Yumi Kawamura

Global analysis of gel mobility of proteins and its use in target identification

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the “mobilitome,” covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.

Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source

Lysine residues can be post-translationally modified by various acyl modifications in bacteria and eukarya. Here, we showed that two major acyl modifications, acetylation and succinylation, were changed in response to the carbon source in the Gram-positive model bacterium Bacillus subtilis. Acetylation was more common when the cells were grown on glucose, glycerol, or pyruvate, whereas succinylation was upregulated when the cells were grown on citrate, reflecting the metabolic states that preferentially produce acetyl-CoA and succinyl-CoA, respectively. To identify and quantify changes in acetylation and succinylation in response to the carbon source, we performed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic analysis of cells grown on glucose or citrate. We identified 629 acetylated proteins with 1355 unique acetylation sites and 204 succinylated proteins with 327 unique succinylation sites. Acetylation targeted different metabolic pathways under the two growth conditions: branched-chain amino acid biosynthesis and purine metabolism in glucose and the citrate cycle in citrate. Succinylation preferentially targeted the citrate cycle in citrate. Acetylation and succinylation mostly targeted different lysine residues and showed a preference for different residues surrounding the modification sites, suggesting that the two modifications may depend on different factors such as characteristics of acyl-group donors, molecular environment of the lysine substrate, and/or the modifying enzymes. Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome. Mutations that modulate protein acylation affected B. subtilis growth. A mutation in acetate kinase (ackA) increased the global acetylation level, suggesting that acetyl phosphate-dependent acetylation is common in B. subtilis, just as it is in Escherichia coli. Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.

Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction

The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as l‐glutamate. During l‐glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor l‐glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label‐free semi‐quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2‐oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate‐dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate‐producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate‐producing condition may reflect metabolic states where the flux through acid‐producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more easily than acetylation in such conditions where the substrates for both acetylation and succinylation are limited. This is the first study investigating the acetylome and succinylome of C. glutamicum, and it provides new insight into the roles of acyl modifications in C. glutamicum biology.

Inhibition of protein SUMOylation by davidiin, an ellagitannin from Davidia involucrata

Conjugation of small ubiquitin-related modifier (SUMO) to lysine residues in target proteins is a multistep enzymatic reaction analogous to ubiquitination.1 Protein SUMOylation regulates numerous biological processes including transcription, the cell cycle, DNA repair and innate immunity.1 In the first step of the reaction, SUMO is cleaved from the SUMO precursor by SUMO-specific proteases. Next, SUMO is bound to the cysteine residue of the SUMO-activating enzyme (E1), forming a thioester linkage in an ATP-dependent manner. SUMO is then transferred from E1 to the cysteine residue of the SUMO-conjugating enzyme (E2). Finally, SUMO ligase (E3) catalyzes the SUMOylation of specific substrates via a direct interaction with E2 and the substrates. Like ubiquitination, SUMOylation is reversible; the deSUMOylation process is mediated by SUMO-specific proteases. Abnormal SUMOylation is implicated in various diseases including neurodegenerative disease,2 viral infection3 and cancer.4,5 Therefore, enzymes responsible for the SUMO conjugation pathway represent potential targets for drug discovery. To date, several natural products including ginkgolic acid,6 anacardic acid,6 kerriamycin B7 and spectomycin B18 as well as synthetic compounds,9 have been reported to inhibit protein SUMOylation. Here, we report another natural product that functions as a SUMOylation inhibitor: davidiin, purified from the plant Davidia involucrata. Although most known SUMOylation inhibitors function in the micromolar range, davidiin is particularly potent, inhibiting at sub-micromolar concentrations. Materials for this study were obtained as follows. Goat polyclonal anti-SUMO-1 (N-19) and goat polyclonal anti-p53 (FL393)-G antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). A mouse monoclonal anti-T7 antibody was from Novagen (Darmstadt, Germany). Mouse monoclonal anti-a-tubulin (B-5-1-2) and anti-FLAG (M2) antibodies were purchased from Sigma (St. Louis, MO, USA). Recombinant Hisand T7-tagged RanGAP1-C2, GST-Aos1-Uba2 fusion protein (E1), His-tagged Ubc9 (E2), and Histagged SUMO-1 proteins were purified as described previously.10 293T, H1299, MKN-45, DU-145 and NCI-H460 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% FBS at 37 1C under 5% CO2. The in vitro SUMOylation reaction was performed as described.6 Briefly, in vitro SUMOylation reaction was performed for 2 h at 30 1C in 20ml buffer (50 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 2 mM ATP and 1 mM dithiothreitol) containing Hisand T7-tagged RanGAP1-C2, GST-Aos1/Uba2 (E1), His-tagged Ubc9 and His-tagged SUMO-1. Samples were separated by 10% SDS–PAGE followed by immunoblotting using an anti-T7 antibody and an anti-SUMO-1 antibody. The reaction for thioester bond formation between SUMO and E1 was performed as described.6 Briefly, the reaction for the thioester bond formation was performed for 20 min at 37 1C in 20ml buffer (50 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 2 mM ATP) containing GSTAos1/Uba2 (E1) and biotinylated SUMO-1 in the absence of dithiothreitol. Samples were separated by 11% SDS–PAGE and the E1–biotinylated SUMO-1 intermediate was detected by avidin-conjugated horseradish peroxidase (Sigma). A screen of 750 samples of botanical and food ingredients extracts using an in situ cell-based SUMOylation assay11 revealed several samples that could inhibit protein SUMOylation, including an extract of D. involucrata (data not shown).6 The inhibitory activity of the D. involucrata extract was confirmed by in vitro SUMOylation assay using RanGAP1-C2 as substrate (Figure 1a). Compound A was isolated by activity-guided fractionation and it was identified by

U2 snRNP is required for expression of the 3' end of genes.

Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3′-end down-regulation. Removal of SSA from the culture media restored expression of the 3′ ends of genes, suggesting that U2 snRNP is required for expression of the 3′ end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5′ end of 3′-end down regulated genes, such as CDK6, SMEK2 and EGFR, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation.

Calcineurin inhibitors suppress the high‐temperature stress sensitivity of the yeast ubiquitin ligase Rsp5 mutant: a new method of screening for calcineurin inhibitors

The ubiquitin/proteasome system plays significant and important roles in the regulation of metabolism of various proteins. The dysfunction of this system is involved in several diseases, for example, cancer, neurogenic diseases and chronic inflammation. Therefore, the compounds, which regulate the ubiquitin/proteasome system, might be candidates for the development use as clinical drugs. The Saccharomyces cerevisiae mutant (rsp5(A401E)) has a single amino acid change, Ala401Glu, in the RSP5 gene, which encodes an essential E3 ubiquitin ligase, is hypersensitive to high-temperature stress. Here, we found that the immunosuppressants FK506 and cyclosporin A, both known as calcineurin inhibitors, complemented the high-temperature stress-induced growth defect of rsp5(A401E) strain. The defect of calcineurin pathway by disrupting the CNB1 and CRZ1 gene also partially complemented the high-temperature stress sensitivity of rsp5(A401E) cells. Thus, these results suggest that inhibition of the calcineurin pathway confers the tolerance to high-temperature stress on rsp5(A401E) cells. Furthermore, some diterpenoid compounds, which restore the growth of rsp5(A401E) cells, showed the activities of calcineurin inhibition and protein phosphatase 2C activation. These results indicate that calcineurin inhibitors suppress the high-temperature stress sensitivity of rsp5(A401E) cells and that analysis of their physiological function is effective for the screening of calcineurin inhibitors in yeast cells.