Archaeal MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain.

MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix-turn-helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1-ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process.

Arginine methylation of REF/ALY promotes efficient handover of mRNA to TAP/NXF1.

The REF/ALY mRNA export adaptor binds TAP/NXF1 via an arginine-rich region, which overlaps with its RNA-binding domain. When TAP binds a REF:RNA complex, it triggers transfer of the RNA from REF to TAP. Here, we have examined the effects of arginine methylation on the activities of the REF protein in mRNA export. We have mapped the arginine methylation sites of REF using mass spectrometry and find that several arginines within the TAP and RNA binding domains are methylated in vivo. However, arginine methylation has no effect on the REF:TAP interaction. Instead, arginine methylation reduces the RNA-binding activity of REF in vitro and in vivo. The reduced RNA-binding activity of REF in its methylated state is essential for efficient displacement of RNA from REF by TAP in vivo. Therefore, arginine methylation fine-tunes the RNA-binding activity of REF such that the RNA-protein interaction can be readily disrupted by export factors further down the pathway.

Shotgun proteome analysis utilising mixed mode (reversed phase-anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis.

The 2-D peptide separations employing mixed mode reversed phase anion exchange (MM (RP-AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP-AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP-AX) compared with the traditional SCX hyphenated to RP-LC-MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP-AX) for 2-D LC proteomic studies was also extended in the analysis of iTRAQ-labelled HeLa and cyanobacterial proteomes using nano-flow chromatography interfaced to the MS/MS. We demonstrate MM (RP-AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.

Structure of the ribosome associating GTPase HflX.

The HflX-family is a widely distributed but poorly characterized family of translation factor-related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0-A resolution in apo- and GDP-bound forms. The enzyme displays a two-domain architecture with a novel "HflX domain" at the N-terminus, and a classical G-domain at the C-terminus. The HflX domain is composed of a four-stranded parallel beta-sheet flanked by two alpha-helices on either side, and an anti-parallel coiled coil of two long alpha-helices that lead to the G-domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX-domain upon GTP-binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24-fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX-domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown.

A cross-species quantitative proteomic study of salt adaptation in a halotolerant environmental isolate using 15N metabolic labelling.

We examine differential protein expression in Euhalothece sp. BAA001, an extremely halotolerant and unsequenced cyanobacterium, under adaptation to low (0% w/v), medium (3% w/v), high (6% w/v) and very high (9% w/v) salt concentrations using cross-species protein identification tools. We combine stable isotope labelling with 1-D SDS-PAGE, and MASCOT protein identification software with MS-driven BLAST searches, to produce an accurate method for protein identification and quantitation. The use of metabolic labelling to improve the confidence in identification of proteins in cross-species proteomics is demonstrated. Three hundred and eighty-three unique proteins were identified, and 72 were deemed to be differentially expressed (average CV for quantitations was 0.10 +/- 0.08), belonging to 24 functional groups. Responses to low salt as well as high salt are discussed in terms of adaptation and evidence shows that Euhalothece cells display 'stress' responses in nonsaline conditions as well as higher salt environments.

A systematic evaluation of chip-based nanoelectrospray parameters for rapid identification of proteins from a complex mixture.

HPLC-MS/MS is widely used for protein identification from gel spots and shotgun fractions. Although HPLC has well recognized benefits, this type of sample infusion also has some undesirable attributes: relatively low sample throughput, potential sample-to-sample carryover, time-varying sample composition, and no option for longer sample infusion for longer MS analyses. An automated chip-based ESI device (CB-ESI) has the potential to overcome these limitations. This report describes a systematic evaluation of the information-dependant acquisition (IDA) and sample preparation protocols for rapid protein identification from a complex mixture using a CB-ESI source compared with HPLC-ESI (gradient and isocratic elutions). Cytochrome c and a six-protein mixture (11-117 kDa) were used to develop an IDA protocol for rapid protein identification and to evaluate the effects of sample preparation protocols. MS (1-10 s) and MS/MS (1-60 s) scan times, sample concentration (50-500 fmol/microL), and ZipTipC(18) cleanup were evaluated. Based on MOWSE scores, protein coverage, experimental run time, number of identified proteins, and reproducibility, a 12.5 min experiment (22 cycles, each with one 3 s MS and eight 10 s MS/MS scans) was determined to be the optimal IDA protocol for CB-ESI. This work flow yielded up to 220% greater peptide coverage compared with gradient HPLC-ESI and provided protein identifications with up to a 2-fold higher throughput rate than either HPLC-ESI approach, whilst employing half the amount of sample over the same time frame. The results from this study support the use of CB-ESI as a rapid alternative to the identification of protein mixtures.

Structural basis for CRISPR RNA-guided DNA recognition by Cascade.

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry.

Arginine and lysine methylation are widespread protein post-translational modifications. Peptides containing these modifications are difficult to retain using traditional reversed-phase liquid chromatography because they are intrinsically basic/hydrophilic and often fragment poorly during collision induced fragmentation (CID). Therefore, they are difficult to analyze using standard proteomic workflows. To overcome these caveats, we performed peptide separations at neutral pH, resulting in increased retention of the hydrophilic/basic methylated peptides before identification using MS/MS. Alternatively trifluoroacetic acid (TFA) was used for increased trapping of methylated peptides. Electron-transfer dissociation (ETD) mass spectrometry was then used to identify and characterize methylated residues. In contrast to previous reports utilizing ETD for arginine methylation, we observed significant amount of side-chain fragmentation. Using heavy methyl stable isotope labeling with amino acids in cell culture it was shown that, similar to CID, a loss of monomethylamine or dimethylamine from the arginine methylated side-chain during ETD can be used as a diagnostic to determine the type of arginine methylation. CID of lysine methylated peptides does not lead to significant neutral losses, but ETD is still beneficial because of the high charge states of such peptides. The developed LC MS/MS methods were successfully applied to tryptic digests of a number of methylated proteins, including splicing factor proline-glutamine-rich protein (SFPQ), RNA and export factor-binding protein 2 (REF2-I) and Sul7D, demonstrating significant advantages over traditional LC MS/MS approaches.

UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

Identification and characterization of sulfolobus solfataricus P2 proteome using multidimensional liquid phase protein separations.

We have identified and characterized the proteome of Sulfolobus solfataricus P2 using multidimensional liquid phase protein separations. Multidimensional liquid phase chromatography was performed using ion exchange chromatography in the first dimension, followed by reverse-phase chromatography using 500 microm i.d. poly(styrene-divinylbenzene) monoliths in the second dimension to separate soluble protein lysates from S. solfataricus. The 2DLC protein separations from S. solfataricus protein lysates enabled the generation of a 2D liquid phase map analogous to the traditional 2DE map. Following separation of the proteins in the second dimension, fractions were collected, digested in solution using trypsin and analyzed using mass spectrometry. These approaches offer significant reductions in labor intensity and the overall time taken to analyze the proteome in comparison to 2DE, taking advantage of automation and fraction collection associated with this approach. Furthermore, following proteomic analysis using 2DLC, the data obtained was compared to previous 2DE and shotgun proteomic studies of a soluble protein lysate from S. solfataricus. In comparison to 2DE, the results show an overall increase in proteome coverage. Moreover, 2DLC showed increased coverage of a number of protein subsets including acidic, basic, low abundance and small molecular weight proteins in comparison to 2DE. In comparison to shotgun studies, an increase in proteome coverage was also observed. Furthermore, 187 unique proteins were identified using 2DLC, demonstrating this methodology as an alternative approach for proteomic studies or in combination with 2DE and shotgun workflows for global proteomics.

Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry.

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.

Small CRISPR RNAs guide antiviral defense in prokaryotes.

Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.

Relative quantification of proteins across the species boundary through the use of shared peptides.

We show that shared peptides of proteins that are encoded in different species are suitable for cross-species relative protein quantification. A 14N-containing proteome from the thermoacidophilic archaeon Sulfolobus tokodaii was mixed with a 15N-labeled proteome from Sulfolobus solfataricus. Using three shared peptides per protein, the relative abundance of six orthologous proteins was calculated. Observed standard deviations were approximately 10%, indicating that the trypsin accessibility to cleavage sites was not altered in the orthologs. The abundance ratios of the and subunits of the Thermosome were 0.64 and 1.24 in Sulfolobus tokodaii compared to Sulfolobus solfataricus, suggesting a different stoichiometry of the complex in both species. In addition, an in silico study was performed on the occurrence of shared peptides. Inter- and intra-species peptide redundancy was investigated in the model organisms Homo sapiens, Mus musculus, Escherichia coli K12, Escherichia coli O157:H7, S. solfataricus, and S. tokodaii. M. musculus and H. sapiens share 30-50% of all peptides (6-15 residues). Moreover, approximately one-third of all proteins shared > or = 40% of their peptides with at least one other protein in the related species, thus offering strong potential for cross-species relative protein quantification. Conversely, approximately 40% of all peptides (6-15 residues) encoded in H. sapiens are encoded multiple times and therefore complicate identification and quantification.

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics.

A method for quantitative protein profiling has been developed utilising multidimensional liquid phase protein separations in conjunction with stable isotope labelling. This approach combines the advantages of high throughput, automated, reproducible protein separations with accurate protein quantitation performed in the mass spectrometer. Escherichia coli cells were grown in the presence and absence of the DNA methylation inhibitor 5-Azacytidine on 14N and 15N enriched media. Protein separations were performed using ion exchange chromatography in the first dimension and RP capillary chromatography in the second dimension. UV absorbance measurements were used for the initial semiquantitative identification of differentially expressed proteins. Selected peaks from the mixed 15N/14N lysates were used for the accurate quantitation performed in the mass spectrometer using the ratios of the stable isotopes. Using this approach, a number of differentially expressed proteins have been identified. Moreover, this approach overcomes a number of caveats associated with multidimensional liquid phase protein separations, including the presence of multiple proteins present in a single chromatographic peak.

Modulation of the oviductal environment by gametes.

The notion of a gamete recognition system that alerts females to the presence of gametes in their reproductive tract profoundly influences our understanding of the physiology of events leading to conception and the bearing of offspring. Here, we show that the female responds to gametes within her tract by modulating the environment in which pregnancy is initially established. We found distinct alterations in oviductal gene expression as a result of sperm and oocyte arrival in the oviduct, which led directly to distinct alterations to the composition of oviductal fluid in vivo. This suggests that either gamete activates a cell-type-specific signal transduction pathway within the oviduct. This gamete recognition system presents a mechanism for immediate and local control of the oviductal microenvironment in which sperm transport, sperm binding and release, capacitation, transport of oocytes, fertilization, and early cleavage-stage embryonic development occur. This may explain the mechanisms involved in postcopulatory sexual selection, where there is evidence suggesting that the female reproductive tract can bias spermatozoa from different males in the favour of the more biologically attractive male. In addition, the presence of a gamete recognition system explains the oviduct's ability to tolerate spermatozoa while remaining intolerant to pathogens.

Shotgun proteomics of cyanobacteria--applications of experimental and data-mining techniques.

Cyanobacteria are photosynthetic bacteria notable for their ability to produce hydrogen and a variety of interesting secondary metabolites. As a result of the growing number of completed cyanobacterial genome projects, the development of post-genomics analysis for this important group has been accelerating. DNA microarrays and classical two-dimensional gel electrophoresis (2DE) were the first technologies applied in such analyses. In many other systems, 'shotgun' proteomics employing multi-dimensional liquid chromatography and tandem mass spectrometry has proven to be a powerful tool. However, this approach has been relatively under-utilized in cyanobacteria. This study assesses progress in cyanobacterial shotgun proteomics to date, and adds a new perspective by developing a protocol for the shotgun proteomic analysis of the filamentous cyanobacterium Anabaena variabilis ATCC 29413, a model for N(2) fixation. Using approaches for enhanced protein extraction, 646 proteins were identified, which is more than double the previous results obtained using 2DE. Notably, the improved extraction method and shotgun approach resulted in a significantly higher representation of basic and hydrophobic proteins. The use of protein bioinformatics tools to further mine these shotgun data is illustrated through the application of PSORTb for localization, the grand average hydropathy (GRAVY) index for hydrophobicity, LipoP for lipoproteins and the exponentially modified protein abundance index (emPAI) for abundance. The results are compared with the most well-studied cyanobacterium, Synechocystis sp. PCC 6803. Some general issues in shotgun proteome identification and quantification are then addressed.

Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable alpha-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for alpha-linked galactosides, which are optimally hydrolyzed at pH 5 and 90 degrees C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable alpha-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.

Reconstruction of central carbon metabolism in Sulfolobus solfataricus using a two-dimensional gel electrophoresis map, stable isotope labelling and DNA microarray analysis.

In the last decade, an increasing number of sequenced archaeal genomes have become available, opening up the possibility for functional genomic analyses. Here, we reconstructed the central carbon metabolism in the hyperthermophilic crenarchaeon Sulfolobus solfataricus (glycolysis, gluconeogenesis and tricarboxylic acid cycle) on the basis of genomic, proteomic, transcriptomic and biochemical data. A 2-DE reference map of S. solfataricus grown on glucose, consisting of 325 unique ORFs in 255 protein spots, was created to facilitate this study. The map was then used for a differential expression study based on (15)N metabolic labelling (yeast extract + tryptone-grown cells (YT) vs. glucose-grown cells (G)). In addition, the expression ratio of the genes involved in carbon metabolism was studied using DNA microarrays. Surprisingly, only 3 and 14% of the genes and proteins, respectively, involved in central carbon metabolism showed a greater than two-fold change in expression level. All results are discussed in the light of the current understanding of central carbon metabolism in S. solfataricus and will help to obtain a system-wide understanding of this organism.

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

Perturbation and interpretation of nitrogen isotope distribution patterns in proteomics.

This study provides a discussion on the applications and limitations of (15)NH(4)(+) metabolic labeling in proteomic studies. The hyperthemophilic crenarchaeon Sulfolobus solfataricus was used as a model organism throughout this study. The distribution of nitrogen was studied in four different experiments in which this distribution was manipulated in a unique way. The experiments included full adaptation to media with relative isotope abundances (RIA) of 0.36%, 50%, and >98% (15)NH(4)(+). The incorporation efficiency was calculated on the basis of a comparison between theoretical and experimental spectra. In the case of full adaptation, incorporation efficiencies reflected the RIA (0.36%, 47.5% and 99% respectively). Labeling efficiencies were calculated on the basis of peak areas in TOF-MS spectra. It is shown that in the case of full adaptation, labeling efficiencies are 100%. In addition, we demonstrate that (15)NH(4)(+) labeling can be used in protein turnover studies, even when labeling is incomplete. In this case, incorporation efficiencies of 88-93% (lower than the RIA) were measured, providing evidence for amino acid recycling. Labeling efficiencies were always between 63% and 94% providing evidence for protein degradation. Finally, it was shown that isotope distributions can be useful in peptide identification.