Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the Gatekeeper Residue is Implicated.

The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as drug targets. The isozyme HDAC10 contributes to chemotherapy resistance and has recently been described to be a polyamine deacetylase, but no studies toward selective HDAC10 inhibitors have been published. Using two complementary assays, we found Tubastatin A, an HDAC6 inhibitor, to potently bind HDAC10. We synthesized Tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10 binding. HDAC10 inhibitors mimicked knockdown by causing dose-dependent accumulation of acidic vesicles in a neuroblastoma cell line. Furthermore, docking into human HDAC10 homology models indicated that a hydrogen bond between a cap group nitrogen and the gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, our data provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the Tubastatin A scaffold.

A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2.

Previous studies proposed a role for the Na/K-ATPase in unconventional secretion of fibroblast growth factor 2 (FGF2). This conclusion was based upon pharmacological inhibition of FGF2 secretion in the presence of ouabain. However, neither independent experimental evidence nor a potential mechanism was provided. Based upon an unbiased RNAi screen, we now report the identification of ATP1A1, the α1-chain of the Na/K-ATPase, as a factor required for efficient secretion of FGF2. As opposed to ATP1A1, down-regulation of the ß1- and ß3-chains (ATP1B1 and ATP1B3) of the Na/K-ATPase did not affect FGF2 secretion, suggesting that they are dispensable for this process. These findings indicate that it is not the membrane potential-generating function of the Na/K-ATPase complex but rather a so far unidentified role of potentially unassembled α1-chains that is critical for unconventional secretion of FGF2. Consistently, in the absence of ß-chains, we found a direct interaction between the cytoplasmic domain of ATP1A1 and FGF2 with submicromolar affinity. Based upon these observations, we propose that ATP1A1 is a recruitment factor for FGF2 at the inner leaflet of plasma membranes that may control phosphatidylinositol 4,5-bisphosphate-dependent membrane translocation as part of the unconventional secretory pathway of FGF2.

Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements.

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho:Sfmbt and human YY1:MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs.

Evaluation of different glutathione S-transferase-tagged protein captures for screening E6/E6AP interaction inhibitors using AlphaScreen.

Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins.

Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line.

A cell line derived from a pleiomorphic liposarcoma, named LSA, was previously reported to secrete (a) factor(s) exhibiting oncotoxic properties. The present article describes the isolation, purification and sequence analysis of a protein released by LSA cells into conditioned culture medium. This protein proved to be a variant isoform of manganese superoxide dismutase (MnSOD), hence its designation as LSA-type-MnSOD. This LSA-type-SOD differed from conventional SODs in its secretion by producer cells, contrasting with the normal localization of SODs in the mitochondrial matrix. Interestingly, during the protein purification process, LSA-type-SOD cosegregated with a cytotoxic activity directed against a number of tumor cell lines, as determined under in vitro conditions. This cytopathic effect was most likely due to LSA-type-SOD, since it could be fully reproduced using recombinant SOD that was expressed from cDNA clones isolated from LSA cells mRNA preparations and henceforth designated L-rSOD. In addition to its manifestation in cell lines kept in tissue culture, the oncotoxicity of LSA-type-SOD was further reflected in a remarkable capacity of this protein for suppression of mammary tumors in Balb-C-FR(III) mice. Animals subcutaneously injected with L-rSOD in the tumor area showed a complete disruption of established mammary carcinomas, as monitored by nuclear magnetic resonance (NMR) scanning. Moreover, metastatic spreading, which was readily detected in the control group, was suppressed in the treated animals. Altogether these data suggest that LSA-type-SOD interferes with survival and spreading of neoplastically transformed cells and deserves to be future validated as a therapeutic agent against cancer, either alone or in combination with conventional treatments.