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Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of proteins in the ER. Trichoderma reesei strains displayed a single representative of each PMT subfamily, Trpmt1, Trpmt2 and Trpmt4. In this work, two knockout strains ΔTrpmt1and ΔTrpmt4were obtained. Both mutants showed retarded growth, defective cell walls, reduced conidiation and decreased protein secretion. Additionally, the ΔTrpmt1strain displayed a thermosensitive growth phenotype, while the ΔTrpmt4 strain showed abnormal polarity. Meanwhile, OETrpmt2 strain, in which the Trpmt2 was over-expressed, exhibited increased conidiation, enhanced protein secretion and abnormal polarity. Using a lectin enrichment method and MS/MS analysis, 173 O-glycoproteins, 295 O-glycopeptides and 649 O-mannosylation sites were identified as the targets of PMTs in T. reesei. These identified O-mannoproteins are involved in various physiological processes such as protein folding, sorting, transport, quality control and secretion, as well as cell wall integrity and polarity. By comparing proteins identified in the mutants and its parent strain, the potential specific protein substrates of PMTs were identified. Based on our results, TrPMT1 is specifically involved inO-mannosylation of intracellular soluble proteins and secreted proteins, specially glycosidases. TrPMT2 is involved inO-mannosylation of secreted proteins and GPI-anchor proteins, and TrPMT4 mainly modifies multiple transmembrane proteins. The TrPMT1-TrPMT4 complex is responsible for O-mannosylation of proteins involved in cell wall integrity. Overexpression of TrPMT2 enhances protein secretion, which might be a new strategy to improve expression efficiency in T. reesei.

Plant plasma membrane (PM) plays important roles in immune response. Here, we utilized quantitative mass spectrometry to explore rice PM protein composition and dynamic changes during Magnaporthe oryzae infection. We report, thus far, the largest rice PM proteome dataset with 3,906 identified proteins, among which 484 proteins were differentially expressed after M. oryzae infection. One third of the identified proteins are predicted to have at least one transmembrane domain. Half of the identified proteins are predicted to have binding functions and over one third of the proteins have enzyme-related functions. In addition, Gene Ontology analyses revealed that abscisic acid (ABA) and cytokinin (CK) signaling were sequentially activated after M. oryzae infection in rice. We found that the activation of ABA signaling and the suppression of rice immune response occurred at the early infection stage, while the activation of CK signaling, the upregulation of sugar transporter genes expression, and the nutrient efflux of infected rice cells occurred at later infection stage. Thus, we further propose that M. oryzae activates ABA signaling to repress rice immune signaling for initial invasion and redirects nutrient efflux of infected cells for massive growth at the later infection stage.

Lignocelluloses contained in animal forage cannot be digested by pigs or poultry with 100% efficiency. On contrary, Aspergillus fumigatus, a saprophytic filamentous fungus, is known to harbor 263 glycoside hydrolase encoding genes, suggesting that A. fumigatus is an efficient lignocellulose degrader. Hence the present study uses corn, wheat, or soybean as a sole carbon source to culture A. fumigatus under animal physiological condition to understand how cellulolytic enzymes work together to achieve an efficient degradation of lignocellulose. Our results showed that A. fumigatus produced different sets of enzymes to degrade lignocelluloses derived from corn, wheat, or soybean cell wall. In addition, the cellulolytic enzymes produced by A. fumigatus were stable under acidic condition or at higher temperatures. Using isobaric tags for a relative and absolute quantification (iTRAQ) approach, a total of ∼600 extracellular proteins were identified and quantified, in which ∼50 proteins were involved in lignocellulolysis, including cellulases, hemicellulases, lignin-degrading enzymes, and some hypothetical proteins. Data are available via ProteomeXchange with identifier PXD004670. On the basis of quantitative iTRAQ results, 14 genes were selected for further confirmation by RT-PCR. Taken together, our results indicated that the expression and regulation of lignocellulolytic proteins in the secretome of A. fumigatus were dependent on both nature and complexity of cellulose, thus suggesting that a different enzyme system is required for degradation of different lignocelluloses derived from plant cells. Although A. fumigatus is a pathogenic fungus and cannot be directly used as an enzyme source, as an efficient lignocellulose degrader its strategy to synergistically degrade various lignocelluloses with different enzymes can be used to design enzyme combination for optimal digestion and absorption of corn, wheat, or soybean that are used as forage of pig and poultry.

Protein O-mannosyltransferases (PMTs) have been identified in fungi but not in plants and nematodes, which makes PMTs become attractive targets for developing a new strategy against phytopathogens. Three PMTs have been identified in Fusarium oxysporum, a fungal pathogen that causes vascular wilt in a broad range of economical crops. By deletion or suppression of the pmt genes, we showed that all mutants displayed retarded growth, reduced conidiation, cell wall defects, ER stress and attenuated virulence in F. oxysporum f.sp. cucumerinum. In addition, the Δpmt1 exhibited reduced thermotolerance, while the Δpmt4 and the pmt2 conditional mutant exhibited abnormal polarized growth. Comparative glycoproteome analysis of these pmt mutants revealed that PMTs preferentially modified random coils with flanking regions rich in Ser, Thr, Ala, Glu, Asp and Lys at the stem region of membrane proteins, the N-terminal region close to signal peptide of secreted proteins, or surface of soluble proteins. PMT1 specifically acted on nuclear proteins and proteins that are responsible for protein folding, which might contribute to thermotolerance. PMT4 specifically acted on the membrane and soluble proteins in secretory pathways, especially the GPI anchoring pathway, which might contribute to synthesis and transportation of GPI anchored proteins and thus polarized growth. PMT2 was responsible for modification of proteins that are required for protein folding and cell wall synthesis, which might make PMT2 essential. Our results gave an insight to understanding of the roles of each O-mannosyltransferase in F. oxysporum f.sp. cucumerinum and provide a new perspective to prevent Fusarium wilt.

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

The rumen bacterium Cellulosilyticum ruminicola H1 efficiently hydrolyzes cellulose. To gain insights into the regulatory mechanisms of cellulase synthesis, comparative transcriptome analysis was conducted for cultures grown on 2% filter paper, 0.5 and 0.05% cellobiose, and 0.5% birchwood xylan. It was found that cellulose induced a majority of (hemi)cellulases, including 33 cellulases and a cellulosomal scaffoldin (1.3- to 22.7-fold); seven endoxylanases, two mannanases, and two pectatelyases (2- to 16-fold); and pyruvate formate-lyase (PFL, 1.5- to 7-fold). Noticeably, 3- and 2.5-fold increased transcription of a cellobiohydrolase and the cellulosomal scaffoldin precursor were detected in 0.05% than in 0.5% cellobiose. Consistently, 9- and 4-fold higher specific cellobiohydrolase activities were detected in the filter paper and 0.05% cellobiose culture. SDS- and native-PAGE zymograms of cellulose-enriched proteins from the filter paper culture displayed cellulase activities, and cellulolytic “complexes” were enriched from the filter paper- and 0.05% cellobiose-cultures, but not from the 0.5% cellobiose culture. LC-MS/MS identified the cellulosomal scaffoldin precursor in the “complexes” in addition to cellulase, hemicellulase, and PFL proteins. The addition of 0.5% cellobiose, but not 0.05% cellobiose remarkably inhibited strain H1 to degrade filter paper. Therefore, this work reveals a cellobiose-dose related regulatory mechanism of cellulase synthesis by lower for induction and higher for repression, which has extended our understanding of the regulation of microbial cellulase synthesis.

Lysine acetylation is a reversible and highly regulated post-translational modification that plays a critical role in regulating many aspects of cellular processes, both in bacteria and in eukaryotes. However, this modification has not been systematically studied in archaea. Herein, we report the lysine acetylome of a model haloarchaeon, Haloferax mediterranei. Using immunoaffinity enrichment and LC-MS/MS analysis, we identified 1017 acetylation sites in 643 proteins, accounting for 17.3% of the total proteins in this haloarchaeon. Bioinformatics analysis indicated that lysine acetylation mainly distributes in cytoplasm (94%) and participates in protein biosynthesis and carbon metabolism. Specifically, the acetylation of key enzymes in PHBV biosynthesis further suggested that acetylation plays a key role in the energy and carbon storage. In addition, a survey of the acetylome revealed a universal rule in acetylated motifs: a positively charged residue (K, R, or H) located downstream of acetylated lysine at the positions +1, +2, or +3. Interestingly, we identified acetylation in several replication initiation proteins Cdc6; mutation on the acetylated site of Cdc6A destroyed the Autonomous Replication Sequence (ARS) activity of its adjacent origin oriC1. Our study indicates that lysine acetylation is an abundant modification in H. mediterranei, and plays key roles in the processes of replication, protein biosynthesis, central metabolism, and carbon storage. This acetylome of H. mediterranei provides opportunities to explore the physiological role of acetylation in halophilic archaea.

Protein lysine methylation occurs extensively in the Crenarchaeota , a major kingdom in the Archaea . However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus . The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate protein using S -adenosyl- l -methionine (SAM) as the methyl donor. aKMT, a non-SET domain protein, is highly conserved among crenarchaea, and distantly related homologs also exist in Bacteria and Eukarya . aKMT was active over a wide range of temperatures, from ∼25 to 90°C, with an optimal temperature at ∼60 to 70°C. Amino acid residues Y9 and T12 at the N terminus appear to be the key residues in the putative active site of aKMT, as indicated by sequence conservation and site-directed mutagenesis. Although aKMT was identified based on its methylating activity on Cren7, the crenarchaeal chromatin protein, it exhibited broad substrate specificity and was capable of methylating a number of recombinant Sulfolobus proteins overproduced in Escherichia coli . The finding of aKMT will help elucidate mechanisms underlining extensive protein lysine methylation and the functional significance of posttranslational protein methylation in crenarchaea.

Clostridium thermocellum, a thermophilic anaerobe, has the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. In this work, the cell wall proteome of C. thermocellum was investigated. The proteins in the cell wall fraction of C. thermocellum prepared by the boiling SDS method were released by mutanolysin digestion and resolved on two-dimensional (2D) gel. One hundred and thirty-two proteins were identified by mass spectrometry, among which the extracellular solute-binding protein (CbpB/cthe_1020), enolase, glyceraldehyde-3-phosphate dehydrogenase and translation elongation factor EF-Tu were detected as highly abundant proteins. Besides the known surface localized proteins, including FtsZ, MinD, GroEL, DnaK, many enzymes involved in bioenergetics, such as alcohol dehydrogenases and hydrogenases were also detected. By glycan stain and MS analysis of glycopeptides, we identified CbpB as a glycoprotein, which is the second glycoprotein from C. thermocellum characterized. The fact that CbpB was highly abundant in the cell wall region and glycosylated, reflects its importance in substrate assimilation. Our results indicate cell wall proteins constitute a significant portion of cellular proteins and may play important physiological roles (i.e. bioenergetics) in this bacterium. The insights described are relevant for the development of C. thermocellum as a biofuel producer.

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in S. clavuligerus .

Acyl homoserine lactone (AHL)-based quorum sensing commonly refers to cell density-dependent regulatory mechanisms found in bacteria. However, beyond bacteria, this cell-to-cell communication mechanism is poorly understood. Here we show that a methanogenic archaeon, Methanosaeta harundinacea 6Ac, encodes an active quorum sensing system that is used to regulate cell assembly and carbon metabolic flux. The methanogen 6Ac showed a cell density-dependent physiology transition, which was related to the AHL present in the spent culture and the filI gene-encoded AHL synthase. Through extensive chemical analyses, a new class of carboxylated AHLs synthesized by FilI protein was identified. These carboxylated AHLs facilitated the transition from a short cell to filamentous growth, with an altered carbon metabolic flux that favoured the conversion of acetate to methane and a reduced yield in cellular biomass. The transcriptomes of the filaments and the short cell forms differed with gene expression profiles consistent with the physiology. In the filaments, genes encoding the initial enzymes in the methanogenesis pathway were upregulated, whereas those for cellular carbon assimilation were downregulated. A luxI–luxR ortholog filI–filR was present in the genome of strain 6Ac. The carboxylated AHLs were also detected in other methanogen cultures and putative filI orthologs were identified in other methanogenic genomes as well. This discovery of AHL-based quorum sensing systems in methanogenic archaea implies that quorum sensing mechanisms are universal among prokaryotes.

Rapid identification of glycosylation sites of glycoproteins is urgently needed in the study of glycoproteomics. In the present work, a rapid and simple method based on non-specific digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry was described, which can efficiently identify the N-linked glycosylation sites. One-step in-gel digestion of ribonuclease B (RNase B) by proteinase K was employed to generate glycopeptides with short and discrepant peptide composition. When compared with glycopeptides prepared by two-step in-gel digestion using trypsin–proteinase K or trypsin-pronase, the direct proteinase K treatment showed obvious superiority in both glycopeptide recovery and preparation simplicity. Most importantly, it helps to generate greater variety of glycopeptide series with rich information for glycosylation site identification. In addition, binary matrices 5-chloro-2-mercaptobenzothiazole/2,5-dihydroxybenzoic acid were found to form homogeneous microcrystals on the target with the purified glycopeptides, leading to improved detection sensitivity. Thus, the present work provides an optimized solution to speed up the characterization of N-linked glycosylation sites in glycoproteins.

Feruloyl esterases (Faes) constitute a subclass of carboxyl esterases that specifically hydrolyze the ester linkages between ferulate and polysaccharides in plant cell walls. Until now, the described microbial Faes were mainly from fungi. In this study, we report that Cellulosilyticum ruminicola H1, a previously described fibrolytic rumen bacterium, possesses three different active feruloyl esterases, FaeI, FaeII, and FaeIII. Phylogenetic analysis classified the described bacterial Faes into two types, FaeI and FaeII in type I and FaeIII in type II. Substrate specificity assays indicated that FaeI is more active against the ester bonds in natural hemicelluloses and FaeIII preferentially attacks the ferulate esters with a small moiety, such as methyl groups, while FaeII is active on both types of substrates. Among the three feruloyl esterase genes, faeI was the only one induced significantly by xylose and xylan, while pectin appeared to moderately induce the three genes during the late log phase to stationary phase. Western blot analysis determined that FaeI and FaeIII were secreted and cytoplasmic proteins, respectively, whereas FaeII seemed to be cell associated. The addition of FaeI and FaeII but not FaeIII enhanced the activity of a xylanase on maize cob, suggesting a synergy of the former two with xylanase. Hence, we propose that the three feruloyl esterases work in concert to hydrolyze ferulate esters in natural hemicelluloses.

Purpose: The 20S proteasome is a multicatalytic protein complex, which plays a major role in intracellular protein degradation. In mammalian cells, it consists of 28 subunits arranged in four stacked rings (α1-7β1-7β1-7α1-7). The aim of this study is to characterize and compare subunit composition and heterogeneity (or subtypes) of the 20S proteasome from four human pancreatic cancer cell lines. Experimental design: To study subunit compositions and heterogeneity of 20S proteasome from human pancreatic cancer cell lines, in the present study, 20S proteasome from four different pancreatic cancer cell lines (SW1990, a human exocrine adenocarcinoma, derived from spleen metastasis; PANC-1, a human ductal carcinoma in situ; BxPC-3, a human ductal carcinoma in situ; and CFPAC-1, a human ductal adenocarcinoma, derived from liver metastasis) were subjected to a gel-based proteomics analysis, respectively. Results: It was found that the differences in the subunit compositions and subtypes of the 20S proteasomes among four pancreatic cancer cell lines exist. Gel-based proteomics analysis showed that more than 60 subunits spots were separated and identified by MS. Our study revealed the presence of various isoforms for each of the subunits and different subtypes of the 20S proteasome. The significant differences among four cell lines are the relative abundances of immunoproteasome subunits, β1i and β2i, indicating that different subtypes of immunoproteasome among four cell lines exist. Conclusions and clinical relevance: The 20S proteasome from four human pancreatic cancer cell lines was characterized. The different expression levels of immunoproteasome subunits, β1i and β2i, indicate that the 20S proteasome may have different subtypes among four cell lines, which may be related to cancer cell property and be useful for the establishment of personalized therapy using proteasome inhibitors in future.

An efficient system is presented for pre-delignifying cereal straw in vitro using laccase produced by Pycnoporus sanguineus H275. Under our experimental conditions, over 97% of the klason lignin was removed from 1 g wheat straw lignocellulose, without loss of Somogyi carbohydrate (reducing sugar). Microscopic observation of birefringence changes further indicated that the structure of the cellulose building blocks (microfibrils) of wheat straw scattered after pre-degradation, compared with the tightly arranged microfibrils in untreated control straw. The P . sanguineus H275-laccase-encoding gene was then cloned and successfully expressed in Pichia pastoris X-33, with a production of 3000 U/L after 6 days of cultivation. The identity of the expressed laccase was further confirmed by using MALDI-TOF-MS/MS. Homology modeling revealed that the native H275-laccase, predicted with five possible N -glycosylation sites, has extra cupredoxine domains and three common Cu-oxidase domains. Both kinetic characterization on the substrate ABTS and pre-delignification investigation on wheat straw lignocellulose showed that the activity of the recombinant laccase was nearly the same as that of the native laccase.

Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps.

The mammalian ER/cytosolic alpha-mannosidase (Man2C1p), yeast vacuolar alpha-mannosidase (Ams1p) and the Aspergillus nidulans alpha-mannosidase are members of Class IIC subgroup, which is involved in oligosaccharide catabolism and N-glycan processing. Unlike their mammalian counterparts, the yeast Ams1p and A. nidulans Class IIC alpha-mannosidase are not essential for morphogenesis and cellular function. In this study, the Afams1, a gene encoding a member of Class IIC alpha-mannosidases, was identified in the opportunistic pathogen Aspergillus fumigatus. Deletion of the Afams1 led to a severe defect in conidial formation, especially at a higher temperature. In addition, abnormalities of polarity and septation were associated with the DeltaAfams1 mutant. Our results showed that the Afams1 gene, in contrast to its homolog in yeast or A. nidulans, was required for morphogenesis and cellular function in A. fumigatus.

Background: Senescence-accelerated mice (SAM) are commonly used as animal models for studying aging. To date, aging-related proteomes in the livers and brains of SAMP8 mice have been reported; however, spleen of SAM has not yet been used as material for studying the aging-related proteome despite its extremely important role in the progress of aging or aging-related diseases. Objective: To identify proteins associated with aging or aging-related diseases in SAM. Methods: Two-dimensional gel electrophoresis (2DE) was used to compare the proteomes of splenic lymphocytes from two strains of SAM (SAMP8 and SAMR1). Differentially expressed spots whose expression altered over twofold (p < 0.01) were identified by LC-MS/MS and database search. Results: Identification results showed that 18 differentially expressed protein spots represented 14 different proteins known to be involved in aging- or aging disease-related pathways, i.e., annexin I, calgranulin B, transaldolase, isocitrate dehydrogenase, and an unnamed protein product were involved in oxidative stress and cell defense, phosphoglycerate mutase I, aldose reductase, carbonic anhydrase I and carbonic anhydrase II were involved in glycolytic energy metabolism and homeostasis, and macrophage capping protein and high mobility group box-1 protein were involved in infection and DNA damage. Conclusion: 2DE combined with MS is effective for comparative proteome analysis of splenic lymphocytes of SAM to identify proteins closely involved in aging or aging-related diseases, especially immune diseases, and further functional studies of these proteins in SAM may throw some light on the study of biomarkers for aging or aging-related diseases.

Haloarcula hispanica is one of members of the Halobacteriaceae, which displays particularly low restriction activity and is therefore important as one of the most tractable haloarchaea for archaeal genetic research. Although the Har. hispanica S-layer protein has been reported glycosylated, the S-layer glycoprotein and its glycosylation have not been investigated yet. In this study, the S-layer proteins of Har. hispanica were extracted and characterized. The S-layer was found containing two different glycoproteins which shared highly similar amino acid sequences. The genes coding for these two S-layer glycoproteins were found next to each other in the genome. Moreover, the N- and O-linked glycans were released from these two S-layer glycoproteins for structural determination. Based on the mass spectrometry and nuclear magnetic resonance, the N-glycan was determined as a branched trisaccharide containing a 225 Da residue corresponded to a 2-amino-6-sulfo-2, 6-dideoxy-quinovose, which was the first time that a naturally occurring form of sulfoquinovosamine was identified. Besides, the O-glycan was characterized as a Glcα-1,4-Gal disaccharide by mass spectrometry combined with monosaccharide composition analysis and glycosidase treatment. The determination of the N- and O-glycan structure will be helpful for studying the diverse protein glycosylation pathways in archaea utilizing H. hispanica as a new model.

Kexin-like proteins belong to the subtilisin-like family of the proteinases that cleave secretory proproteins to their active forms. Several fungal kexin-like proteins have been investigated. The mutants lacking of kexin-like protein display strong phenotypes such as cell wall defect, abnormal polarity, and, in case of Candida albicans, diminished virulence. However, only several proteins have been confirmed as the substrates of kexin-like proteases in these fungal species. It still remains unclear how kexin-like proteins contribute to the morphogenesis in these fungal species. In this study, a kexB-null mutant of the human opportunistic fungal pathogen Aspergillus fumigatus was constructed and analyzed. The ΔkexB mutant showed retarded growth, temperature-sensitive cell wall defect, reduced conidia formation, and abnormal polarity. Biochemical analyses revealed that deletion of the kexB gene resulted in impaired N-glycan processing, activation of the MpkA-dependent cell wall integrity signaling pathway, and ER-stress. Results from in vivo assays demonstrated that the mutant exhibited an attenuated virulence in immunecompromised mice. Based on our results, the kexin-like endoprotease KexB was involved in the N-glycan processing, which provides a novel insight to understand how kexin-like protein affects the cell-wall modifying enzymes and therefore morphogenesis in fungi.

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p

Cold-adaptive methanogens contribute significantly to methane emission from the cold area, while the cold-adaptive mechanisms used by Archaea remain elusive. Methanolobus psychrophilus R15, a cold-adaptive methanogen isolated from a Tibetan plateau wetland, grows at 0-25 °C and optimally at 18 °C when isolated; however, it grows optimally at 30 °C after culturing at 18 °C for several years. Aiming to gain insights into the protein profiles that are involved in optimal growth and cold adaptation of this methanogen, here, we performed a comparative proteomic study using 2D DIGE on the cultures grown at 30, 18 and 4 °C. 1439 protein spots (3167 ORFs annotated in the R15 genome) were detected, and 202 of 322 differentially expressed protein spots were identified by MALDI-TOF/TOF. The protein abundance of most enzymes involved in methanogenesis, energy conservation and central metabolism were increased at 30 °C, while most ribosome proteins were decreased at 30 °C. Proteasome and ROS scavengers increased expressions at 4 °C, suggesting more aberrant proteins and ROS formed at lower temperatures. Different from the cold-adaptive Methanococcoides burtonii, some chaperones were increased at 4 °C, implying that protein folding was impaired at cold in this psychrophilic archaeon. This study indicates that diverse cold-adaptive mechanisms can be used by different methanogenic Archaea.

A novel strategy for design of haptens that were used to produce catalytic antibodies was developed and three monoclonal antibodies, 3G5, 2F3, and 5C9, were generated using this strategy. These monoclonal antibodies were converted into selenium-containing abzymes by chemically modifying the hydroxyl group of serines followed by sodium hydrogen selenide displacement. These selenium-containing abzymes exhibited remarkable glutathione peroxidase activity, which surpasses the activity of some native glutathione peroxidases. The activities of the selenium-containing abzymes Se-3G5, Se-2F3, and Se-5C9 which catalyzed reduction of hydroperoxides by glutathione were 2.23, 4.20, and 3.79 times that of rabbit liver glutathione peroxidase, respectively. Detailed steady-state kinetics study on Se-2F3 was carried out and the value of k(cat)/K(m) (H(2)O(2)) was found to be 2.11 x 10(7) M(-1) min(-1) which was supposed to be one of the highest among the known catalytic antibodies. The data of association constants and glutathione peroxidase activities of these catalytic antibodies and the steady-state kinetics of Se-2F3 showed that the method might be a remarkably efficient one for generating catalytic antibodies with glutathione peroxidase activity.

A novel artificial glutathione peroxidase mimic consisting of a selenocystine-di-beta-cyclodextrin conjugate (selenium-bridged-6, 6'-amino-selenocystine-6,6'-deoxy-di-beta-cyclodextrin), in which selenocystine is bound to the primary side of beta-cyclodextrin through the two amino nitrogen groups of selenocystine, was synthesized. The glutathione peroxidase activities of the mimic-catalyzed reduction of H(2)O(2), tert-butylhydroperoxide, and cumene hydroperoxide by glutathione are 4.1, 2.11, and 5.82 units/micromol, respectively. The first activity was 82 and 4.2 times as much as that of selenocysteine and ebselen, respectively. Studies on the effect of substrate binding on the glutathione peroxidase activity suggest that it is important to consider substrate binding in designing glutathione peroxidase mimics. The detailed steady-state kinetic studies showed that the mimic-catalyzed reduction of H(2)O(2) by glutathione followed a ping-pong mechanism, which was similar to that of the native glutathione peroxidase.

Identification of secreted proteins of lung cancer could provide new candidates of serum biomarkers for cancer diagnosis or targets for therapeutic intervention. In this study, we developed a novel strategy that combined functional monoclonal antibody library screening technique and mass spectrometry to identify functional secreted proteins. BALB/c mice were immunized with cancer cells isolated from fresh human lung cancer tissues. The monoclonal antibody library containing 1160 mAbs was established with the mouse spleen cells, whose serum had most anti-proliferative effect on lung cancer cells. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with secreted proteins in condition medium and lung cancer tissues with high inhibotion of cell proliferation were selected. Antigens that recognized by antibodies were obtained by immunoprecipitation and then identified by mass spectrometry. Mac-2-binding protein (Mac-2BP), the antigen of 13H3 antibody, was identified using this approach. Functional studies demonstrated that the 13H3 antibody suppressed lung cancer cell lines ANIP-973 and A549 proliferation in vitro and inhibit ANIP973 xenograft tumors growth in vivo by inducing cell-cycle arrest at G1 phase, with up-regulation of p27 and down-regulation of cyclin D1. Moreover, the serum level of Mac-2BP was significantly higher in lung cancer patients than healthy controls. At a cutoff value of 6 μg/ml, Mac-2BP might be a diagnostic biomarker of lung cancer, especially for SCLC. Mac-2BP concentrations of 6 μg/ml or higher was associated with poor overall survival in univariate analysis, and was an independent predictor in the multivariate COX analysis. Together, these results firstly demonstrated that Mac-2BP can be used as a therapeutic target and potential biomarker for lung cancer. Our strategy is feasible, which may facilitate the identification of novel secreted biomarkers of lung cancer.

Two new cyclic pentapeptides (1 and 2) and the known blazein (3), ganodesterone (4), ergosterin (5), cerevisterol (6), 24-methylcholesta-4,6,8(14),22-tetraen-3-one (7), 5,8-epidioxyergosta-6,22-dien-3-ol (8), 16-α-d-mannopyranosyloxyisopimar-7-en-19-oic acid (9), and 16-hydroxy isopimar-7-en-19-oic acid (10) have been isolated from the crude extract of an endolichenic Xylaria sp. The structures of 1 and 2 were elucidated primarily by NMR and MS methods. The absolute configurations of 1 and 2 were assigned using Marfey's method on their acid hydrolysate. Compounds 1-10 were evaluated for activity against fungi and for synergistic antifungal activity. Compound 1 showed synergistic antifungal activity against Candida albicans SC5314 with 0.004 μg/mL ketoconazole.

The solventogenic bacterium Clostridium acetobutylicum is an important species of the Clostridium community. To develop a fundamental tool that is useful for biological studies of C. acetobutylicum, we established a high resolution proteome reference map for this species. We identified 1206 spots representing 564 different proteins by mass spectrometry, covering approximately 50% of major metabolic pathways. To better understand the relationship between butanol tolerance and butanol yield, we performed a comparative proteomic analysis between the wild type strain DSM 1731 and the mutant Rh8, which has higher butanol tolerance and higher butanol yield. Comparative proteomic analysis of two strains at acidogenic and solventogenic phases revealed 102 differentially expressed proteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, protein synthesis, nucleotide metabolism, transport, and others. Hierarchical clustering analysis revealed that over 70% of the 102 differentially expressed proteins in mutant Rh8 were either upregulated (e.g., chaperones and solvent formation related) or downregulated (e.g., amino acid metabolism and protein synthesis related) in both acidogenic and solventogenic phase, which, respectively, are only upregulated or downregulated in solventogenic phase in the wild type strain. This suggests that Rh8 cells have evolved a mechanism to prepare themselves for butanol challenge before butanol is produced, leading to an increased butanol yield. This is the first report on the comparative proteome analysis of a mutant strain and a base strain of C. acetobutylicum. The fundamental proteomic data and analyses will be useful for further elucidating the biological mechanism of butanol tolerance and/or enhanced butanol production.

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak ( Bos grunniens ) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.

We attempted to identify membrane proteins associated with the glycoconjugates and cell wall biosynthesis in the total membrane preparations of Aspergillus fumigatus. The total membrane preparations were first run on 1D gels, and then the stained gels were cut and submitted to in-gel digestion followed by 2D LC-MS/MS and database search. A total of 530 proteins were identified with at least two peptides detected with MS/MS spectra. Seventeen integral membrane proteins were involved in N-, O-glycosylation or GPI anchor biosynthesis. Nine membrane proteins were involved in cell wall biosynthesis. Eight proteins were identified as enzymes involved in sphingolipid synthesis. In addition, the proteins involved in cell wall and ergosterol biosynthesis can potentially be used as antifungal drug targets. Our method, for the first time, clearly provided a global view of the membrane proteins associated with glycoconjugates and cell wall biosynthesis in the total membrane proteome of A. fumigatus.

α-Glucosidase I regulates trimming of the terminal α-1,2-glucose residue in the N-glycan processing pathway, which plays an important role in quality control systems in mammalian cells. Previously, we identified the gene encoding α-glucosidase I in the opportunistic human fungal pathogen Aspergillus fumigatus, namely Afcwh41. Deletion of the Afcwh41 gene results in a severe reduction of conidia formation, a temperature-sensitive deficiency of cell wall integrity, and abnormalities of polar growth and septation. An upregulation of the genes encoding Rho-type GTPases was also observed, which suggests activation of the cell wall integrity pathway in the mutant. Using 2D gel analysis, we revealed that the proteins involved in protein assembly, ubiquitin-mediated degradation and actin organization are altered in the ΔAfcwh41 mutant. Evidence was obtained for a defect in the polarized localization of the actin cytoskeleton in the mutant. Our results suggest that blocking of the glucose trimming in A. fumigatus might induce accumulation of misfolded proteins in the endoplasmic reticulum; these misfolded proteins are probably required for cell wall synthesis and thus activate the cell wall integrity pathway, which then causes the abnormal polarity associated with the ΔAfcwh41 mutant.

Four extracellular keratinases (designated KI, KII, KIII, and KIV) were produced during submerged aerobic cultivation in a medium containing native human foot skin (NHFS) for enzyme synthesis. The molecular weights, determined by SDS-PAGE, were 25, 50, 34, and 19 kDa, respectively. Gel filtration of the four purified enzymes in native conditions indicated that active keratinase KI is a novel homo-octamer, KII a homo-dimer, and KIII and KIV monomers. All four keratinases exhibited high activities at pH 8.0-10.0 with an optimal pH of 9.0. The optimal temperature for keratinolytic activity of KI, KII, and KIII was approximately 50, and 60 degrees C for KIV. One millimolar of PMSF completely inhibited the keratinolytic activities of the four enzymes. The N-terminal sequences of KI, KII, and KIII showed that they were different from previously described enzymes, whereas KIV shared an identical N-terminal sequence with two other peptidases from Streptomyces.

Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

In yeast, glycosylphosphatidylinositol (GPI) is essential for viability and plays an important role in biosynthesis and organization of cell wall. Initiation of the GPI anchor biosynthesis is catalysed by the GPI-N-acetylglucosaminyltransferase complex (GPI-GnT). The GPI3 (SPT14) gene is thought to encode the catalytic subunit of GPI-GnT complex. In contrast to Saccharomyces cerevisiae, little is known about the GPI biosynthesis in filamentous fungi. In this study, the afpig-a gene was identified as the homologue of the GPI3/pig-A gene in Aspergillus fumigatus, an opportunistic fungal pathogen. By replacement of the afpig-a gene with a pyrG gene, we obtained the null mutants. Although the Deltaafpig-a mutant exhibited a significant increased cell lysis instead of temperature-sensitive or conditional lethal phenotype associated to the GPI3 mutant of yeast, they could survive at temperatures from 30 degrees C to 50 degrees C. The analysis of the mutants showed that a completely blocking of the GPI anchor synthesis in A. fumigatus led to cell wall defect, abnormal hyphal growth, rapid conidial germination and aberrant conidiation. In vivo assays revealed that the mutant exhibited a reduced virulence in immunocompromised mice. The GPI anchor was not essential for viability, but required for the cell wall integrity, morphogenesis and virulence in A. fumigatus.

The solventogenic bacterium Clostridium acetobutylicum is the most important species of Clostridium used in the fermentation industry. However, the intolerance to butanol hampers the efficient production of solvents. Butanol toxicity has been attributed to the chaotropic effect on the cell membrane, but the knowledge on the effect of butanol on membrane associated proteins is quite limited. Using 2-DE combined with MALDI-TOF MS/MS and 1-DE integrated with LC-MS/MS, 341 proteins in the membrane fractions of cell lysate were identified, thus establishing the first comprehensive membrane proteome of C. acetobutylicum. The identified proteins are mainly involved in transport, cellular membrane/wall machinery, formation of surface coat and flagella, and energy metabolism. Comparative analysis on the membrane proteomes of the wild type strain DSM 1731 and its butanol-tolerant mutant Rh8 revealed 73 differentially expressed proteins. Hierarchical clustering analysis suggested that mutant Rh8 may have evolved a more stabilized membrane structure, and have developed a cost-efficient energy metabolism strategy, to cope with the butanol challenge. This comparative membrane proteomics study, together with our previous published work on comparative cytoplasmic proteomics, allows us to obtain a systemic understanding of the effect of butanol on cellular physiology of C. acetobutylicum.

Background: Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology. Results: A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation. Conclusion: Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes. Background Clostridium thermocellum is a Gram-positive thermophilic anaerobe capable of degrading cellulose and producing ethanol and hydrogen. These qualities render C. thermocellum potentially useful for the production of biofuel from biomass. The cellulytic activities of this organism were well studied, the corresponding enzymes were found to organize into a cell surfaced bound multienzyme complex, termed cellulosome [1]. The arrangement of the enzymatic subunits in the cellulosome complex, made possible by a scaffoldin subunit, promotes enhanced substrate binding and degradation. However, other parts of its cellular functions are not well understood. Recently, a genome scale metabolic model was constructed [2], which provides a good basis for the overall understanding of its metabolism. Since membrane is where many important physiological functions, such as energy generation, protein trafficking, and small molecule transport [3], take place, we focused on membrane protein complexes as a start point to identify unique features of C. thermocellum. Identification of protein complexes in C. thermocellum is an important step toward understanding cellular behavior at an integrative level.