Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans

Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.     

O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.     

Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe,mediates tRNA methylation in response to nutrient signaling

The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNA^Asp and, to a lesser extent, of tRNA^Glu, despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.     

SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52

The Schizosaccharomyces pombe rad31 and hus5 genes are required for the DNA damage response, as mutants defective in these genes are sensitive to DNA damaging agents, such as UV and ionising radiation and to the DNA synthesis inhibitor hydroxyurea (HU). Sequence analysis has suggested that rad31 and hus5 encode components of the Pmt3 (SUMO) modification process in S.pombe. We show here that the rad31 null and hus5.62 mutants display reduced levels of Pmt3 modification. We have initiated a search for proteins required for the DNA damage response, which may be modified by Pmt3 and have identified Rad22, the fission yeast homologue of the recombination protein Rad52. Purification of myc + His-tagged Rad22 protein from cells expressing HA-tagged Pmt3 identifies an 83 kDa species which cross-reacts with anti-HA antisera. We show here that Rad22 interacts with Rhp51 and Rpa70 (the fission yeast homologues of Rad51 and the large subunit of RPA, respectively), but that neither of these proteins appears to be responsible for the 83 kDa species. The 83 kDa species is observed when extracts are prepared under both native and denaturing conditions, and is also observed when myc + His-tagged Rad22 and Pmt3 are expressed at wild type levels, suggesting that Rad22 is modified by Pmt3 in vivo. We have established an S.pombe in vitro Pmt3 modification system and have shown that Rad22 and Rhp51 are modified in vitro, but that Rpa70 is not.     

Identification of O-mannosylated Virulence Factors in Ustilago maydis

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.     

Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.Protein glycosylation, which is a major posttranslational modification, plays essential roles in eukaryotic cells from fungi to mammals (19). N-linked oligosaccharides in glycoproteins that share relatively common structures are structurally classified into high-mannose, complex, and hybrid types (3). O-linked oligosaccharides in glycoproteins are diverse with respect to their sugar components and the mode of sugar linkages among the eukaryotic organisms (8, 19). O mannosylation, which is commonly found in the glycoproteins of fungi, has been extensively studied in the budding yeast Saccharomyces cerevisiae (4, 21, 35). The initial reaction of mannose transfer to serine and threonine residues in proteins is catalyzed by protein O-d-mannosyltransferase (Pmt) in the endoplasmic reticulum (ER), where dolichyl phosphate-mannose is required as an immediate sugar donor (4). In the Golgi complex, O mannosylation in S. cerevisiae is linearly elongated by up to five mannose residues by mannosyltransferases (Mnt) that utilize GDP-mannose as the mannosyl donor. At least six Pmt-encoding genes (PMT1 to -6), three α-1,2-Mnt-encoding genes (KRE2, KTR1, and KTR3), and three α-1,3-Mnt-encoding genes (MNN1, MNT2, and MNT3) are known to be involved in O mannosylation in S. cerevisiae (21, 31, 45).The Pmt family of proteins can be classified into the PMT1, PMT2, and PMT4 subfamilies based on phylogeny (6). Proteins of the PMT1 subfamily form a heteromeric complex with proteins belonging to the PMT2 subfamily, and PMT4 subfamily proteins form a homomeric complex (7). Simultaneous disruptions of three different types of PMT genes were lethal (4), suggesting that each class provided a unique function for O mannosylation. Yeasts other than S. cerevisiae, such as Schizosaccharomyces pombe (38, 41), Candida albicans (29), and Cryptococcus neoformans (28), possess three to five pmt genes, which have been characterized. Several studies provide evidence that protein O mannosylation modulates the functions and stability of secretory proteins and thereby affects the growth and morphology of these yeasts. O mannosylation by Pmt2 in S. cerevisiae (ScPmt2) provides protection from ER-associated degradation and also functions as a fail-safe mechanism for ER-associated degradation (11, 13, 23). Likewise, in C. albicans, CaPmt1- and CaPmt4-mediated O mannosylation specifically protects CaSec20 from proteolytic degradation in the ER (40). Cell wall integrity is maintained in S. cerevisiae by increased stabilization and correct localization of the sensor proteins ScWsc and ScMid2 due to O mannosylation by ScPmt2 and ScPmt4 (20). Similarly, the stability and localization to the plasma membrane of axial budding factor ScAxl2/Bud10 is enhanced by ScPmt4-mediated O mannosylation, increasing its activity (32). ScPmt4-mediated O glycosylation also functions as a sorting determinant for cell surface delivery of ScFus1 (30). CaPmt4-mediated O glycosylation is required for environment-specific morphogenetic signaling and for the full virulence of C. albicans (29).With respect to filamentous fungi like Aspergillus that develop hyphae in a highly ordered manner, which then differentiate to form conidiospores, little is known about the function and synthetic pathway of the O-mannose-type oligosaccharides. O-Glycans in glycoproteins of Aspergillus include sugars other than mannose, and their structures have been determined (8). The initial mannosylation catalyzed by Pmts is found in Aspergillus and occurs as in yeasts (8).We characterized the pmtA gene of Aspergillus nidulans (AnpmtA), belonging to the PMT2 subfamily, and found that the mutant exhibited a fragile cell wall phenotype and alteration in the carbohydrate composition, with a reduction in the amount of skeletal polysaccharides in the cell wall (26, 33). Recently, the Afpmt1 gene belonging to the PMT1 family of Aspergillus fumigatus, a human pathogen, was characterized. AfPmt1 is crucial for cell wall integrity and conidium morphology (46).In this study, we characterize the pmtB and pmtC genes of A. nidulans to understand their contribution to the cell morphology of this filamentous fungus. We also demonstrate that the PmtA, PmtB, and PmtC proteins have distinct specificities for protein substrates and function differently during cell growth of filamentous fungi.     

Expression of catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of the key problems in immuno-biotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively performed in yeast cells. We obtained expression strains of the methylotrophic yeast Pichia pastoris producing single-chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab), and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant K _d and the first order constant (k ₂) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and to the single-chain antibody A.17 expressed in CHO and E. coli cells, respectively.     

Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.Filamentous fungi use highly polar growth to explore their environments. Except for a brief period of isotropic expansion just after spores break dormancy, filamentous fungi add new cell wall material exclusively at the tips of tubular hyphal cells. Such polar growth involves a high degree of coordination between signals from the environment and the secretory apparatus. In fungi, O mannosylation of specific target proteins has been shown to be important for sensing environmental stress, stabilizing the cell wall, and proper development (18, 28). The assembly of protein linked O-mannosyl glycans in the endoplasmic reticulum lumen is catalyzed by protein O-mannosyltransferases (Pmts), which transfer a single mannosyl residue to the hydroxyl group of serine or threonine residues to form an α-d-mannosyl linkage (30). The addition of further carbohydrate residues to the first O-linked mannose occurs in the Golgi apparatus and involves a range of enzymes (35). Modification by Pmts seems to be specific to proteins that are synthesized and sorted in the secretory pathway; however, the only motif so far identified is that Ser/Ter-rich membrane-bound proteins are O mannosylated by Pmt4 in Saccharomyces cerevisiae (15). This lack of a clear motif makes identification of Pmt targets by computational methods challenging. All of the fungal Pmt-modified proteins identified so far are localized to the cell membrane or cell wall or are secreted. At least 23 target proteins have been described in yeasts (15). Only three Pmt target proteins have been described in filamentous fungi (12, 23, 37).Pmts have been found in both prokaryotes and eukaryotes (33), but not in plants (8). The lengths and compositions of O-mannosyl glycans are different among species. In fungi, O-glycosyl chains range from 2 to 7 residues. In S. cerevisiae, the mannosyl chain can be modified by mannosyl phosphate (6). In Schizosaccharomyces pombe, the O-linked glycan is capped with 1 or 2 galactose residues (6). In the filamentous fungi so far examined, O-glycans are linear and branched, with 3 to 5 monosaccharide residues (4).In fungi, the Pmts are classified into the Pmt1, Pmt2, and Pmt4 subfamilies, with each species having three to seven members. S. cerevisiae and Candida albicans Pmts are the most redundant, with subfamilies 1 and 2 containing two or three members (7, 26). S. pombe and many filamentous fungi, including Aspergillus nidulans, have one representative from each subfamily. In S. cerevisiae, the enzymatic activity of Pmts requires interaction among members of the Pmt1 and Pmt2 subfamilies, while Pmt4 forms homomeric complexes (8). Heteromeric complexes between Pmt1 and Pmt2 subfamily members have also been reported in S. pombe (34).O mannosylation appears to be required for the stability, localization, and function of target proteins (18, 28, 32), and in vivo consequences of Pmt loss range from limited to lethal. In S. cerevisiae, O mannosylation is essential for cell integrity and cell wall rigidity (7). In C. albicans and Cryptococcus neoformans, Pmt mutation affects morphogenesis and virulence (24, 26, 27). In S. cerevisiae, strains with single Pmt subfamily representatives deleted are viable; however, deletion of subfamily 2 representatives is lethal in S. pombe and C. albicans (7, 34). In filamentous fungi, deletion of individual Pmts has been reported. Deletions of Trichoderma reesei pmtI, Aspergillus fumigatus pmt1, A. nidulans pmtA, and Aspergillus awamori pmtA were not lethal but affected growth and development (10, 22, 23, 37).In previous work, we identified the swoA mutant from a collection of temperature-sensitive polarity mutants and showed that the swoA allele encoded a Pmt2 subfamily member (PmtA) (21, 29). In this study, we use Δpmt strains to show that each of the three Pmts in A. nidulans (pmtA, pmtB, and pmtC) is nonessential but that all play distinct roles in cell wall integrity and developmental patterning. We also demonstrate that PmtA and PmtC modify an ortholog of S. cerevisiae Wsc1, a known Pmt target. Because of redundancy, all Pmt1 and Pmt2 subfamily members have not been deleted in S. cerevisiae. Because of lethality, the effects of loss of the Pmt2 subfamily cannot be addressed in S. pombe or C. albicans. This makes A. nidulans the first fungus in which the phenotypes of deleted strains for each Pmt subfamily have been reported.     

Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants

Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Delta mutant. We found that pmt2 pmt4Delta cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Delta mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Delta mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.     

The Telomere-Binding Protein Taz1p as a Target for Modification by a SUMO-1 Homologue in Fission Yeast

In fission yeast (Schizosaccharomyces pombe) the homologue of the mammalian SUMO-1 ubiquitin-like modifier is encoded by the pmt3 gene. A two-hybrid screen using the telomere-binding protein Taz1p as bait identified Pmt3p as an interacting factor. In vitro experiments using purified components of the fission yeast Pmt3p modification system demonstrated that Taz1p could be modified directly by Pmt3p. The amino acid sequence of Taz1p contains a close match to the consensus modification site for SUMO-1, and a PEST sequence similar to those found in established SUMO-1 targets. Although previous experiments have identified an increase in telomere length as one consequence of the pmt3– genotype, we could not detect Pmt3p modification of Taz1p in protein extracts made from exponentially growing haploid cells or any effect of Pmt3p on the localization of GFP-Taz1p at discrete foci in the haploid cell nucleus.     

Pmt-mediated O mannosylation stabilizes an essential component of the secretory apparatus, Sec20p, in Candida albicans

Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.     

Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen candida albicans

Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.     

SUMO Chain Formation Is Required for Response to Replication Arrest in S. pombe

SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved ββαββαβ fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe.     

A conserved acidic motif is crucial for enzymatic activity of protein O-mannosyltransferases

Protein O-mannosylation is an essential modification in fungi and mammals. It is initiated at the endoplasmic reticulum by a conserved family of dolichyl phosphate mannose-dependent protein O-mannosyltransferases (PMTs). PMTs are integral membrane proteins with two hydrophilic loops (loops 1 and 5) facing the endoplasmic reticulum lumen. Formation of dimeric PMT complexes is crucial for mannosyltransferase activity, but the direct cause is not known to date. In bakers' yeast, O-mannosylation is catalyzed largely by heterodimeric Pmt1p-Pmt2p and homodimeric Pmt4p complexes. To further characterize Pmt1p-Pmt2p complexes, we developed a photoaffinity probe based on the artificial mannosyl acceptor substrate Tyr-Ala-Thr-Ala-Val. The photoreactive probe was preferentially cross-linked to Pmt1p, and deletion of the loop 1 (but not loop 5) region abolished this interaction. Analysis of Pmt1p loop 1 mutants revealed that especially Glu-78 is crucial for binding of the photoreactive probe. Glu-78 belongs to an Asp-Glu motif that is highly conserved among PMTs. We further demonstrate that single amino acid substitutions in this motif completely abolish activity of Pmt4p complexes. In contrast, both acidic residues need to be exchanged to eliminate activity of Pmt1p-Pmt2p complexes. On the basis of our data, we propose that the loop 1 regions of dimeric complexes form part of the catalytic site.     

Blockade of Fas Signaling in Breast Cancer Cells Suppresses Tumor Growth and Metastasis via Disruption of Fas Signaling-initiated Cancer-related Inflammation

Mechanisms for cancer-related inflammation remain to be fully elucidated. Non-apoptotic functions of Fas signaling have been proposed to play an important role in promoting tumor progression. It has yet to be determined if targeting Fas signaling can control tumor progression through suppression of cancer-related inflammation. In the current study we found that breast cancer cells with constitutive Fas expression were resistant to apoptosis induction by agonistic anti-Fas antibody (Jo2) ligation or Fas ligand cross-linking. Higher expression of Fas in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. To determine whether blockade of Fas signaling in breast cancer could suppress tumor progression, we prepared an orthotopic xenograft mouse model with mammary cancer cells 4T1 and found that blockade of Fas signaling in 4T1 cancer cells markedly reduced tumor growth, inhibited tumor metastasis in vivo, and prolonged survival of tumor-bearing mice. Mechanistically, blockade of Fas signaling in cancer cells significantly decreased systemic or local recruitment of myeloid derived suppressor cells (MDSCs) in vivo. Furthermore, blockade of Fas signaling markedly reduced IL-6, prostaglandin E2 production from breast cancer cells by impairing p-p38, and activity of the NFκB pathway. In addition, administration of a COX-2 inhibitor and anti-IL-6 antibody significantly reduced MDSC accumulation in vivo. Therefore, blockade of Fas signaling can suppress breast cancer progression by inhibiting proinflammatory cytokine production and MDSC accumulation, indicating that Fas signaling-initiated cancer-related inflammation in breast cancer cells may be a potential target for treatment of breast cancer.     

From transcriptional landscapes to the identification of biomarkers for robustness

Background

Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.

Results

The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.

Conclusions

Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.     

The role of l-phenylalanine in the production of isobutene by Rhodotorula minuta

The role of l-phenylalanine and its synergistic effect on the production of isobutene were investigated with both the living cells and a cell-free system of Rhodotorula minuta IFO 1102. Many aromatic carboxylic acids also had the same effect on the production of isobutene as l-phenylalanine. Cycloheximide, an inhibitor of protein synthesis, inhibited the synergistic effect of l-phenylalanine on the production of isobutene. Furthermore, the cell-free extract prepared from cells cultivated in the medium which contained l-phenylalanine had isobutene-forming activity. These results confirm that l-phenylalanine is an inducer of a tentative “isobutene-forming enzyme” in Rhodotorula minuta.     

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pmt1 for tRNA^Asp methylation, which distinguishes Pmt1 from other Dnmt2 homologs. The present analysis also revealed a novel Pmt1- and Q-independent tRNA methylation site in S. pombe, C34 of tRNA^Pro. Notably, queuine is a micronutrient that is scavenged by higher eukaryotes from the diet and gut microflora. This work therefore reveals an unanticipated route by which the environment can modulate tRNA modification in an organism.     

Genomic, regulatory and epigenetic mechanisms underlying duplicated gene evolution in the natural allotetraploid Oryza minuta

Background

Polyploid species contribute to Oryza diversity. However, the mechanisms underlying gene and genome evolution in Oryza polyploids remain largely unknown. The allotetraploid Oryza minuta, which is estimated to have formed less than one million years ago, along with its putative diploid progenitors (O. punctata and O. officinalis), are quite suitable for the study of polyploid genome evolution using a comparative genomics approach.

Results

Here, we performed a comparative study of a large genomic region surrounding the Shattering4 locus in O. minuta, as well as in O. punctata and O. officinalis. Duplicated genomes in O. minuta have maintained the diploid genome organization, except for several structural variations mediated by transposon movement. Tandem duplicated gene clusters are prevalent in the Sh4 region, and segmental duplication followed by random deletion is illustrated to explain the gene gain-and-loss process. Both copies of most duplicated genes still persist in O. minuta. Molecular evolution analysis suggested that these duplicated genes are equally evolved and mostly manipulated by purifying selection. However, cDNA-SSCP analysis revealed that the expression patterns were dramatically altered between duplicated genes: nine of 29 duplicated genes exhibited expression divergence in O. minuta. We further detected one gene silencing event that was attributed to gene structural variation, but most gene silencing could not be related to sequence changes. We identified one case in which DNA methylation differences within promoter regions that were associated with the insertion of one hAT element were probably responsible for gene silencing, suggesting a potential epigenetic gene silencing pathway triggered by TE movement.

Conclusions

Our study revealed both genetic and epigenetic mechanisms involved in duplicated gene silencing in the allotetraploid O. minuta.