Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF). KAT6A has essential roles in normal haematopoietic stem cells and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus, a function that requires its KAT activity. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.
We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.
Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle
T
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castaneum
and the mosquito
Aedes aegypti
. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET–TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that
T
.
castaneum
JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.
Previously, we have presented an expedited strategy for sustained delivery of bone morphogenetic protein-2 (BMP-2) to bone lesions based on the implantation of gene-activated fat and muscle fragments. The aim of the present in vitro experiments was to evaluate the potential of muscle with fascia as a BMP-2 delivering osteo-regenerative implant in comparison to fat tissue and muscle alone. Subcutaneous fat, muscle, and muscle with fascia were harvested from Fischer 344 rats. The tissues were cut into small pieces and cultured for up to 90 days after direct transduction with adenoviral BMP-2 or green fluorescence protein vectors. Different vector doses were applied, and proliferation, long-term BMP-2 production, and osteogenic differentiation of the 3 different tissues were investigated in vitro. Muscle with fascia produced the largest amounts of BMP-2. Expression of the transgene was detected for up to 90 days. Proliferation was reduced with increased vector doses. Muscle with fascia showed a higher potential for osteogenic differentiation than fat, but it was not improved as compared to muscle alone. A dose of 4 × 10 plaque forming units of the adenoviral BMP-2 vector appeared to be the optimal dose for transduction of muscle with fascia. Because muscle with fascia produced higher amounts of BMP-2 as compared to muscle alone or fat tissue grafts, showing a high potential for osteogenic differentiation, it might represent an improved osteo-regenerative implant facilitating endogenous repair. Future studies should investigate the effect of muscle with fascia transduced with 4 × 10 plaque forming units on bone healing in vivo.
The homodimer BMP-2 induced osteoblastogenesis in muscle faster, at a lower concentration and with a higher potency than the heterodimers BMP-2/6 or BMP-2/7. The findings of this in vitro study encourage bone repair by muscle implants in combination with BMP-2 single growth factor delivery, which might be beneficial with respect to clinical translation.
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