Posttranslational modification of Elongation Factor P from Staphylococcus aureus.

Antibiotic-resistant Staphylococcus aureus is becoming a major burden on health care systems in many countries, necessitating the identification of new targets for antibiotic development. Elongation Factor P (EF-P) is a highly conserved elongation protein factor that plays an important role in protein synthesis and bacteria virulence. EF-P undergoes unique posttranslational modifications in a stepwise manner to function correctly, but experimental information on EF-P posttranslational modifications is currently lacking for S. aureus. Here, we expressed EF-P in S. aureus to analyze its posttranslational modifications by mass spectrometry and report experimental proof of 5-aminopentanol modification of S. aureus EF-P.

Expression, purification, crystallization and preliminary crystallographic study of the cytoplasmic domain of the mitochondrial dynamics protein MiD51.

Mitochondria play central roles in many cellular and physiological processes. They are highly dynamic organelles and continually undergo fusion and fission. Mitochondrial dynamics protein 51 kDa (MiD51), an integral mitochondrial outer membrane protein, recruits dynamin-related protein 1 (Drp1; a mitochondrial fission protein) to mitochondria and facilitates Drp1-directed mitochondrial fission. In this study, the cytoplasmic domain of MiD51 was overexpressed in Escherichia coli, purified and crystallized. An X-ray diffraction data set was collected to a resolution of 3.1 Å and the crystal belonged to space group P41212, with unit-cell parameters a = b = 90.1, c = 124.7 Å, α = ß = γ = 90°. The asymmetric unit had the highest probability of containing one molecule, with a Matthews coefficient of 3.32 Å(3) Da(-1) and a solvent content of 63.0%.

Cloning and characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa.

We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 µM, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) µM(-1). In the presence of EF-Ts, these values were shifted to K M = 2 µM, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) µM(-1). The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30-75 nM and to GTP (K GTP) were 125-200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNA(Phe) at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

Noninvasive detection of HER2 amplification with plasma DNA digital PCR.

PURPOSE: Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number. EXPERIMENTAL DESIGN: The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort. RESULTS: In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86-0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2-positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2-negative, with a positive and negative predictive value of 70% and 92%, respectively. CONCLUSION: Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer.

Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli.

The C5aR antagonist (C5aRA)(1), which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli. To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37°C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496mg/L of NusA-fused C5aRA could be produced.

Rpn13p and Rpn14p are involved in the recognition of ubiquitinated Gcn4p by the 26S proteasome.

The 26S proteasome, composed of the 20S core and 19S regulatory complexes, is important for the turnover of polyubiquitinated proteins. Each subunit of the complex plays a special role in proteolytic function, including substrate recruitment, deubiquitination, and structural contribution. To assess the function of some non-essential subunits in the 26S proteasome, we isolated the 26S proteasome from deletion strains of RPN13 and RPN14 using TAP affinity purification. The stability of Gcn4p and the accumulation of ubiquitinated Gcn4p were significantly increased, but the affinity in the recognition of proteasome was decreased. In addition, the subcomplexes of the isolated 26S proteasomes from deletion mutants were less stable than that of the wild type. Taken together, our findings indicate that Rpn13p and Rpn14p are involved in the efficient recognition of 26S proteasome for the proteolysis of ubiquitinated Gcn4p.

Purification of components of the translation elongation factor complex of Plasmodium falciparum by tandem affinity purification.

Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1beta harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1beta-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1beta, the G-protein PfEF-1alpha, and two new proteins that we named PfEF-1gamma and PfEF-1delta based on their homology to other eukaryotic gamma and delta translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1beta. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity.

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.

Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA.

In bacteria, the selenocysteine-specific elongation factor SelB is necessary for incorporation of selenocysteine, the 21st amino acid, into proteins by the ribosome. SelB binds to an mRNA hairpin formed by the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA (Sec-tRNASec) at the ribosomal A site. The minimum fragment (residues 512-634) of Moorella thermoacetica SelB (SelB-M) required for mRNA binding has been overexpressed and purified. The complex of SelB-M with 23 nucleotides of the SECIS mRNA hairpin was crystallized at 293 K using the hanging-drop vapour-diffusion or oil-batch methods. The crystals diffract to 2.3 A resolution using SPring-8 BL41XU and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 81.69, b = 169.58, c = 71.69 A.

Purification and crystallization of the yeast translation elongation factor eEF3.

A Saccharomyces cerevisiae strain expressing full-length histidine-tagged translation elongation factor 3 (eEF3) as the only form of the protein facilitated purification of the factor for both structural and functional studies. Additionally, an identical full-length form has been successfully expressed in Escherichia coli and a C-terminally truncated form of histidine-tagged eEF3 has been successfully expressed in E. coli and S. cerevisiae. Both forms have been crystallized and crystals of the truncated protein expressed in yeast diffract synchrotron radiation to a maximum resolution of 2.3 A. A density-modified map derived from low-resolution SIRAS phases allows model building.

Isolation and characterization of Candida albicans homologue of RAP1, a repressor and activator protein gene in Saccharomyces cerevisiae.

To study the function of RAP1, a Candida albicans gene (CaRAP1) that shows sequence similarity to RAP1 of Saccharomyces cerevisiae was isolated by colony hybridization. DNA sequencing predicted an open reading frame of 429 amino acids with an overall identity of 24% to the ScRap1p. The DNA binding domain (DBD) was highly conserved, and EMSA using a GST-CaRap1p fusion protein confirmed its binding ability to the RPG-box of S. cerevisiae ENO1. In contrast, the N-terminus was less conserved and a moderate homology was observed in the BRCT domain. Interestingly, CaRap1p did not contain the C-terminal activation/repression region of ScRap1p.

Purification and characterization of hexahistidine-tagged elongation factor SelB.

The cotranslational incorporation of selenocysteine into proteins is mediated by a specialized elongation factor, named SelB. Its amino-terminal three domains show homology to elongation factor EF-Tu and accordingly bind GTP and selenocysteyl-tRNASec. In addition, SelB exhibits a long carboxy-terminal extension that interacts with a secondary structure of selenoprotein mRNAs (SECIS element) positioned immediately downstream of the in-frame UGA codons specifying the sites of selenocysteine insertion. In this report, a fast and efficient method for the purification of large amounts of hexahistidine-tagged SelB is presented. After two chromatographic steps, 10 mg pure protein was isolated from 12 g wet cell pellet. Biochemical analysis of the purified protein showed that the tag does not influence the interaction of SelB with guanine nucleotides, SECIS elements, and selenocysteyl-tRNASec. In addition, the fusion protein is fully functional in mediating UGA read-through in vivo. It therefore represents an excellent model for studying the function of SelB and the mechanisms of selenocysteine incorporation.

Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu.

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.

Isolation, crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes.

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.

Preparation of a crystallizable mRNA-binding fragment of Moorella thermoacetica elongation factor SelB.

SelB is a bacterial elongation factor required for the decoding of a UGA stop codon together with a specific mRNA hairpin to selenocysteine. In attempts to crystallize Moorella thermoacetica SelB, a proteolysis process occurred and crystals of a proteolytic fragment were observed. The crystals, which appeared after a year, contained a C-terminal 30 kDa fragment containing the mRNA-binding domain. This fragment was reproduced through recloning. Crystals diffracting to 2.7 A were obtained.

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach.

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions.

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a approximately 150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.

Differential protein expression in phenotypic variants of Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, whereas the opaque variants show increased virulence during systemic infections. To gain insight into the pathogenesis of pneumococcal disease at the molecular level, protein expression patterns of the phenotypic variants of two pneumococcal strains were compared by high-resolution two-dimensional protein electrophoresis. In comparison with transparent variants, the opaque variants reduced the expression of two proteins and overexpressed one protein. The proteins were identified by mass spectrometric analysis. The protein overexpressed in the opaque phenotype revealed significant homology to elongation factor Ts of Helicobacter pylori. One of the two proteins that were underexpressed in the opaque variants revealed significant homology to the proteinase maturation protein PrtM of Lactocobacillus paracasei, a member of the family of peptidyl-prolyl cis/trans isomerases. A consensus lipoprotein signal sequence suggests that the putative proteinase maturation protein A, designated PpmA, is located at the surface of the pneumococcus and may play a role in the maturation of surface or secreted proteins. The second underexpressed protein was identified as pyruvate oxidase, SpxB. The lower SpxB expression in opaque variants most probably explains the reduced production of hydrogen peroxide, a reaction product of SpxB, in this variant. Since a spxB-defective pneumococcal mutant has decreased ability to colonize the nasopharynx (B. Spellerberg, D. R. Cundell, J. Sandros, B. J. Pearce, I. Idanpaan-Heikkila, C. Rosenow, and H. R. Masure, 1996. Mol. Microbiol. 19:803-813, 1996), our data suggest that SpxB plays an important role in enhancing the ability of transparent variants to efficiently colonize the nasopharynx.

A protein phosphatase functions to recycle RNA polymerase II.

Transcription is regulated by the state of phosphorylation of a heptapeptide repeat known as the carboxy-terminal domain (CTD) present in the largest subunit of RNA polymerase II (RNAPII). RNAPII that associates with transcription initiation complexes contains an unphosphorylated CTD, whereas the elongating polymerase has a phosphorylated CTD. Transcription factor IIH has a kinase activity specific for the CTD that is stimulated by the formation of a transcription initiation complex. Here, we report the isolation of a cDNA clone encoding a 150-kD polypeptide, which, together with RNAPII, reconstitutes a highly specific CTD phosphatase activity. Functional analysis demonstrates that the CTD phosphatase allows recycling of RNAPII. The phosphatase dephosphorylates the CTD allowing efficient incorporation of RNAPII into transcription initiation complexes, which results in increased transcription. The CTD phosphatase was found to be active in ternary elongation complexes. Moreover, the phosphatase stimulates elongation by RNAPII; however, this function is independent of its catalytic activity.