Robin Wait

A modified silver staining protocol for visualization of proteins compatible with matrix‐assisted laser desorption/ionization and electrospray ionization‐ mass spectrometry

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two‐dimensional gel electrophoresis (2‐D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2‐D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix‐assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: Implications for autoimmunity in rheumatoid arthritis

OBJECTIVE To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA). METHODS The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and α-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry. RESULTS Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or α-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry. CONCLUSION Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis-mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.

Identification of citrullinated alpha-enolase as a candidate autoantigen in rheumatoid arthritis.

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.

Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with γ-32P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule–associated phosphoproteins after incubation of intact amyloplasts with γ-32P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein–protein interactions in the control of starch anabolism and catabolism.

Antibodies to citrullinated α‐enolase peptide 1 are specific for rheumatoid arthritis and cross‐react with bacterial enolase

OBJECTIVE To map the antibody response to human citrullinated alpha-enolase, a candidate autoantigen in rheumatoid arthritis (RA), and to examine cross-reactivity with bacterial enolase. METHODS Serum samples obtained from patients with RA, disease control subjects, and healthy control subjects were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with citrullinated alpha-enolase peptides. Antibodies specific for the immunodominant epitope were raised in rabbits or were purified from RA sera. Cross-reactivity with other citrullinated epitopes was investigated by inhibition ELISAs, and cross-reactivity with bacterial enolase was investigated by immunoblotting. RESULTS An immunodominant peptide, citrullinated alpha-enolase peptide 1, was identified. Antibodies to this epitope were observed in 37-62% of sera obtained from patients with RA, 3% of sera obtained from disease control subjects, and 2% of sera obtained from healthy control subjects. Binding was inhibited with homologous peptide but not with the arginine-containing control peptide or with 4 citrullinated peptides from elsewhere on the molecule, indicating that antibody binding was dependent on both citrulline and flanking amino acids. The immunodominant peptide showed 82% homology with enolase from Porphyromonas gingivalis, and the levels of antibodies to citrullinated alpha-enolase peptide 1 correlated with the levels of antibodies to the bacterial peptide (r2=0.803, P<0.0001). Affinity-purified antibodies to the human peptide cross-reacted with citrullinated recombinant P gingivalis enolase. CONCLUSION We have identified an immunodominant epitope in citrullinated alpha-enolase, to which antibodies are specific for RA. Our data on sequence similarity and cross-reactivity with bacterial enolase may indicate a role for bacterial infection, particularly with P gingivalis, in priming autoimmunity in a subset of patients with RA.

The 3′ Untranslated Region of Tumor Necrosis Factor Alpha mRNA Is a Target of the mRNA-Stabilizing Factor HuR

ABSTRACT Posttranscriptional regulation is important for tumor necrosis factor alpha (TNF-α) expression in monocytes and macrophages, and an AU-rich element (ARE) in the 3′ untranslated region (UTR) of TNF-α mRNA is implicated in control of its translation and mRNA stability. Regulation of mRNA turnover is thought to be mediated bytrans-acting proteins, which bind the ARE and stabilize or destabilize the transcript. However, with the exception of the destabilizing factor tristetraprolin, the identity and function of the proteins binding the TNF-α mRNA ARE have not been established. To identify other proteins involved in the posttranscriptional control of TNF-α, the subcellular location of TNF-α mRNA was determined in the macrophage-like cell line RAW 264.7. TNF-α mRNA was located in the pellet following centrifugation of cytoplasm at 100,000 ×g (P100 fraction). This fraction also contained proteins which formed two distinct ARE-specific complexes with the TNF-α mRNA 3′ UTR in electrophoretic mobility shift assays (EMSAs). A protein present in these two complexes was purified and identified by peptide mass mapping and tandem mass spectrometry as HuR. In EMSAs both complexes were supershifted by an anti-HuR antibody, while Western blotting also demonstrated the presence of HuR in the P100 extract. A HeLa cell tetracycline-regulated reporter system was used to determine the effect of HuR on mRNA stability. In this system, overexpression of HuR resulted in stabilization of an otherwise unstable reporter-mRNA containing the TNF-α ARE. These results demonstrate that the TNF-α ARE is a target of the mRNA-stabilizing factor HuR.

The estrogen receptor-α-induced microRNA signature regulates itself and its transcriptional response

Following estrogenic activation, the estrogen receptor-α (ERα) directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) modulated by ERα have the potential to fine tune these regulatory systems and also provide an alternate mechanism that could impact on estrogen-dependent developmental and pathological systems. Through a microarray approach, we identify the subset of microRNAs (miRNAs) modulated by ERα, which include upregulation of miRNAs derived from the processing of the paralogous primary transcripts (pri-) mir-17–92 and mir-106a-363. Characterization of the mir-17–92 locus confirms that the ERα target protein c-MYC binds its promoter in an estrogen-dependent manner. We observe that levels of pri-mir-17–92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17–92 is immediately cleaved by DROSHA to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursor. The clinical implications of this novel regulatory system were confirmed by demonstrating that pre-miR-18a was significantly upregulated in ERα-positive compared to ERα-negative breast cancers. Mechanistically, miRNAs derived from these paralogous pri-miRNAs (miR-18a, miR-19b, and miR-20b) target and downregulate ERα, while a subset of pri-miRNA-derived miRNAs inhibit protein translation of the ERα transcriptional p160 coactivator, AIB1. Therefore, different subsets of miRNAs identified act as part of a negative autoregulatory feedback loop. We propose that ERα, c-MYC, and miRNA transcriptional programs invoke a sophisticated network of interactions able to provide the wide range of coordinated cellular responses to estrogen.

Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs

Strains of Deinococcus geothermalis sp. nov. were isolated from the hot spring and runoff at Agnano, Naples, Italy, and from the hot spring at São Pedro do Sul in central Portugal, while strains of Deinococcus murrayi sp. nov. were isolated from the hot springs at São Pedro do Sul, São Gemil, and Alcafache in central Portugal. The strains of D. geothermalis and D. murrayi produce orange-pigmented colonies and have an optimum growth temperature of about 45 to 50 degrees C. The type strains of the two new species are extremely gamma radiation resistant. The fatty acids of these new species are primarily branched-chain fatty acids. The two new species can be distinguished from each other by the lower pH range of D. geothermalis than of D. murrayi, by their fatty acid compositions, and by several biochemical parameters, including the ability of D. geothermalis to grow in minimal medium without yeast extract. 16S rRNA gene sequencing also showed that the isolates constitute two species and that these species are distinct from the other species of the genus Deinococcus. The type strain of D. geothermalis is AG-3a (= DSM 11300), and the type strain of D. murrayi is ALT-1b (= DSM 11303).

Synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis.

OBJECTIVE To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated alpha-enolase, in rheumatoid arthritis (RA). METHODS Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, alpha-enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated alpha-enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay. RESULTS Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-enolase was detected in all of the samples, with mean levels of 6.4 ng/microl in RA samples, 4.3 ng/microl in SpA samples, and <0.9 ng/microl in OA samples. Two-dimensional electrophoresis provided evidence that the alpha-enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample. CONCLUSION These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.

Analysis of Protein Complexes in Wheat Amyloplasts Reveals Functional Interactions among Starch Biosynthetic Enzymes

Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with γ-32P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis.