclonal antibody (Mab) C7. The immunogen is a major target of secretory IgA and its expression is regulated by different environmental conditions including temperature, pH, glucose concentration and ammonium sulphate in the culture medium. Mab C7 reacted with a peptide epitope present in the > 200 kDa antigen as well as in a number of antigens from the blastoconidium and germ tube cell wall, including enolase. In addition to its reactivity with C. albicans, Mab C7 also reacted with antigens present in C. krusei, C, tropicalis, C. glabrata, C. dubliniensis and C. lusitaniae, as well as in Cryptococcus neoformans, Scedosporium prolificans and Aspergillus fumigatus. Mab C7 exhibited four important biological activities, namely inhibition of adhesion of C. albicans to a variety of surfaces, inhibition of germination of C. albicans, direct candidacidal activity and direct tumoricidal activity. In tumor cells, Mab C7 reacted with nucleoporin Nup88, a reactivity that can be utilized for diagnostic and prognostic purposes.

Using a rabbit model of systemic infection, we show that it is possible to differentiate infections caused by Candida dubliniensis and other Candida species by detecting the antibody response mounted by the infected animals. These results confirm our previous observation in a patient with C. dubliniensis candidemia and suggest that detection of C. dubliniensis -specific antibodies is useful in the diagnosis of invasive candidiasis caused by this yeast.