BACKGROUND: The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. RESULTS: We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. CONCLUSIONS: The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

• Klíčová slova
• Comparative genomics, Diplonemea, Euglenida, Evolution, Kinetochores, Kinetoplastea, Metabolism, Trypanothione,
• MeSH
• biologická evoluce * MeSH
• Euglenida genetika   metabolismus   MeSH
• Euglenozoa genetika   metabolismus   MeSH
• genom protozoální * MeSH
• Kinetoplastida genetika   metabolismus   MeSH
• molekulární evoluce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Soybean (Glycine max (L.) Merr.) plants were exposed to various Cd concentrations from background and low non-toxic (0.5-50 nM) via sublethally toxic (< 550 nM) to highly, ultimately lethally toxic (3 µM) concentrations. Plants were cultivated hydroponically for 10 weeks until pod development stage of the control plants. The threshold and mechanism of sublethal Cd toxicity was investigated by metabolomics and metalloproteomics (HPLC-ICP-MS) measuring metal binding to proteins in the harvested roots. Spatial distribution of Cd was revealed by µXRF-CT. Specific binding of Cd to proteins already at 50 nM Cd revealed the likely high-affinity protein binding targets in roots, identified by protein purification from natural abundance. This revealed allantoinase, aquaporins, peroxidases and protein disulfide isomerase as the most likely high-affinity targets of Cd binding. Cd was deposited in cortex cell vacuoles at sublethal and bound to the cell walls of the outer cortex and the vascular bundle at lethal Cd. Cd binding to proteins likely inhibits them, and possibly induces detoxification mechanisms, as verified by metabolomics: allantoic acid and allantoate increased due to sublethal Cd toxicity. Changes of the Cd binding pattern indicated a detoxification strategy at lower Cd, but saturated binding sites at higher Cd concentrations.

• Klíčová slova
• Metabolites, Metal stress, Metalloproteomics, Sublethal toxicity, µXRF-CT,
• MeSH
• Glycine max * metabolismus   MeSH
• kadmium * toxicita   metabolismus   MeSH
• kořeny rostlin metabolismus   MeSH
• metabolom MeSH
• peroxidasy metabolismus   MeSH
• proteindisulfidisomerasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kadmium * MeSH
• peroxidasy MeSH
• proteindisulfidisomerasy MeSH

Most of the organs of the digestive tract comprise secretory epithelia that require specialized molecular machines to achieve their functions. As such anterior gradient (AGR) proteins, which comprise AGR1, AGR2, and AGR3, belong to the protein disulfide isomerase family, and are involved in secretory and transmembrane protein biogenesis in the endoplasmic reticulum. They are generally expressed in epithelial cells with high levels in most of the digestive tract epithelia. To date, the vast majority of the reports concern AGR2, which has been shown to exhibit various subcellular localizations and exert pro-oncogenic functions. AGR2 overexpression has recently been associated with a poor prognosis in digestive cancers. AGR2 is also involved in epithelial homeostasis. Its deletion in mice results in severe diffuse gut inflammation, whereas in inflammatory bowel diseases, the secretion of AGR2 in the extracellular milieu participates in the reshaping of the cellular microenvironment. AGR2 thus plays a key role in inflammation and oncogenesis and may represent a therapeutic target of interest. In this review, we summarize the already known roles and mechanisms of action of the AGR family proteins in digestive diseases, their expression in the healthy digestive tract, and in digestive oncology. At last, we discuss the potential diagnostic and therapeutic implications underlying the biology of AGR proteins.

• MeSH
• gastrointestinální nádory * genetika   MeSH
• karcinogeneze genetika   MeSH
• mukoproteiny genetika   MeSH
• myši MeSH
• nádorové mikroprostředí MeSH
• onkogenní proteiny * genetika   MeSH
• proteindisulfidisomerasy MeSH
• zánět genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• mukoproteiny MeSH
• onkogenní proteiny * MeSH
• proteindisulfidisomerasy MeSH

Francisella tularensis is a highly infectious facultative intracellular bacterium and aetiological agent of tularaemia. The conserved hypothetical lipoprotein with homology to thiol/disulphide oxidoreductase proteins (FtDsbA) is an essential virulence factor in F. tularensis. Its protein sequence has two different domains: the DsbA_Com1_like domain (DSBA), with the highly conserved catalytically active site CXXC and cis-proline residue; and the domain amino-terminal to FKBP-type peptidyl-prolyl isomerases (FKBP_N). To establish the role of both domains in tularaemia infection models, site-directed and deletion mutagenesis affecting the active site (AXXA), the cis-proline (P286T) and the FKBP_N domain (ΔFKBP_N) were performed. The generated mutations led to high attenuation with the ability to induce full or partial host protective immunity. Recombinant protein analysis revealed that the active site CXXC as well as the cis-proline residue and the FKBP_N domain are necessary for correct thiol/disulphide oxidoreductase activity. By contrast, only the DSBA domain (and not the FKBP_N domain) seems to be responsible for the in vitro chaperone activity of the FtDsbA protein.

• MeSH
• faktory virulence genetika   metabolismus   MeSH
• Francisella tularensis enzymologie   genetika   MeSH
• molekulární chaperony genetika   metabolismus   MeSH
• mutační analýza DNA MeSH
• mutageneze cílená MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• proteindisulfidisomerasy genetika   metabolismus   MeSH
• sekvenční delece MeSH
• thioredoxiny genetika   metabolismus   MeSH
• virulence MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• molekulární chaperony MeSH
• mutantní proteiny MeSH
• proteindisulfidisomerasy MeSH
• thioredoxiny MeSH

Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture-based LC-MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2-PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein-protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells.

• Klíčová slova
• anterior gradient protein 2, mass spectrometry, protein disulfide isomerase, protein–protein interactions, secretory pathway,
• MeSH
• chromatografie kapalinová MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• mukoproteiny * metabolismus   MeSH
• nádory * MeSH
• onkogenní proteiny * metabolismus   MeSH
• proteindisulfidisomerasy * MeSH
• simulace molekulového dockingu MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AGR2 protein, human MeSH Prohlížeč
• mukoproteiny * MeSH
• onkogenní proteiny * MeSH
• proteindisulfidisomerasy * MeSH

Francisella tularensis subspecies tularensis is a highly virulent intracellular bacterial pathogen, causing the disease tularemia. However, a safe and effective vaccine for routine application against F. tularensis has not yet been developed. We have recently constructed the deletion mutants for the DsbA homolog protein (ΔdsbA/FSC200) and a hypothetical protein IglH (ΔiglH/FSC200) in the type B F. tularensis subsp. holarctica FSC200 strain, which exerted different protection capacity against parental virulent strain. In this study, we further investigated the immunological correlates for these different levels of protection provided by ΔdsbA/FSC200 and ΔiglH/FSC200 mutants. Our results show that ΔdsbA/FSC200 mutant, but not ΔiglH/FSC200 mutant, induces an early innate inflammatory response leading to strong Th1-like antibody response. Furthermore, vaccination with ΔdsbA/FSC200 mutant, but not with ΔiglH/FSC200, elicited protection against the subsequent challenge with type A SCHU S4 strain in mice. An immunoproteomic approach was used to map a spectrum of antigens targeted by Th1-like specific antibodies, and more than 80 bacterial antigens, including novel ones, were identified. Comparison of tularemic antigens recognized by the ΔdsbA/FSC200 post-vaccination and the SCHU S4 post-challenge sera then revealed the existence of 22 novel SCHU S4 specific antibody clones.

• Klíčová slova
• antibody response, cytokines, immunoproteomics, protection, tularemia,
• MeSH
• atenuované vakcíny aplikace a dávkování   genetika   imunologie   MeSH
• bakteriální vakcíny aplikace a dávkování   genetika   imunologie   MeSH
• cytokiny metabolismus   MeSH
• faktory virulence nedostatek   MeSH
• Francisella tularensis klasifikace   enzymologie   imunologie   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• proteindisulfidisomerasy nedostatek   MeSH
• Th1 buňky imunologie   MeSH
• tularemie imunologie   prevence a kontrola   MeSH
• tvorba protilátek * MeSH
• zkřížená ochrana * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• atenuované vakcíny MeSH
• bakteriální vakcíny MeSH
• cytokiny MeSH
• faktory virulence MeSH
• proteindisulfidisomerasy MeSH

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

• Klíčová slova
• DsbA, Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, moonlighting,
• MeSH
• delece genu * MeSH
• faktory virulence analýza   MeSH
• Francisella tularensis enzymologie   imunologie   patogenita   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy nedostatek   metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• mikrobiální viabilita MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteindisulfidisomerasy nedostatek   MeSH
• proteom analýza   MeSH
• salmonelová infekce u zvířat mikrobiologie   patologie   MeSH
• vazba proteinů MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• krevní proteiny MeSH
• proteindisulfidisomerasy MeSH
• proteom MeSH

BACKGROUND: Green tea is a rich source of polyphenols, mainly catechins (flavanols), which significantly contribute to the beneficial health effects of green tea in the prevention and treatment of various diseases. In this study the effects of four green tea catechins on protein ERp57, also known as protein disulfide isomerase isoform A3 (PDIA3), have been investigated in an in vitro model. METHODS: The interaction of catechins with ERp57 was explored by fluorescence quenching and surface plasmon resonance techniques and their effect on ERp57 activities was investigated. RESULTS: A higher affinity was observed for galloylated cathechins, which bind close to the thioredoxin-like redox-sensitive active sites of the protein, with a preference for the oxidized form. The effects of these catechins on ERp57 properties were also investigated and a moderate inhibition of the reductase activity of ERp57 was observed as well as a strong inhibition of ERp57 DNA binding activity. CONCLUSIONS: Considering the high affinity of galloylated catechins for ERp57 and their capability to inhibit ERp57 binding to other macromolecular ligands, some effects of catechins interaction with this protein on eukaryotic cells may be expected. GENERAL SIGNIFICANCE: This study provides information to better understand the molecular mechanisms underlying the biological activities of catechins and to design new polyphenol-based ERp57-specific inhibitors.

• MeSH
• DNA chemie   MeSH
• enzymatické testy MeSH
• fluorescenční spektrometrie MeSH
• katalytická doména MeSH
• katechin analogy a deriváty   chemie   MeSH
• kinetika MeSH
• lidé MeSH
• ligandy MeSH
• molekulární modely MeSH
• oxidace-redukce MeSH
• povrchová plasmonová rezonance MeSH
• proteindisulfidisomerasy chemie   MeSH
• rekombinantní proteiny chemie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• epicatechin gallate MeSH Prohlížeč
• epigallocatechin gallate MeSH Prohlížeč
• gallocatechol MeSH Prohlížeč
• katechin MeSH
• ligandy MeSH
• PDIA3 protein, human MeSH Prohlížeč
• proteindisulfidisomerasy MeSH
• rekombinantní proteiny MeSH

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

• MeSH
• bakteriální proteiny * chemie   genetika   metabolismus   MeSH
• buněčné linie MeSH
• chromatografie kapalinová metody   MeSH
• faktory virulence chemie   genetika   metabolismus   MeSH
• Francisella tularensis * genetika   metabolismus   patogenita   MeSH
• hmotnostní spektrometrie metody   MeSH
• isoelektrická fokusace MeSH
• lidé MeSH
• makrofágy cytologie   metabolismus   MeSH
• membránové proteiny * chemie   genetika   metabolismus   MeSH
• míra přežití MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidy chemie   genetika   metabolismus   MeSH
• proteindisulfidisomerasy * chemie   genetika   metabolismus   MeSH
• proteom analýza   MeSH
• proteomika metody   MeSH
• tularemie metabolismus   mortalita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• faktory virulence MeSH
• membránové proteiny * MeSH
• peptidy MeSH
• proteindisulfidisomerasy * MeSH
• proteom MeSH

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

• Klíčová slova
• DacD, Francisella, carboxypeptidase, membrane defects, penicillin binding proteins, phagosomal escape, virulence,
• MeSH
• antibakteriální látky farmakologie   MeSH
• buněčná stěna metabolismus   MeSH
• Francisella tularensis účinky léků   růst a vývoj   patogenita   MeSH
• karboxypeptidasa štěpící D-Ala-D-Ala vazby serinového typu genetika   metabolismus   MeSH
• kultivované buňky MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidoglykan biosyntéza   MeSH
• proteindisulfidisomerasy genetika   MeSH
• proteiny vázající penicilin genetika   metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• tularemie mikrobiologie   patologie   MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• antibakteriální látky MeSH
• karboxypeptidasa štěpící D-Ala-D-Ala vazby serinového typu MeSH
• peptidoglykan MeSH
• proteindisulfidisomerasy MeSH
• proteiny vázající penicilin MeSH