Structure and Dimerization of the Kinase Domain from Yeast Snf1, a Member of the Snf1/AMPK Protein Family

SummaryThe Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved αC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds

Histone H2B deubiquitylation and transcriptional control in the yeast Saccharomyces cerevisiae

Eukaryotic DNA is condensed into a structure referred to as chromatin. The basic unit of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around an octamer of histone proteins. Histone proteins are highly conserved, as are many of the covalent modifications that alter their function. Known histone modifications include acetylation, phosphorylation, methylation, and ubiquitylation, which have been implicated in a variety of genomic processes including DNA replication, repair, recombination, and transcription. The most extensively studied histone modification to date is lysine acetylation. Histone acetylation is believed to have a positive role in transcription, whereas removal of the acetyl group represses transcription. These observations have led to a model in which genes are regulated by the addition and removal of different histone modifications that act as on/off switches for the expression of genes. Histone modifications occur in patterns, and there are instances known in which one modification is required for subsequent modification of another residue. Recently, it was reported that monoubiquitylation of histone H2B on Lysine-123 is required for activation-associated methylation on histone H3 at Lysine-4 and Lysine-79. While the connection to H3 methylation suggests a role for H2B ubiquitylation in activation, there is no direct evidence for the function of this modification in the regulation of gene expression. Here we provide evidence that monoubiquitylation of H2B and subsequent deubiquitylation are both involved in transcriptional activation. H2B deubiquitylation is mediated by the ubiquitin-specific protease, Ubp8, which is a component of the SAGA co-activator complex. Perturbation of the dynamic ubiquitylation/deubiquitylation cycle results in altered levels of H3 methylation at Lysine-4 and Lysine-36, which we partially attribute to defective recruitment of downstream factors to the gene. We propose that H2B ubiquitylation serves, in part, as a barrier to transcription factor recruitment during gene induction, and that subsequent deubiquitylation by Ubp8 removes this barrier at the appropriate point in the activation pathway. Finally, we provide evidence that this specific function for Ubp8 occurs within the context of an elongation-specific SAGA-related complex

Histone H2B deubiquitylation and transcriptional control in the yeast Saccharomyces cerevisiae

Eukaryotic DNA is condensed into a structure referred to as chromatin. The basic unit of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around an octamer of histone proteins. Histone proteins are highly conserved, as are many of the covalent modifications that alter their function. Known histone modifications include acetylation, phosphorylation, methylation, and ubiquitylation, which have been implicated in a variety of genomic processes including DNA replication, repair, recombination, and transcription. The most extensively studied histone modification to date is lysine acetylation. Histone acetylation is believed to have a positive role in transcription, whereas removal of the acetyl group represses transcription. These observations have led to a model in which genes are regulated by the addition and removal of different histone modifications that act as on/off switches for the expression of genes. Histone modifications occur in patterns, and there are instances known in which one modification is required for subsequent modification of another residue. Recently, it was reported that monoubiquitylation of histone H2B on Lysine-123 is required for activation-associated methylation on histone H3 at Lysine-4 and Lysine-79. While the connection to H3 methylation suggests a role for H2B ubiquitylation in activation, there is no direct evidence for the function of this modification in the regulation of gene expression. Here we provide evidence that monoubiquitylation of H2B and subsequent deubiquitylation are both involved in transcriptional activation. H2B deubiquitylation is mediated by the ubiquitin-specific protease, Ubp8, which is a component of the SAGA co-activator complex. Perturbation of the dynamic ubiquitylation/deubiquitylation cycle results in altered levels of H3 methylation at Lysine-4 and Lysine-36, which we partially attribute to defective recruitment of downstream factors to the gene. We propose that H2B ubiquitylation serves, in part, as a barrier to transcription factor recruitment during gene induction, and that subsequent deubiquitylation by Ubp8 removes this barrier at the appropriate point in the activation pathway. Finally, we provide evidence that this specific function for Ubp8 occurs within the context of an elongation-specific SAGA-related complex

Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer

Abstract Background Inhibitors of Poly (ADP-Ribose) Polymerases (PARP) provide clinical benefit to patients with breast and ovarian cancers, by compromising the DNA repair activity of cancer cells. Although these agents extend progression-free survival in many patients, responses can be short lived with many patients ultimately progressing. Identification of combination partners that increase dependence of cancer cells to the DNA repair activity of PARPs may represent a strategy to increase the utility of PARP inhibitors. Protein arginine methyltransferase 5 (PRMT5) regulates DNA damage response pathways through splicing and protein modification, and inhibitors of PRMT5 have recently entered clinical trials. Methods The effect of PRMT5 inhibition on the levels of DNA damage and repair markers including γH2AX, RAD51, and 53BP1 was determined using high content immunofluorescent imaging. The anti-proliferative activity of the combination of PRMT5 and PARP inhibitors was evaluated using in vitro models of breast and ovarian cancers using both cell lines and ex vivo patient derived xenografts. Finally, the combinations of PRMT5 and PARP inhibitors were evaluated in cell line xenograft models in vivo. Results Inhibition of PRMT5 by GSK3326595 led to increased levels of markers of DNA damage. The addition of GSK3326595 to the PARP inhibitor, niraparib, resulted in increased growth inhibition of breast and ovarian cancer cell lines and patient derived spheroids. In vivo, the combination improved the partial effects on tumor growth inhibition achieved by either single agent, producing complete tumor stasis and regression. Conclusion These data demonstrate that inhibition of PRMT5 induced signatures of DNA damage in models of breast and ovarian cancer. Furthermore, combination with the PARP inhibitor, Niraparib, resulted in increased anti-tumor activity in vitro and in vivo. Overall, these data suggest inhibition of PRMT5 as a mechanism to broaden and enhance the clinical application of PARP inhibitors

Additional file 6 of Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer

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Additional file 3 of Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer

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Additional file 5 of Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer

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Additional file 2 of Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer

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Additional file 1 of Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer

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