Ryoko Takasawa

Differential apoptotic pathways in human keratinocyte HaCaT cells exposed to UVB and UVC

The induction of apoptosis in keratinocytes by ultraviolet (UV)-irradiation is considered to be a protective function against skin cancer. UV-induced DNA damage is a crucial event in UVB- and UVC-mediated apoptosis. However, the differences between the UVB- and UVC-induced apoptotic pathways remain unclear. Here we examine the differential mechanisms by which UVB and UVC irradiations induce keratinocyte apoptosis using human keratinocyte HaCaT cells. Differences in the production of (6-4)photoproducts ((6-4)PPs) and cyclobutane pyrimidine dimers (CPDs) were measured following irradiation with UVB and UVC at doses causing the same extent of apoptotic cell death. In addition, main apoptotic features, such as caspase activation and its regulation, were compared between UVB- and UVC-induced apoptosis. Exposures of 500 J/m2 UVB and 100 J/m2 UVC resulted in apoptosis to almost the same extent. At these apoptotic doses, the amounts of both (6-4)PPs and CPDs were significantly larger in the case of UVC irradiation than UVB irradiation; in parallel, the release of cytochrome c and Smac/DIABLO and the activation of caspases-9 following UVC irradiation were greater than after UVB irradiation. Importantly, caspase-8 activation occurred only in UVB-irradiated cells. Furthermore, the activation of caspase-8 was not inhibited by caspases-9 and -3 specific tetrapeptide inhibitors, indicating that the caspase-8 cleavage is not due to feedback from activation of caspases-9 and -3. Thus, these results clearly suggest that the reason apoptosis is induced to the same extent by UVB irradiation as by UVC irradiation, despite the lower production of photoproducts in DNA by UVB irradiation, is attributable to the additional activation of the caspase-8 pathway. Thus, UVB irradiation induces apoptosis through both mitochondrial (intrinsic) and caspase-8 activation (extrinsic) pathways, while UVC induces apoptosis only via the intrinsic pathway.

Structure-activity relationship of human GLO I inhibitory natural flavonoids and their growth inhibitory effects.

Glyoxalase I (GLO I) is the rate-limiting enzyme for detoxification of methylglyoxal (MG), a side product of glycolysis, which is able to induce apoptosis. Since GLO I is known to be highly expressed in the most tumor cells and little in normal cells, specific inhibitors of this enzyme have been expected as effective anticancer drugs. The purpose of this study is a good construction of the human GLO I/inhibitor pharmacophore to obtain unique human GLO I inhibitory seed compounds for the development of useful anticancer drugs. Here, we selected natural flavonoid compounds that possess a plane configuration of cis C-4 ketone and C-5 hydroxy groups as the substrate (MG) transition-state mimetic structure. These compounds were examined the inhibitory abilities to human GLO I activity and analyzed their structure-activity relationships to determine an important pharmacophore of flavonoids for the human GLO I binding. Our results point to the contribution of hydroxy groups at the B ring of flavonoids to the effective inhibition of the human GLO I. Based on the binding mode of flavonoids, we constructed the human GLO I/inhibitor pharmacophore. This work delivers the first three-dimensional (3D) structural data and explains certain flavonoids interact specifically with the human GLO I.

Sustained release of Smac/DIABLO from mitochondria commits to undergo UVB-induced apoptosis

Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m2, but not at 150 J/m2, induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m2 UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m2 UVB, but not at 150 J/m2. However, with both 150 and 500 J/m2 UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m2 UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.

Delphinidin, a dietary anthocyanidin in berry fruits, inhibits human glyoxalase I.

Glyoxalase I (GLO I) is the rate-limiting enzyme for detoxification of methylglyoxal (MG), a side-product of glycolysis, which is able to induce apoptosis. Since GLO I is known to be highly expressed in the most tumor cells and little in normal cells, inhibitors of this enzyme has been expected to be new anticancer drugs. Here, we examined the inhibitory abilities to the human GLO I of anthocyanidins, such as delphinidin, cyanidin and pelargonidin. Among them, delphinidin was found to have the most potent inhibitory effect on human GLO I. Also, only delphinidin-induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Furthermore, we determined a pharmacophore for delphinidin binding to the human GLO I by computational simulation analyses of the binding modes of delphinidin, cyanidin and pelargonidin to the enzyme hot spot. These results suggest that delphinidin could be a useful lead compound for the development of novel GLO I inhibitory anticancer drugs.

Structural insights into the hot spot amino acid residues of mushroom tyrosinase for the bindings of thujaplicins.

Tyrosinase inhibitors are important agents for cosmetic products. We examined here the inhibitory effects of three isomers of thujaplicins (α, β and γ) on mushroom tyrosinase and analyzed their binding modes using a homology model from the crystal structure of Streptomyces castaneoglobisporus tyrosinase (PDB ID: 1wx2). All the thujaplicins were found to be competitive inhibitors and γ-thujaplicin has the most potent inhibitory activity (IC(50)=0.07μM). It is noted that there are good correlations between their observed IC(50) values and their binding free energies calculated by MM-GB/SA. The binding modes of thujaplicins were predicted to be similar to that of Tyr98 of caddie protein (ORF378), which was co-crystallized with S. castaneoglobisporus tyrosinase. Furthermore, free energy decomposition analysis indicated that the potent inhibitory activity of γ-thujaplicin is due to the interactions with His242, Val243 and Pro257 (hot spot amino acid residues) at the active site of tyrosinase. These results provide a novel structural insight into the hot spot of mushroom tyrosinase for the specific binding of γ-thujaplicin.

Structural and functional definition of the specificity of a novel caspase-3 inhibitor, Ac-DNLD-CHO

BackgroundThe rational design of peptide-based specific inhibitors of the caspase family members using their X-ray crystallographies is an important strategy for chemical knockdown to define the critical role of each enzyme in apoptosis and inflammation. Recently, we designed a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 using a new computational screening system named the Amino acid Positional Fitness (APF) method (BMC Pharmacol. 2004, 4:7). Here, we report the specificity of the DNLD sequence against caspase-3 over other major caspase family members that participate in apoptosis by computational docking and site-directed mutagenesis studies.ResultsAc-DNLD-CHO inhibits caspases-3, -7, -8, and -9 activities with Kiapp values of 0.68, 55.7, >200, and >200 nM, respectively. In contrast, a well-known caspase-3 inhibitor, Ac-DEVD-CHO, inhibits all these caspases with similar Kiapp values. The selective recognition of a DNLD sequence by caspase-3 was confirmed by substrate preference studies using fluorometric methylcoumarin-amide (MCA)-fused peptide substrates. The bases for its selectivity and potency were assessed on a notable interaction between the substrate Asn (N) and the caspase-3 residue Ser209 in the S3 subsite and the tight interaction between the substrate Leu (L) and the caspase-3 hydrophobic S2 subsite, respectively, in computational docking studies. Expectedly, the substitution of Ser209 with alanine resulted in loss of the cleavage activity on Ac-DNLD-MCA and had virtually no effect on cleaving Ac-DEVD-MCA. These findings suggest that N and L residues in Ac-DNLD-CHO are the determinants for the selective and potent inhibitory activity against caspase-3.ConclusionOn the basis of our results, we conclude that Ac-DNLD-CHO is a reliable, potent and selective inhibitor of caspase-3. The specific inhibitory effect on caspase-3 suggests that this inhibitor could become an important tool for investigations of the biological function of caspase-3. Furthermore, Ac-DNLD-CHO may be an attractive lead compound to generate novel effective non-peptidic pharmaceuticals for caspase-mediated apoptosis diseases, such as neurodegenerative disorders and viral infection diseases.

A novel method for evaluation and screening of caspase inhibitory peptides by the amino acid positional fitness score

BackgroundSince caspases are key executioners of apoptosis in cases of severe diseases including neurodegenerative disorders such as Alzheimers disease and Huntingtons disease, and viral infection diseases such as AIDS and hepatitis, potent and specific inhibitors of caspases have clinical potential. A series of peptide inhibitors has been designed based on cleavage sites of substrate proteins. However, these peptides are not necessarily the most potent to each caspase. Moreover, so far, it has proved to be difficult to design potent and specific peptide inhibitors of each caspase from sequence data of known cleavage sites in substrate proteins. We have attempted to develop a computational screening system for rapid selection of potent and specific peptide inhibitors from a comprehensive peptide library.ResultsWe developed a new method for rapid evaluation and screening of peptide inhibitors based on Amino acid Positional Fitness (APF) score. By using this score, all known peptide inhibitors of each caspases-3,-7,-8, and -9 were rapidly selected in their enriched libraries. In this libraries, there were good correlations between predicted binding affinities of the known peptide inhibitors and their experimental Ki values. Furthermore, a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 was able to be designed by this method. To our knowledge, DNLD is a first reported caspase-3 inhibitory peptide identified by using the computational screening strategy.ConclusionOur new method for rapid screening of peptide inhibitors using APF score is an efficient strategy to select potent and specific peptide inhibitors from a comprehensive peptide library. Thus, the APF method has the potential to become a valuable approach for the discovery of the most effective peptide inhibitors. Moreover, it is anticipated that these peptide inhibitors can serve as leads for further drug design and optimization of small molecular inhibitors.

The release of high mobility group box 1 in apoptosis is triggered by nucleosomal DNA fragmentation.

High mobility group box 1 (HMGB1) initially identified as a non-histone chromosomal protein, which mainly functions as chromatin structure and transcriptional regulation, has been recently reported to be secreted into extracellular milieu in necrosis and apoptosis, and act as a proinflammatory mediator. However, the mechanism by which apoptotic cells release HMGB1 is not clear. In this study, we found that staurosporine (apoptosis-inducer)-induced HMGB1 release was associated with nucleosomal DNA fragmentation catalyzed by caspase-activated DNase (CAD) in WEHI-231 cells. Importantly, this event was effectively attenuated by the treatment of a pan-caspase inhibitor, Z-VAD-fmk, and by the inhibition of CAD-mediated DNA fragmentation by the expression of caspase-resistant inhibitor of CAD (ICAD-CR). In WEHI-231/ICAD-CR and WEHI-231/Puro cells, DNase γ-catalyzed nucleosomal DNA fragmentation occurred by anti-IgM antibody treatment was critical for HMGB1 release. Furthermore, in DNase γ stably-expressing HeLa S3 cells (HeLa S3/γ), the release of HMGB1 accompanied with nucleosomal DNA fragmentation was more apparent than that in parental HeLa S3 cells in which DNA fragmentation was scarcely observed. Taken together, these date suggest that nucleosomal DNA fragmentation catalyzed by CAD or DNase γ plays a pivotal role in HMGB1 release.

Identification of a novel gene family, paralogs of inhibitor of apoptosis proteins present in plants, fungi, and animals.

AbstractOnly few orthologs of animal apoptosis regulators have been found in plants. Recently, the ectopic expression of mammalian inhibitor of apoptosis proteins (IAPs) has been shown to affect plant programmed cell death. Here, we identified two novel proteins homologous to ArabidopsisthalianaIAP-like protein (AtILP) 1 and 2 by applying an improved motif searching method. Furthermore, homologs of AtILP1 were found to occur as a novel gene family in other organisms such as fungi and animals including Homo sapiens (HsILP1). Like baculovirus IAP repeats (BIRs) in IAPs, ILPs contain two highly conserved BIR-like domains (BLDs) with a putative C2HC-type zinc finger. Phylogenetic analyses indicated that ILPs are putative paralogs of IAPs. Homology modeling revealed that the three-dimensional structure of BLD in HsILP1 is similar to that of BIR. Transient expression of HsILP1 resulted in inhibition of etoposide-induced apoptosis in HEK293 and HeLaS3 cells. These findings suggest that ILPs are conserved in a wide range of eukaryotes including plants, and that their functions are closely related to those of IAPs.

Development of HER2-antagonistic peptides as novel anti-breast cancer drugs by in silico methods

An antagonistic peptide called HRAP that binds to the human HER2 molecule was designed by our computational method. In silico docking study demonstrated the specific interaction of HRAP with the dimerization domain in the HER2 molecule. Interestingly, HRAP inhibited proliferation of HER2-overexpressed human breast cancer cell lines. However, it had little cellular cytotoxicity (apoptosis inducibility). The cell proliferation inhibition was associated with the suppression of phosphorylation of PTEN and Akt. Thus, HRAP is the first HER2-binding small peptide antagonist rationally designed by a computer-aided SBDD method and is useful for the development of peptide mimetics to generate novel anti-breast cancer drugs.