Yasuhiko Komatsu

Histone deacetylase as a new target for cancer chemotherapy

Abstract. Trichostatin A (TSA) and trapoxin (TPX), inhibitors of the eukaryotic cell cycle and inducers of morphological reversion of transformed cells, inhibit histone deacetylase (HDAC) at nanomolar concentrations. Recently, FK228 (also known as FR901228 and depsipeptide) and MS-275, antitumor agents structurally unrelated to TSA, have been shown to be potent HDAC inhibitors. These inhibitors activate the expression of p21Waf1 in a p53-independent manner. Changes in the expression of regulators of the cell cycle, differentiation, and apoptosis with increased histone acetylation may be responsible for the cell cycle arrest and antitumor activity of HDAC inhibitors. TSA has been suggested to block the catalytic reaction by chelating a zinc ion in the active site pocket through its hydroxamic acid group. On the other hand, an epoxyketone has been suggested to be the functional group of TPX capable of alkylating the enzyme. We synthesized a novel TPX analogue containing a hydroxamic acid instead of the epoxyketone. The hybrid compound, called cyclic hydroxamic-acid-containing peptide 1 (CHAP1) inhibited HDAC at low nanomolar concentrations. The HDAC1 inhibition by CHAP1 was reversible, as is that by TSA, in contrast to irreversible inhibition by TPX. Interestingly, HDAC6, but not HDAC1 or HDAC4, was resistant to TPX and CHAP1, while TSA inhibited these HDACs to a similar degree. CHAP31, the strongest HDAC inhibitor obtained from a variety of CHAP derivatives, exhibited antitumor activity in BDF1 mice bearing B16/BL6 tumor cells. These results suggest that CHAP31 is promising as a novel therapeutic agent for cancer treatment, and that CHAP may serve as a basis for new HDAC inhibitors and be useful for combinatorial synthesis and high-throughput screening.

From discovery to the coming generation of histone deacetylase inhibitors.

Trichostatin A (TSA) is a Streptomyces metabolite that causes differentiation of murine erythroleukemia cells as well as specific inhibition of the cell cycle of some lower eukaryotes and mammalian cells. The targeted molecule of TSA has been shown by genetic and biochemical analyses to be histone deacetylases (HDACs). Histone acetylation is a key modification to control transcription, and HDACs are profoundly involved in pathogenesis of cancer through removing acetyl groups from histones and other transcriptional regulators. Trapoxin (TPX) and FK228 (also known as FR901228 and depsipeptide because FK228 = FR901228 = depsipeptide), structurally unrelated microbial metabolites, were also shown to inhibit HDACs. These HDAC inhibitors cause cell cycle arrest, differentiation and/or apoptosis of many tumors, suggesting their usefulness for chemotherapy and differentiation therapy. In addition, HDAC inhibitors play important roles in identifying the specific function of the enzymes. Indeed, we identified tubulin as one of the substrates of HDAC6 by means of differential sensitivity to HDAC inhibitors. Since recent studies have revealed that HDACs are structurally and functionally diverse, it should be important to develop inhibitors specific to individual enzymes as more promising agents for cancer therapy. We have synthesized novel TSA/TPX hybrids, which will serve as a basis for developing enzyme-specific HDAC inhibitors.

Global analysis of gel mobility of proteins and its use in target identification

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the “mobilitome,” covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

HIV‐1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP‐associated E4‐type ubiquitin‐ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60‐dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV‐1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.

Regulation of SV40 large T-antigen stability by reversible acetylation

Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as α-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as α-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.

REEVALUATING THE EFFECTS OF TYROSINE IODINATION OF RECOMBINANT HIRUDIN ON ITS THROMBIN INHIBITION KINETICS

To reevaluate the effects of iodination of hirudin on its thrombin-inhibiting activity, we iodinated a recombinant hirudin analog, CX-397, by a chloramine-T method and isolated the eight kinds of iodinated derivatives by reverse-phase HPLC. Their structure were different combinations of non-, mono-, or di-iodinated residues at Tyr-3 and Tyr-64. Their ability to inhibit alpha-thrombin were determined and compared with each other. Iodination at Tyr-64 slightly increased the association rate constant (Kon) 1.26- to 1.79-fold; this may be explained by the increase in the negative charge at C-terminal region of CX-397 caused by the electrophilic ortho substitution of Tyr-64 with iodine. On the contrary, iodination at Tyr-3 caused dual effects on the interaction between CX-397 and alpha-thrombin; namely, mono-iodination at Tyr-3 caused a small decrease in Koff (1.29- to 1.63-fold) than non-iodinated ones, whilst di-iodination at Tyr-3 brought about 3.04- to 3.88-fold increase in Koff. Since the N-terminal region of hirudin has been reported to bind with thrombin by hydrophobic interaction, the destabilizing effect of di-iodination at Tyr-3 can be explained by the increase in its polar nature, whereas the opposite effect of mono-iodination can not. These results indicate that it is not appropriate to use a mixture of variably radioiodinated hirudins as a radiotracer. We also provide a simple method to obtain well characterized radioiodinated hirudins.

Erratum: Regulation of SV40 large T-antigen stability by reversible acetylation

Correction to: Oncogene (2006) 25, 7391–7400; doi:10.1038/sj.onc.1209731; published online 12 June 2006 Since the publication of this article, the authors found that Figures 1c and 3c had been incorrectly presented. The corrected figures are shown below. These changes do not affect any of the results or conclusions of the study.

Design of Analogs of Trapoxin, Cyl-1, and Chlamydocin for MHC Class-I Molecule Up-Regulation

Hydroxamic acid analogs of trapoxin, Cyl-1, and chlamydocin have been designed and synthesized as targeting molecules toward histone deacetylases (HDAC) [1,2]. These analogs resemble each other except the configurations of component amino acids. Trapoxin analog has LLLD configuration from its functional amino acid (L-Asu(NHOH)), while Cyl-1 analog and chlamydocin analog have LDLL and LxLD configuration, respectively. In order to apply these HD AC inhibitors for the regulation of cell affiliation and cell cycles, the efficient transportation into the cell nucleus is important. Using an assaying method [3] in which up-regulation of MHC class-I molecule could be measured as a result of HDAC inhibition, we found more active stereoisomers than the natural cyclic tetrapeptides.