BRK phosphorylates SMAD4 for proteasomal degradation and inhibits tumor suppressor FRK to control SNAIL, SLUG, and metastatic potential.

The tumor-suppressing function of SMAD4 is frequently subverted during mammary tumorigenesis, leading to cancer growth, invasion, and metastasis. A long-standing concept is that SMAD4 is not regulated by phosphorylation but ubiquitination. Our search for signaling pathways regulated by breast tumor kinase (BRK), a nonreceptor protein tyrosine kinase that is up-regulated in ~80% of invasive ductal breast tumors, led us to find that BRK competitively binds and phosphorylates SMAD4 and regulates transforming growth factor-ß/SMAD4 signaling pathway. A constitutively active BRK (BRK-Y447F) phosphorylates SMAD4, resulting in its recognition by the ubiquitin-proteasome system, which accelerates SMAD4 degradation. Activated BRK-mediated degradation of SMAD4 is associated with the repression of tumor suppressor gene FRK and increased expression of mesenchymal markers, SNAIL, and SLUG. Thus, our data suggest that combination therapies targeting activated BRK signaling may have synergized the benefits in the treatment of SMAD4 repressed cancers.

Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits.

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex. To understand fully the mechanism by which these large protein complexes perform their functions in the cell, it is crucial that all the subunits of these complexes are identified. In an attempt to identify new subunits associated with SAGA and Swi/Snf, we used tandem affinity purification, followed by a multidimensional protein identification technology to analyse the subunit composition. Our analysis identified two novel proteins, one associated with SAGA, YPL047W (Sgf11), and another associated with Swi/Snf, Rtt102.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion.

Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.

An automated multidimensional protein identification technology for shotgun proteomics.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

Analysis of the microbial proteome.

Proteomics has begun to provide insight into the biology of microorganisms. The combination of proteomics with genetics, molecular biology, protein biochemistry and biophysics is particularly powerful, resulting in novel methods to analyse complex protein mixtures. Emerging proteomic technologies promise to increase the throughput of protein identifications from complex mixtures and allow for the quantification of protein expression levels.

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase.

Bovine erythrocyte glutathione (GSH) peroxidase (GPX, EC 1.11.1.9) was examined for GSH-dependent dehydroascorbate (DHA) reductase (EC 1.8.5.1) and thioltransferase (EC 1.8.4.1) activities. Using the direct assay method for GSH-dependent DHA reductase activity, GPX had a kcat (app) of 140 +/- 9 min-1 and specificity constants (kcat/Km(app)) of 5.74 +/- 0.78 x 10(2) M-1s-1 for DHA and 1.18 +/- 0.17 x 10(3) M-1s-1 for GSH based on the monomer Mr of 22,612. Using the coupled assay method for thioltransferase activity, GPX had a kcat (app) of 186 +/- 9 min-1 and specificity constants (app) of 1. 49 +/- 0.14 x 10(3) M-1s-1 for S-sulfocysteine and 1.51 +/- 0.18 x 10(3) M-1s-1 for GSH based on the GPX monomer molecular weight. GPX has a higher specificity constant for S-sulfocysteine than DHA, and both assay systems gave nearly identical specificity constants for GSH. The DHA reductase and thioltransferase activities of GPX adds to the repertoire of functions of this enzyme as an important protector against cellular oxidative stress.

The catalytic mechanism of the glutathione-dependent dehydroascorbate reductase activity of thioltransferase (glutaredoxin).

The catalytic mechanism of the glutathione (GSH)-dependent dehydroascorbic acid (DHA) reductase activity of recombinant pig liver thioltransferase (RPLTT) was investigated. RPLTT and the C25S mutant protein had equivalent specificity constants (kcat/Km) for both DHA and GSH. Iodoacetamide (IAM) inactivated the DHA reductase activities of RPLTT and C25S, confirming the essential role of cysteine in the reaction mechanism. When preincubated with DHA, RPLTT but not C25S was protected against IAM inactivation, suggesting that RPLTT has the ability to chemically reduce DHA forming ascorbic acid (AA) and the intramolecular disulfide form of the enzyme. Electrochemical detection of AA demonstrated the ability of both reduced RPLTT and C25S to chemically reduce DHA to AA in the absence of GSH. However, RPLTT had an initial rate of DHA reduction which was 4-fold greater than that of C25S, and after 10 min, RPLTT resulted in an AA concentration 11-fold greater than that of C25S. Isoelectric focusing analysis revealed that the product of reaction of reduced RPLTT but not C25S with DHA was consistent with the oxidized form of the enzyme. This result suggested that even though both RPLTT and the C25S mutant had equivalent specificity constants for DHA and GSH, they may have different catalytic mechanisms. On the basis of the experimental results, a catalytic mechanism for the DHA reductase activity of RPLTT is proposed. This is the first description of a catalytic mechanism of a glutathione:dehydroascorbate oxidoreductase (EC 1.8.5.1).

Glutathione dependent reduction of alloxan to dialuric acid catalyzed by thioltransferase (glutaredoxin): a possible role for thioltransferase in alloxan toxicity.

Recombinant pig liver thioltransferase (rPLTT) catalyzes the reduction of alloxan to dialuric acid by glutathione (GSH). This is the second non-disulfide substrate, after dehydroascorbic acid, described for thioltransferase. The reaction kinetics, measured by a coupled assay including glutathione disulfide reductase and NADPH yielded a Km = 82 microM for alloxan, a k(cat) = 37 s(-1), and a k(cat)/Km = 4.5 x 10(5) M(-1) s(-1). The presence of rPLTT suppressed the competitive formation of compound 305, an alloxan-GSH conjugate of unknown structure, and at GSH concentrations between 0.05 mM and 1.5 mM, oxygen consumption was greater than that recorded in the uncatalyzed reaction. Both superoxide dismutase and catalase inhibited oxygen consumption in 1.0 mM GSH and 0.2 mM alloxan in the presence of rPLTT. This study suggests that thioltransferase (glutaredoxin) plays a significant role in the cytotoxicity of alloxan in vulnerable tissues.

Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes.

A GSH-dependent dehydroascorbate reductase (EC 1.8.5.1) was purified to homogeneity from human erythrocytes. The enzyme was a monomer of 32 kDa and was purified 133-fold from a crude DEAE-Sepharose fraction with a 25% yield. The reduced protein had a pI of 5.1 as judged by isoelectric focusing. Kinetic analysis gave a Kcat of 316 min-1, a Km of 0.21 mM for DHA with a Kcat/Km of 2.47 x 10(4) M-1 sec-1, and a Km of 3.5 mM for GSH with a Kcat/Km of 1.51 x 10(3) M-1 sec-1. This is the second DHA reductase (after thioltransferase) isolated from human erythrocytes, but unlike thioltransferase, it has no thiol-disulfide oxido-reductase activity.