Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation

The Saccharomyces cerevisiae WD-40 repeat protein Swd2p associates with two functionally distinct multiprotein complexes: the cleavage and polyadenylation factor (CPF) that is involved in pre-mRNA and snoRNA 3′ end formation and the SET1 complex (SET1C) that methylates histone 3 lysine 4. Based on bioinformatic analysis we predict a seven-bladed β-propeller structure for Swd2p proteins. Northern, transcriptional run-on and in vitro 3′ end cleavage analyses suggest that temperature sensitive swd2 strains were defective in 3′ end formation of specific mRNAs and snoRNAs. Protein–protein interaction studies support a role for Swd2p in the assembly of 3′ end formation complexes. Furthermore, histone 3 lysine 4 di-and tri-methylation were adversely affected and telomeres were shortened in swd2 mutants. Underaccumulation of the Set1p methyltransferase accounts for the observed loss of SET1C activity and suggests a requirement for Swd2p for the stability or assembly of this complex. We also provide evidence that the roles of Swd2p as component of CPF and SET1C are functionally independent. Taken together, our results establish a dual requirement for Swd2p in 3′ end formation and histone tail modification.

Protein interactions within the SET1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5′ end of active coding regions but a decrease of H3K4 dimethylation at the 3′ end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3′ end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

Phenylalanine-544 plays a key role in substrate and inhibitor binding by providing a hydrophobic packing point at the active site of insulin-regulated aminopeptidase

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn2+ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn2+ ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the Ki in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.

Repeated social defeat selectively increases ΔFosB expression and histone H3 acetylation in the infralimbic medial prefrontal cortex

Exposure to social stress has been linked to the development and maintenance of mood-related psychopathology; however, the underlying neurobiological changes remain uncertain. In this study, we examined numbers of ΔFosB-immunoreactive cells in the forebrains of rats subjected to 12 episodes of social defeat. This was achieved using the social conflict model whereby animals are introduced into the home cage of older males (“residents”) trained to attack and defeat all such “intruders”; importantly, controls were treated identically except that the resident was absent. Our results indicated that the only region in which ΔFosB-positive cells were found in significantly higher numbers in intruders than in controls was the infralimbic medial prefrontal cortex (mPFC). This same effect was not apparent using another psychological stressor, noise stress. Cells of the infralimbic mPFC also displayed evidence of chromatin remodeling. We found that exposure to repeated episodes of social defeat increased numbers of cells immunoreactive for histone H3 acetylation, but not for histone H3 phosphoacetylation, in the infralimbic mPFC. Collectively, these findings highlight the importance of the infralimbic mPFC in responding to social stress—a finding that provides insight into the possible neurobiological alterations associated with stress-induced psychiatric illness.

Multicentre phase I/II study of PI-88, a heparanase inhibitor in combination with docetaxel in patients with metastatic castrate-resistant prostate cancer

Background: Docetaxel (Taxotere) improve survival and prostate-specific antigen (PSA) response rates in patients with metastatic castrate-resistant prostate cancer (CRPC). We studied the combination of PI-88, an inhibitor of angiogenesis and heparanase activity, and docetaxel in chemotherapy-naive CRPC.

Patients and methods: We conducted a multicentre open-label phase I/II trial of PI-88 in combination with docetaxel. The primary end point was PSA response. Secondary end points included toxicity, radiologic response and overall survival. Doses of PI-88 were escalated to the maximum tolerated dose; whereas docetaxel was given at a fixed 75 mg/m2 dose every three weeks

Results: Twenty-one patients were enrolled in the dose-escalation component. A further 35 patients were randomly allocated to the study to evaluate the two schedules in phase II trial. The trial was stopped early by the Safety Data Review Board due to a higher-than-expected febrile neutropenia of 27%. In the pooled population, the PSA response (50% reduction) was 70%, median survival was 61 weeks (6–99 weeks) and 1-year survival was 71%.

Conclusions: The regimen of docetaxel and PI-88 is active in CRPC but associated with significant haematologic toxicity. Further evaluation of different scheduling and dosing of PI-88 and docetaxel may be warranted to optimise efficacy with a more manageable safety profile.

Use of electrochemical impedance spectroscopy for study of CO2 corrosion prevention by batch treatment inhibitor films

Electrochemical impedance spectroscopy (EIS) was used to study and evaluate commercial batch treatment inhibitors which are used for protecting oil wells, gas wells, and pipelines from CO2 corrosion, focusing on the evaluation of inhibitor film persistency. It was found that theformation and deterioration of batch treatment inhibitor films were accompanied by typical impedance spectral changes. During the formation of inhibitor films, electrode impedance showed a rapid increase and the Bode phase angle plots also showed a sudden change. Thus, the formation of inhibitor film was a very fast process. During the deterioration of inhibitor films, electrode impedance showed a gradual decrease and the Bode phase angle plots showed changes which characterised the three stages of the inhibitor film deterioration process. The relationships between EIS and corrosion rate are discussed, including comparisons with weight loss measurements. Based on the experimental findings in the present work, a method is suggested for estimating the persistency of inhibitor films by monitoring the characteristic changes in the Bode phase angle plots and by measuring electrochemical charge transfer resistance at the second and third stages of the inhibitor film deterioration process.

Monitoring batch treatment inhibitor performance continuously using electrochemical noise analysis

Electrochemical noise analysis (ENA) was used to monitor continuously the formation and deterioration processes of a commercial batch treatment inhibitor film of the type used for protecting against CO₂ corrosion in oilfields; ENA was shown to be able to follow effectively the formation and deterioration processes of batch treatment inhibitor films. As an inhibitor film formed, the current noise amplitude decreased rapidly and the noise resistance R_n, which is deducible from the voltage and current noise records, was found to increase sharply. Conversely, as the inhibitor film deteriorated, the current noise amplitude increased rapidly and R_n decreased rapidly. In the corrosion inhibition system studied, the noise resistance was confirmed to be similar to the linear polarisation resistance. Based on the calculation of R_n on a continuous basis, a technique is proposed to study fast corrosion processes.