Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling

We describe an improved Phos-tag SDS-PAGE (Zn(2+)-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a Bis-tris-buffered neutral-pH gel system to detect shifts in the mobility of phosphoproteins. An existing technique (Mn(2+)-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn(2+)-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant Tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole-cell lysates. Additionally, the Zn(2+)-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn(2+)-Phos-tag SDS-PAGE gels. We can therefore provide a simple, convenient, and more reliable homemade gel system for phosphate-affinity SDS-PAGE.     

Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. The phosphate-affinity site is a polyacrylamide-bound Phos-tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c-Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr-394, Tyr-197, or Tyr-18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two-component system. FixL is autophosphorylated at the His-285 with ATP, and the phosphate group is transferred to the Asp-54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well-known isotype phosphorylated at the Asp-54. The other is a novel isotype phosphorylated at the His-84.     

Mobility shift detection of phosphorylation on large proteins using a Phos‐tag SDS‐PAGE gel strengthened with agarose

We describe a novel technique of phosphate‐affinity SDS‐PAGE using Phos‐tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up‐shifted migration bands in a 3% w/v polyacrylamide gel containing 20 μM Phos‐tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high‐molecular‐mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).     

Interpreting the SDS-PAGE protein patterns with self-organizing maps: application for the characterization of mosquito-pathogenic Bacillus strains

Aims: To present the pairwise comparison of potential mosquito‐pathogenic Bacillus strains based on their SDS‐PAGE protein patterns and to evaluate their characteristic toxicity patterns. Methods and Results: In this work, 20 Bacillus strains were subjected to qualitative toxicity tests against Aedes aegypti and Culex quinquefasciatus larvae. The selected strains were then characterized by SDS‐PAGE protein profiles. The highly heterogeneous multiple protein components of protein patterns were analysed using self‐organizing map (SOM), a ‘visualization and clustering’ tool. Members of mosquitocidal Bacillus species were classified in four distinct clusters, and then toxicity patterns were examined. Cluster (1, 1) comprised of three highly toxic strains of Bacillus sphaericus: SPH88, 1593 and KSD‐4; cluster (1, 2) consisted of two B. sphaericus strains: SSII‐1 and Bsp‐R that showed weak larvicidal activity; cluster (2, 1) constituted two B. sphaericus strains: WHO2297 and ISPC‐5 that possessed moderate toxicity; and cluster (2, 2) contained four B. thuringiensis ssp. israelensis strains: ONR‐60A, HD500, IPS70 and IPS82 belonging to serotype H14 but exhibited moderate to high mosquito larvicidal toxicity. Conclusions: SOM served as a colour‐coded alternate for easy visualization of similarities or dissimilarities between the strains even at the infra subspecies level. Furthermore, characteristic toxicity patterns of Bacillus strains of different clusters were determined. Significance and Impact of the Study: Analysis of electrophoretic protein patterns using SOM provides a better insight into the inter‐relationships of bacterial strains through similarity‐based clustering and pairwise comparison of two strains.     

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.     

HO1 and PcyA proteins involved in phycobilin biosynthesis form a 1:2 complex with ferredoxin-1 required for photosynthesis

The HO1 and PcyA genes, encoding heme oxygenase-1 (HO1) and phycocyanobilin (PCB):ferredoxin (Fd) oxidoreductase (PcyA), respectively, are required for chromophore synthesis in photosynthetic light-harvesting complexes, photoreceptors, and circadian clocks. In the PCB biosynthetic pathway, heme first undergoes cleavage to form biliverdin. I confirmed that Fd1 induced the formation of a stable and functional HO1 complex by the gel mobility shift assay. Furthermore, analysis by a chemical cross-linking technique designed to detect protein-protein interactions revealed that HO1 and PcyA directly interact with Fd in a 1:2 ratio. Thus, Fd1, a one-electron carrier protein in photosynthesis, drives the phycobilin biosynthetic pathway.

Structured summary

MINT-7014657: Fd1 (uniprotkb:P0A3C9) and HO1 (uniprotkb:Q8DLW1) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)MINT-7014666: HO1 (uniprotkb:Q8DLW1 and Fd1 (uniprotkb:P0A3C9) bind (MI:0407) by cross-linking studies (MI:0030)MINT-7014675: PcyA (uniprotkb:P59288) and Fd1 (uniprotkb:P0A3C9) bind (MI:0407) by cross-linking studies (MI:0030)     

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry

Yersinia encodes a protein kinase, YpkA, which disrupts the actin cytoskeleton. Using an approach termed chemical genetics, we identified a 36-kDa substrate for YpkA in both J774 lysates and bovine brain cytosol. Mass spectrometry analysis identified this substrate as FLJ20113, an open reading frame that corresponds to otubain 1, a deubiquitinating enzyme implicated in immune cell clonal anergy. We demonstrate that otubain 1 is phosphorylated by YpkA in vitro and interacts with YpkA and actin in vivo. Identification of otubain 1 as a YpkA substrate suggests that regulation of immune cell anergy may be a survival mechanism for Yersinia.     

Self-association of isolated large cytoplasmic domain of plasma membrane H-ATPase from Saccharomyces cerevisiae: Role of the phosphorylation domain in a general dimeric model for P-ATPases

Large cytoplasmic domain (LCD) plasma membrane H⁺-ATPase from S. cerevisiae was expressed as two fusion polypeptides in E. coli: a DNA sequence coding for Leu353-Ileu674 (LCDh), comprising both nucleotide (N) and phosphorylation (P) domains, and a DNA sequence coding for Leu353-Thr543 (LCDΔh, lacking the C-terminus of P domain), were inserted in expression vectors pDEST-17, yielding the respective recombinant plasmids. Overexpressed fusion polypeptides were solubilized with 6 M urea and purified on affinity columns, and urea was removed by dialysis. Their predicted secondary structure contents were confirmed by CD spectra. In addition, both recombinant polypeptides exhibited high-affinity 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP) binding (K_d = 1.9 μM and 2.9 μM for LCDh and LCDΔh, respectively), suggesting that they have native-like folding. The gel filtration profile (HPLC) of purified LCDh showed two main peaks, with molecular weights of 95 kDa and 39 kDa, compatible with dimeric and monomeric forms, respectively. However, a single elution peak was observed for purified LCDΔh, with an estimated molecular weight of 29 kDa, as expected for a monomer. Together, these data suggest that LCDh exist in monomer-dimer equilibrium, and that the C-terminus of P domain is necessary for self-association. We propose that such association is due to interaction between vicinal P domains, which may be of functional relevance for H⁺-ATPase in native membranes. We discuss a general dimeric model for P-ATPases with interacting P domains, based on published crystallography and cryo-electron microscopy evidence.     

Characterization of two pathogenic mutations in cystathionine beta-synthase: Different intracellular locations for wild-type and mutant proteins

Cystathionine β-synthase (CBS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by a deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C > A (p.T87N)) and a previously reported variant (c.700G > A (p.D234N)) found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B₆. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype.