Naoyuki Okita

Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks

Replication protein A2 (RPA2), a component of the RPA heterotrimer, is hyperphosphorylated and forms nuclear foci in response to camptothecin (CPT) that directly induces replication‐mediated DNA double‐strand breaks (DSBs). Ataxia‐telangiectasia mutated and Rad3‐related kinase (ATR) and DNA‐dependent protein kinase (DNA‐PK) are activated by CPT, and RPA2 is hyperphosphorylated in a DNA‐PK‐dependent manner. To distinguish the roles of phosphatidylinositol 3‐kinase‐related protein kinases including DNA‐PK, ataxia‐telangiectasia mutated (ATM), and ATR, in the response to replication‐mediated DSBs, we analyzed RPA2 focus formation and hyperphosphorylation during exposure to CPT. ATR knock‐down with siRNA suppressed CPT‐induced RPA2 hyperphosphorylation and focus formation. CPT‐induced RPA2 focus formation was normally observed in DNA‐PK‐ or ATM‐deficient cells. Comparison between CPT and hydroxyurea (HU) indirectly inducing DSBs showed that RPA2 hyperphosphorylation is DNA‐PK‐dependent in CPT‐treated cells and DNA‐PK‐independent in HU‐treated cells. Although RPA2 foci rapidly formed in response to HU and CPT, the RPA2 hyperphosphorylation in HU‐treated cells occurred later than in the CPT‐treated cells, indicating that the DNA‐PK dependency of RPA2 hyperphosphorylation is likely to be related to the mode of DSB induction. These results suggest that DNA‐PK is responsible for the RPA2 hyperphosphorylation following ATR‐dependent RPA2 focus formation in response to replication‐mediated DSBs directly induced by CPT.

Differential responses of white adipose tissue and brown adipose tissue to caloric restriction in rats

Caloric restriction (CR) slows the aging process and extends longevity, but the exact underlying mechanisms remain debatable. It has recently been suggested that the beneficial action of CR may be mediated in part by adipose tissue remodeling. Mammals have two types of adipose tissue: white adipose tissue (WAT) and brown adipose tissue (BAT). In this study, proteome analysis using two-dimensional gel electrophoresis combined with MALDI-TOF MS, and subsequent analyses were performed on both WAT and BAT from 9-month-old male rats fed ad libitum or subjected to CR for 6 months. Our findings suggest that CR activates mitochondrial energy metabolism and fatty acid biosynthesis in WAT. It is likely that in CR animals WAT functions as an energy transducer from glucose to energy-dense lipid. In contrast, in BAT CR either had no effect on, or down-regulated, the mitochondrial electron transport chain, but enhanced fatty acid biosynthesis. This suggests that in CR animals BAT may change its function from an energy consuming system to an energy reservoir system. Based on our findings, we conclude that WAT and BAT cooperate to use energy effectively via a differential response of mitochondrial function to CR.

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.

DNA damage-induced CHK1 autophosphorylation at Ser296 is regulated by an intramolecular mechanism

CHK1 phosphorylates CHK1 by protein kinase assay (View Interaction: 1, 2, 3)

Caloric restriction-associated remodeling of rat white adipose tissue: effects on the growth hormone/insulin-like growth factor-1 axis, sterol regulatory element binding protein-1, and macrophage infiltration

The role of the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis in the lifelong caloric restriction (CR)-associated remodeling of white adipose tissue (WAT), adipocyte size, and gene expression profiles was explored in this study. We analyzed the WAT morphology of 6–7-month-old wild-type Wistar rats fed ad libitum (WdAL) or subjected to CR (WdCR), and of heterozygous transgenic dwarf rats bearing an anti-sense GH transgene fed ad libitum (TgAL) or subjected to CR (TgCR). Although less effective in TgAL, the adipocyte size was significantly reduced in WdCR compared with WdAL. This CR effect was blunted in Tg rats. We also used high-density oligonucleotide microarrays to examine the gene expression profile of WAT of WdAL, WdCR, and TgAL rats. The gene expression profile of WdCR, but not TgAL, differed greatly from that of WdAL. The gene clusters with the largest changes induced by CR but not by Tg were genes involved in lipid biosynthesis and inflammation, particularly sterol regulatory element binding proteins (SREBPs)-regulated and macrophage-related genes, respectively. Real-time reverse-transcription polymerase chain reaction analysis confirmed that the expression of SREBP-1 and its downstream targets was upregulated, whereas the macrophage-related genes were downregulated in WdCR, but not in TgAL. In addition, CR affected the gene expression profile of Tg rats similarly to wild-type rats. Our findings suggest that CR-associated remodeling of WAT, which involves SREBP-1-mediated transcriptional activation and suppression of macrophage infiltration, is regulated in a GH–IGF-1-independent manner.

An Mdm2 antagonist, Nutlin-3a, induces p53-dependent and proteasome-mediated poly(ADP-ribose) polymerase1 degradation in mouse fibroblasts

Nutlin-3a (Nutlin) is an Mdm2 inhibitor and is potent to stabilize p53, which is a tumor-suppressor involved in various biological processes such as cell cycle regulation, DNA repair, and apoptosis. Here we demonstrate that Nutlin treatment in mouse fibroblast cell lines reduces the protein levels of poly(ADP-ribose) polymerase1 (Parp1). Parp1 functions in DNA repair, replication, and transcription and has been regarded as a target molecule for anti-cancer therapy and protection from ischemia/reperfusion injury. In this study, first we found that Nutlin, but not DNA damaging agents such as camptothecin (Cpt), induced a decrease in the Parp1 protein levels. This reduction was not associated with cell death and not observed in p53 deficient cells. Next, because Nutlin treatment did not alter Parp1 mRNA levels, we expected that a protein degradation pathway might contribute to this phenomenon. Predictably, a proteasome inhibitor, MG132, inhibited the Nutlin-induced decrease in the levels of Parp1 protein. These results show that Nutlin induces the proteasomal degradation of Parp1 in a p53-dependent manner. Thus, this study demonstrates characterization of a novel regulatory mechanism of Parp1 protein. This novel regulatory mechanism of Parp1 protein level could contribute to development of inhibitors of the Parp1 signaling pathway.

Involvement of lysosomal dysfunction in autophagosome accumulation and early pathologies in adipose tissue of obese mice

ABSTRACT Whether obesity accelerates or suppresses autophagy in adipose tissue is still debatable. To clarify dysregulation of autophagy and its role in pathologies of obese adipose tissue, we focused on lysosomal function, protease maturation and activity, both in vivo and in vitro. First, we showed that autophagosome formation was accelerated, but autophagic clearance was impaired in obese adipose tissue. We also found protein and activity levels of CTSL (cathepsin L) were suppressed in obese adipose tissue, while the activity of CTSB (cathepsin B) was significantly enhanced. Moreover, cellular senescence and inflammasomes were activated in obese adipose tissue. In 3T3L1 adipocytes, downregulation of CTSL deteriorated autophagic clearance, upregulated expression of CTSB, promoted cellular senescence and activated inflammasomes. Upregulation of CTSB promoted additional activation of inflammasomes. Therefore, we suggest lysosomal dysfunction observed in obese adipose tissue leads to lower autophagic clearance, resulting in autophagosome accumulation. Simultaneously, lysosomal abnormalities, including deteriorated CTSL function and compensatory activation of CTSB, caused cellular senescence and inflammasome activation. Our findings strongly suggest lysosomal dysfunction is involved in early pathologies of obese adipose tissue.

Autophagosomes accumulate in differentiated and hypertrophic adipocytes in a p53-independent manner.

Autophagy is induced by several kinds of stress, including oxidative, genotoxic, endoplasmic reticulum and nutrient stresses. The tumor suppressor p53, which is a stress sensor, plays a critical role in the regulation of autophagy. Although p53 is required for starvation (nutrient deficient stress)-induced autophagy, it is still not clear whether p53 is also required for the autophagy observed in differentiated and hypertrophic adipocytes, which accumulate excessive amounts of nutrients in the form of triglycerides. In this study, we demonstrated that starvation induces autophagy in p53-proficient adipocytes, but not in p53-deficient adipocytes as previously reported. On the other hand, autophagy was equally observed in both p53-deficient and -proficient differentiated and hypertrophic adipocytes. Similar results were obtained by in vivo analysis using white adipose tissue of high-fat diet-induced obese mice. Moreover, unexpectedly, the autophagy observed in the differentiated and hypertrophic adipocytes involved increased accumulation of autophagosomes and decreased autophagic flux. Thus, we concluded that in differentiated and hypertrophic adipocytes autophagosomes accumulate in a p53-independent manner, and this accumulation is caused by reduced autophagic flux.

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose).

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (Vmax) and the michaelis constant (km) of PARG reaction were 4.46 microM and 128.33 micromol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 microM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

Structure-based discovery of a novel non-peptidic small molecular inhibitor of caspase-3.

Ac-DNLD-CHO is a novel caspase-3 specific peptide inhibitor that was rationally designed by our computational strategy. The specificity was shown to be due to the specific interaction of NLD moiety with the active site of caspase-3 on the basis of docking mode and site-directed mutagenesis analyses. Here, we computationally screened non-peptidic small molecular inhibitors of caspase-3 from our chemical library using a reliable pharmacophore derived from the specific binding mode of NLD. Through in vitro enzyme assay of the screened candidate compounds, we discovered a novel caspase-3 specific small molecular inhibitor, CS4566, which has a unique scaffold structure. The binding mode of CS4566 to caspase-3 mimics that of NLD, especially LD moiety. This represents a promising lead compound for creating non-peptidic pharmaceuticals for caspase-mediated diseases, such as neurodegenerative disorders.